Crystallographic Investigation of the Ubiquinone binding site of Respiratory Complex II and its Inhibitors

Li-shar Huang; Peter Lümmen; Edward A Berry

doi:10.1016/j.bbapap.2021.140679

. Author manuscript; available in PMC: 2022 Sep 1.

Published in final edited form as: Biochim Biophys Acta Proteins Proteom. 2021 Jun 3;1869(9):140679. doi: 10.1016/j.bbapap.2021.140679

Crystallographic Investigation of the Ubiquinone binding site of Respiratory Complex II and its Inhibitors

Li-shar Huang ¹, Peter Lümmen ¹, Edward A Berry ¹

PMCID: PMC8516616 NIHMSID: NIHMS1742189 PMID: 34089891

Abstract

The quinone binding site (Q-site) of Mitochondrial Complex II (succinate-ubiquinone oxidoreductase) is the target for a number of inhibitors useful for elucidating the mechanism of the enzyme. Some of these have been developed as fungicides or pesticides, and species-specific Q-site inhibitors may be useful against human pathogens. We report structures of chicken Complex II with six different Q-site inhibitors bound, at resolutions 2.0 - 2.4 Å. These structures show the common interactions between the inhibitors and their binding site. In every case a carbonyl or hydroxyl oxygen of the inhibitor is H-bonded to Tyr58 in subunit SdhD and Trp173 in subunit SdhB. Two of the inhibitors H-bond Ser39 in subunit SdhC directly, while two others do so via a water molecule. There is a distinct cavity that accepts the 2-substituent of the carboxylate ring in flutolanil and related inhibitors. A hydrophobic “tail pocket” opens to receive a side-chain of intermediate-length inhibitors. Shorter inhibitors fit entirely within the main binding cleft, while the long hydrophobic side chains of ferulenol and atpenin A5 protrude out of the cleft into the bulk lipid region, as presumably does that of ubiquinone. Comparison of mitochondrial and Escherichia coli Complex II shows a rotation of the membrane-anchor subunits by 7° relative to the iron-sulfur protein. This rotation alters the geometry of the Q-site and the H-bonding pattern of SdhB:His216 and SdhD:Asp57. This conformational difference, rather than any active-site mutation, may be responsible for the different inhibitor sensitivity of the bacterial enzyme.

Keywords: Protein structure, active site, membrane protein, succinate-quinone reductase, ubiquinone, oxidoreductase, ligand binding, X-ray crystallography

1. Introduction:

1.1. Complex II

Respiratory Complex II is an important mitochondrial oxidoreductase that links the Krebs cycle with the respiratory chain by oxidizing succinate to fumarate in the mitochondrial matrix and reducing ubiquinone to ubiquinol in the mitochondrial inner membrane [1-3]. Complex II also exists in proteobacteria, and more distant but evolutionarily related homologs are spread through all branches of life [4-7].

The structure of E. coli Complex II was determined in 2003 [8], and that of mitochondrial Complex II is known from later crystallographic studies of the porcine, avian, and nematode complexes [9-14]. Later structures from an improved crystal form of the E. coli complex [15] resolve a number of apparent differences between the earlier E. coli structures and the mitochondrial enzyme.

Figure 1 shows the subunit structure of Complex II. It consists of four subunits: A flavoprotein (chain SdhA, yellow and brown in Figure 1), an iron-sulfur protein (SdhB, green), and two small transmembrane subunits (SdhC and SdhD) that anchor the complex to the membrane with SdhA and SdhB extrinsic on the matrix side. Two horizontal amphipathic helices, one on the distal surface of each of the anchor proteins, contribute to a hydrophilic patch which is exposed to the intermembrane space and presumably serves to keep the transmembrane domain vertical in the membrane. Additional proteins are required for assembly and co-factor incorporation [2, 16, 17].

Succinate is oxidized at the dicarboxylate site in the flavoprotein, transferring electrons to the covalently bound flavin moiety (FAD). From FAD the electrons proceed through SdhB via a series of three different iron-sulfur clusters to a quinone binding site (Q-site) near the membrane interface, where quinone is reduced. A heme moiety within the anchor subunits is located within electron transfer distance of both the Q-site and the third iron-sulfur cluster, but seems not to be required for succinate-quinone reductase activity [18-20].

In humans, genetic defects in Complex II have been linked to disease [21-24]. Defects in subunits SdhB, C, or D; or in factors required for their assembly, result in formation of tumors (paraganglioma and pheochromocytoma). For the most part, mutations in Subunit A do not result in tumorigenesis, but rather give rise to recessive traits of myopathy and neurodegeneration ("bioenergetic disease") [21]. However exceptions in which mutations in SdhA result in tumors have been reported [25-28], and a mutation in the SDH assembly factor 2 (SdhAF2), involved in flavinylation of SdhA, results in familial paraganglioma [17, 29-31].

The name “Complex II” was coined for a “particulate” (not soluble in the absence of detergents) preparation from bovine mitochondria that catalyzed succinate-quinone reductase (SQR) activity [32-34]. A soluble form can be prepared [35-40], that oxidizes succinate using artificial electron acceptors (succinate dehydrogenase activity) but does not reduce ubiquinone. In its purified form [38-40] it consists of two subunits: the flavoprotein SdhA and iron-sulfur protein SdhB (the "catalytic subunits"). When precautions are taken to prevent oxidation of the iron-sulfur center S3 [41, 42], the soluble SDH can be reconstituted with stripped membranes to regenerate succinate oxidase [43], or with solubilized membrane preparations containing the anchor peptides, to generate functional succinate-cytochrome c reductase [44, 45] or SQR [46-50]. The forces holding the catalytic subunits onto the anchor domain appear to be mainly hydrophobic, as chaotropes favor dissociation while ammonium sulfate reverses the dissociation brought about by chaotropes [51].

Historically the term succinate dehydrogenase (SDH) refers to the soluble subcomplex, while Complex II refers to the intact heterotetramer. More recently SDH has come to be used for the heterotetramer, and in particular the four subunits and the genes that encode them are called SdhA, SdhB, SdhC, and SdhD; or SDH1, SDH2, SDH3, and SDH4.

Complex II is a member of a larger “superfamily” of membrane-bound oxidoreductases coupling the succinate/fumarate redox pair to a hydrophobic quinone, the succinate:quinone oxidoreductases (SQORs) . These enzymes share very similar Flavo- and iron-sulfur- proteins, but differ in the membrane anchor subunits. SQORs can be classified into six classes, types A through F, based on the number of subunits and heme moieties in their anchor domains [4, 7, 52, 53]. Mitochondrial Complex II falls into class C, with two subunits and one heme in the anchor. In this report we use “Complex II” to refer specifically to mitochondrial Complex II or very similar proteobacterial Type C SQRs.

Some members of the SQOR family function normally in the opposite direction, as quinol:fumarate reductases (QFRs) rather than SQRs. QFRs are also called by the more general term Fumarate Reductase (FRD), and the subunits of QFRs are FrdA, FrdB, etc. In some di-heme SQORs with a “distal” Q-site (on the external side of the membrane), the overall reaction is proton-motive, allowing the proton gradient to drive the redox reaction in the physiologically relevant direction [54-58]. Electron transfer in Complex II is not coupled to proton translocation [56, 59, 60]

1.2. Q-site inhibitors.

One class of Complex II inhibitors, including oxaloacetate (OAA) and malonate, inhibits the SDH activity of the 2-subunit complex and both the SDH and SQR activity of the 4-subunit complex. These inhibitors compete with succinate for the dicarboxylate binding site, which is located in the SdhA subunit. The residues making up this site are highly conserved, and these inhibitors inhibit SQRs and QFRs from a wide phylogenetic range of organisms.

In contrast to the dicarboxylate site inhibitors, another class of inhibitors typified by 2-thenoyltrifluoroacetone (TTFA) and carboxin, can be identified because while inhibiting the SQR activity, they do not inhibit, or only partially inhibit [61], SDH activity with artificial electron acceptors. Activity of the soluble 2-subunit SDH preparation is not inhibited [61]. These compounds are thus inhibitors of ubiquinone reduction by the enzyme. The Q-site inhibitors carboxin and derivatives (flutolanil, boscalid, penthiopyrad, fluopyram) are commercially important fungicides, thiapronil has been investigated as an insecticide [62], and flutolanil has been suggested as a scaffold for design of ascaricides [63]. Acquired resistance to SQR inhibitors by the target organisms is a problem [64, 65], and the SQR Q-site is the target of active investigations in drug design [62, 66-69]. Understanding their mode of binding can aid in developing improved compounds and in forestalling the development of resistance in the organisms being controlled, as well as providing insight into, and experimental tools for investigating, the mechanism of the enzyme.

Kinetic studies do not consistently show competitive inhibition with respect to substrate quinone by these inhibitors [62, 70-76], but Matsson et al. [74] found competitive inhibition by TTFA with respect to pentyl-ubiquinone in the Pc. denitrificans complex, and Maklashina et al. [75] found Q₁ reduction by E. coli Complex II was inhibited by alkyldinitrophenols or pentachlorophenol in a competitive manner. TTFA or Carboxin eliminates the epr signal due to a stable semiquinone in bovine Complex II [77]. It seems likely that these inhibitors block the binding of ubiquinone at the active site.

Early studies sought to identify residues in SQR and QFR involved in quinone binding by using site-directed mutagenesis [76, 78-80], inhibitor-resistant mutations [81-83], and affinity photolabeling [79, 84, 85]. The residues involved were located in SdhB, C, and D. and were attributed to two quinone binding sites, with one site near either side of the membrane. While evidence for a site on the proximal side of the membrane has solidified and been confirmed by crystallographic studies, recent work has failed to provide strong evidence for the distal Q-site of Complex II or E. coli QFR, and it has been suggested that these enzymes have only the proximal site, located at the interface of SdhB with the anchor subunits SdhC and D [3, 86, 87].

The Complex II Q-site seems to be rather unique with respect to its inhibitors. Carboxin and TTFA do not inhibit other quinone-binding sites, including that of E. coli [75] or W. succinogenes QFR [88]; or B. subtilis SQR [89]. The general Q-site inhibitor heptyl-hydroxyquinoline-N-oxide (HQNO), which inhibits Q sites in E. coli QFR [75], B. subtilis SQR, Complex III, the photosynthetic b6f complex, Photosystem II, and cytochrome bd and bo quinol oxidases, does not inhibit Complex II [75, 89, 90].

Detailed comparison of the Q-sites of E. coli SQR and QFR [3, 86, 91] shows little in common, and lack of conservation of Complex II Q-site residues in W. succinogenes QFR has previously been reported [52]. Figure 2 aligns portions of SdhB, C, and D contributing to the Q site, from Complex II of various species. Residues contributing to the Q site are highlighted in yellow. Examples of SQORs of types A, B, and D are also included as the last three lines for each subunit. For SDHB there is significant identity due to the common fold and conserved cluster-ligand cysteines, but very little identity among the highlighted Q-site residues. For the anchor subunits SDHC and SDHD there is even less identity, although the Type A SQOR (M. tuberculosis) SDH2 conserves the critical C:Arg43 and D:Asp57-Tyr58 as well as several other highlighted residues in each subunit. The possibility that mycobacterial QCOR Type A complexes have a proximal Q site similar to that of complex II will be considered in section S2.4.6 in regard to the anti-mycobacterial action of ferulenol.

Figure 2. — Alignment of Complex II sequences in the region of the Q-site.

Rhodoquinol-fumarate reductase of A. suum does have the conserved residues of the SQR Q-site, and the Xray structure [14, 92] shows its Q-site to be much the same as that of SQR. This is consistent with its phylogeny deduced from the flavoprotein sequence [93], which places it among mitochondrial SQR enzymes, and with its presumed role as an SQR in certain stages of the parasite’s life cycle [94, 95].

Figure 3 shows the structure of the natural substrate ubiquinone and a number of Q-site inhibitors. Panel A shows ubiquinone and molecules that more or less resemble ubiquinone or ubiquinol, having an aromatic ring with two O atoms on opposite sides. Atpenin A5 (AA5) somewhat resembles ubiquinol, with two methoxy groups and (in one tautomeric state) two OH groups on the ring. Ferulenol resembles AA5, but the two methoxy groups are replaced by a second ring, resembling not ubiquinone but naphthoquinone. 1,4-Naphthoquinols such as menaquinol-1 [75, 96] and reduced plumbagin [97] or lapachol [98] can be used as substrates for Complex II in studying the reverse reaction (fumarate reduction). E. coli SQR can function as a fumarate reductase in anaerobically grown cells [99], in which the endogenous quinone is menaquinone. But 2-OH-3-alkyl-1,4 naphthoquinones such as SN5949 (2-hydroxy-,3-(2-Me-octyl)-1,4-naphthoquinone) [100], better known as inhibitors of Complex III [101-103], also inhibit vertebrate Complex II [90, 101]. and MQ₁ is a competitive inhibitor of the SQR activity of E. coli complex II [75]. Perhaps the way in which hydroxynapthoquinones inhibit SQR activity is by acting as a substrate with a redox potential so low that they cannot be reduced by succinate. In any case it seems likely that menaquinone derivatives bind at the Q-site of Complex II, and the failure of HQNO to inhibit is thus unexpected. It would be helpful to have a structure of Complex II with menadione or a 2-hydroxy-1,4-napthoquinone inhibitor bound^*.

The four inhibitors in panel B of Figure 3 have structural similarity to benzanilide, and they all bind in a similar way. Flutolanil is a substituted benzanilide, with 2-trifluoromethyl on the ring of the benzoate and a 3-isopropoxy group on the ring of aniline. The 2- substituent on the carboxylate ring is important, as structure-activity studies of benzanilide inhibitors [104] show that such an ortho- substituent on the benzoate moiety is required for significant inhibition. Carboxin is likewise an anilide, but the ring of the carboxylate is dihydro-1,4-oxathiin, with a methyl group ortho to the carboxylate. In TTFA and thiapronil the carbonyl is a keto group rather than amide. Thiapronil has 2-chlorobenzoate for the carboxylate, while the ring of TTFA is 5-membered thiophene and lacks the ortho substituent.

The other end of these molecules is variable, although generally hydrophobic. It would seem the specificity in this class of inhibitors lies mainly in the ortho-substituted ring, and the carbonyl group. We will refer to this ring as the “head”, and the other end as the “tail” of the inhibitor. Interestingly this class of inhibitors, which includes most of the commercially important fungicides targeting SQR, does not much resemble the natural substrate ubiquinone, in which the carbonyl oxygens attach directly to carbons in the ring. The difference may contribute to the ease with which resistance mutations have been found to arise, in which affinity for the inhibitor is greatly reduced while maintaining affinity for the substrate. Panel C of Figure 3 shows some optimized commercial fungicides related to flutolanil and carboxin: boscalid, fluopyram, and penthiopyrad, illustrating some of the modifications that can be made to this basic design.

1.3. X-ray studies.

Location of the binding site of Q-site inhibitors at the interface between SdhB and the two small membrane anchor subunits was confirmed crystallographically by location of DNP-17 [8] and later atpenin A5 (AA5) [105] in the E. coli enzyme; TTFA in the porcine enzyme [9], and carboxin in the chicken enzyme [12]. As of September 2019 (prior to release of the new structures described here), there were 50 structures in the Protein Database [106] of Complex II (excluding bacterial fumarate reductases, but including the Ascaris suum rhodoquinol-fumarate reductase). The structures include ten from the E. coli enzyme, 23 from the porcine [9, 107], five from chicken [12, 13, 108], and 12 from A. suum [14, 63, 92]. Of these, 39 have a specific inhibitor bound at the Q site. Among those carboxin is represented six times, flutolanil four times, AA5 three times, and pentachlorophenol twice. Thirteen of the porcine structures contain variants of flutolanil. However most of the 39 inhibitor structures were determined at relatively low resolution, >2.8 Å (exceptions being carboxin at 2.1 [12] or 2.4 [15] Å, pentachlorophenol at 2.61 Å [107], and NN23 in the A. suum enzyme at 2.25Å [92]). At such low resolution water molecules cannot be reliably placed, and details of binding and even orientation of the inhibitors can be quite unclear.

The structures of chicken Complex II tend to have higher resolution, which allowed us to correctly identify for the first time [12] the non-proline cis-peptide in SdhA, the correct structure of the catalytic base arginine and OAA at the dicarboxylate site, and the nature of the covalent adduct in the 3-nitropropionate-inhibited active site of Complex II. The presence of the non-Pro cis-peptide has been confirmed in subsequent E. coli [15] and A. suum [92] structures. The E. coli structures also confirmed our model of the dicarboxylate site (which is the same as that in the earlier high-resolution Shewanella FCc structure PDB ID: 1QJD). The nature of the nitropropionate adduct has since been confirmed and further characterized in E. coli QFR [109].

In this report we describe structures of chicken Complex II with six different Q-site inhibitors, at resolutions from 2.0 to 2.4 Å, including the first structures with Ferulenol and Thiapronil. We also discuss a further refinement of our previous structure with carboxin at 2.1 Å and of the uninhibited protein from two different crystal forms at 2.2 and 1.8 Å.

2. Results and Discussion

2.1. Structure in the region of the Q-site

Location of the Q site and structural elements involved in its formation.

As mentioned in the introduction, the Q site is located at the interface of three of the four subunits of Complex II: SdhB, C, and D. Figure 2 shows alignment of the sequences of relevant portions of these subunits from a wide phylogenetic range of organisms. Those residues with atoms within 4 Å of the inhibitor in at least one of the structures are highlighted in yellow. Most of these residues are strictly conserved, but some are variable and might result in species-specificity of the inhibitors. The role of individual binding-site residues will be discussed below. In comparing these residues in structures from different organisms, we will adhere to the numbering used in the chicken structures. The corresponding sequence numbers used in structures from the porcine, A. suum, and E. coli structures are given, for key residues, in the Table. Numbering of corresponding residues in human and yeast Complex I are also given. When the subunit is not explicitly given in the text, residues will be denoted by subunit letter followed by 3-letter code for residue type, and residue number as in B:His216.

Table.

Key residues of the SQR Q-site as numbered in different species

Species	example	SdhB				SdhC						SdhD
Chicken:	1YQ3	P169	W173	H216	I218	I27	W32	M36	S39	I40	R43	D57	Y58
porcine:	1ZOY	P169	W173	H216	I218	I30	W35	M39	S42	I43	R46	D90	Y91
A.suum:	5C2T	P193	W197	H240	I242	L60	P65	W69	S72	G73	R76	D106	Y107
E.coli:	2WDQ	P160	W164	H207	I209	L15	F20	A24	S27	I28	R31	D82	Y83
M. smegmatis: 6LUM		P185	S189	R231	T233	---	G23	M27	W30	V31	R34	D118	Y119
Human:		P197	W201	H244	I246	I56	W61	M65	S68	I69	R72	D113	Y114
S. cerevisiae:		P190	W194	H237	I239	L81	P86	W90	S93	S94	R97	D119	Y120
U. maydis:		P206	W210	H253	I255	F82	F87	W91	S94	I95	R98	D122	Y123
M. graminicola:		P220	W224	H267	I269	L65	P70	W74	S77	I78	R81	D129	Y130
P. denitrificans:		P181	W185	H228	I230	L15	L20	A24	S27	I28	R31	D82	Y83

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For the species having a known SQR structure, an example PDB ID is given and the numbering is that used in the structure. In the chicken or porcine cases this is the numbering of the mature or presumptive mature protein, otherwise it is the sequence of the gene product. The gene product sequence is also used for those complexes with no known structure. In some literature, S. cerevisiae mutations have been reported using the sequences of mature Sdh3 and 4. To obtain the mature numbering subtract 50 and 31, respectively) from the values given here.

Figure 4 shows enlarged views of mitochondrial Complex II around the Q-site, with space-filling models of bound AA5 or thiapronil to locate the site. The backbone is shown as secondary-structure cartoon, color-coded by subunit as in Figure 1, with selected pertinent side chains rendered. The mouth of the Q site is framed by elements of SdhB, C, and D in an inverted triangular arrangement, highlighted by more saturated colors in Figure 4.

SdhC (salmon-pink) has an N-terminal extra-membranous extension of 32 residues before the start of the first transmembrane helix (TMH). Residues 4 to 18 form an α-helix (labeled “N-term helix” in Figure 4A) which interacts with SdhA and SdhB. Between that helix and the start of the first transmembrane helix at residue 33 is extended random coil (supported by SdhB) except for a loop, tied off by a single α-helical H-bond between residues 26 and 30, and further stabilized by a water molecule H-bonding the amide nitrogens of residues 26, 27,28 and the side chain of C:Ser28. This loop is just above the Q-site and forms part of the top of the above-mentioned triangle framing the site. After two more residues including C:Trp32, the first transmembrane helix starts. This helix angles back under the Q-site, making the left side of the triangle and contributing four more residues to the binding site (Figure 4B). The C-terminal end of the second TMH of SdhD (dark blue in Figure 4) forms the right side of the triangle, with D:Asp57 and D:Tyr58 contributing to the binding site.

The top of the triangle includes, in addition to the above-mentioned loop 26-30 of SdhC, a short (two-turn) helix of SdhB (residues 168 - 175, green). Deeper in, a loop of SdhB including residues 215 to 219 contributes to the lining of the pocket. These two segments of SdhB are linked by the iron-sulfur cluster 3 (Fe₃S₄), with cluster ligands B:Cys168 from the former and B:Cys215 from (and B:Cys221 just outside) the latter. This positions the cluster (olive-green and orange space-filling model) deeper in than, and just above, the binding pocket (as seen in the orientation of Figure 4B).

Figures 4B and C show AA5 in a frontal and side view of the binding site, respectively. Panels D and E show thiapronil in the same two orientations. The Q-site inhibitors can be divided into three groups depending on how much of the binding site they occupy and whether part of the molecule extends out into the surrounding lipid/detergent phase when they are bound. The “head” group of all the inhibitors is positioned most deeply in the pocket, approaching B:His216 and the heme propionates. This end is to the right in the side views of Figures 4C and E.

The long tails of Inhibitors AA5 and ferulenol extend out through the mouth of the main pocket as shown in Figure 4C for AA5. Presumably ubiquinone with its ten isoprenoid units does the same. The small inhibitors TTFA and carboxin fit entirely within the main pocket of the Q site, between SdhB His216 on one end and SdhC: Ile27, Trp32, and Met36 on the other. The site does not close over the inhibitor, the tail of which is exposed to the solvent.

With the intermediate-length inhibitors flutolanil and thiapronil, the tail extends between the three SdhC residues mentioned above, displacing them and projecting into a “tail pocket”, in the elbow between the start of the first transmembrane helix of SdhC and its N-terminal extension. This can be seen with thiapronil in Figure 4E: The “tail” of thiapronil ends in a phenyl ring, located at the upper left end of the inhibitor in Figure 4E. This tail pocket will be further described in Section 2.2.7 below.

2.2. Overview of binding interactions: the role of common features of the inhibitors.

Figure 5 shows stereo views of the different inhibitors superimposed based on the protein structure, to illustrate common features of their binding and help explain results from structure-activity relationship studies [61, 64, 65, 104, 110-116]. Figure 5a shows structures of TTFA, carboxin, flutolanil, thiapronil, and AA5. Ferulenol and AA5, which bind somewhat differently, are shown separately in Figure 5b. The protein shown is from the structures with flutolanil (5a) or ferulenol (5b), with C:Ser39 also drawn from the AA5 structure in 5b to show the H-bond in that structure. Numbered black circles indicate different features or positions discussed below. The head rings and midsections of the inhibitors in Figure 5a lie in nearly the same plane so that the superposed structures form a flat ribbon with a left-hand twist. Figure 6 shows electrostatically colored surfaces of the protein in structures with AA5 and Flutolanil, showing the shape of the binding pocket and the areas described below.

Figure 6. — The surface is made with the inhibitor removed, so the inhibitor is outside and the protein is inside the surface. The outside is colored in bright colors and the inside (protein side) dark. The intersection of the surface with the clipping plane is outlined in green. The clipping plane was raised over some parts of the structure to show pieces above that plane. **(a)** and **(b)** compare structures with AA5 and flutolanil. The view is like panels c and e of Figure 4, but rotated 180° about the vertical so that the head of the inhibitors is to the left and the tail to the right; looking into the pocket from outside. The tail of AA5 hangs out into the “solvent” **(a)** while that of Flutolanil inserts into the tail pocket **(b)**. At the left end of the inhibitors are the head ring of Flutolanil and the methoxy groups of AA5. Behind these the head pocket opens into the ortho cavity. On the left the pocket is terminated by His216 (above, behind), a heme propionate (in front below) and Arg43 below. Trp173 is directly below (behind) the inhibitor, hidden by the surface; Tyr58 is also below the surface, partly exposed in A, the bulge due to OH atom is visible in B (arrow). **(c)**. Another view of the structure with flutolanil, rotated about 90° about the horizontal so that the view is from below (in the orientation of Figure 4), looking up through Ile40 (which has been removed by the front clipping plane) at the inhibitor and beyond to the Fe₃S₄ cluster. The ortho cavity containing the trifluoromethyl group, which was behind the inhibitor in B, projects below it in C (labeled 1). The water-filled cavity extending from the head pocket is visible (2). **(d)**. Flutolanil again, rotated about 90° about the vertical from B so that we are looking along the length of the inhibitor, from head end, at the residues encircling the midsection and the Fe₃S₄ cluster above. The surface is sectioned through the head pocket, at the level of the ortho cavity enclosing MeF₃ group of flutolanil.

2.2.1. Universal interactions with inhibitor keto or hydroxyl group.

One feature common to all these structures is a carbonyl oxygen or phenolic OH H-bonding with D:Tyr58 and B:Trp173 (region 1 in Figure 5). In the new chicken structures, the bonds to D:Tyr58 are in the range 2.48-3.05 Å, while those to B:Trp173 are longer and more variable: 2.77-3.53 Å. D:Tyr58 seems to be completely conserved among Complex II (Type C) SQORs. It is also conserved in di-heme SDH2 of Actinomycetes. In Complex II it has been suggested to donate one of the protons for quinone reduction [8, 117]. Mutations of the corresponding residue in yeast [117] or E.coli [118] result in complete or major loss of activity. Surprisingly in E.coli mutations of this residue are less deleterious than mutations in C:Arg43, C:Ser39, or D:Asp57, resulting in loss of only 75-85% of the activity, and do not prevent growth on non-fermentable substrates [118]. Mutation to Cys in humans is associated with paraganglioma [119].

Trp173 is conserved in mitochondrial and proteobacterial Complex II. It is replaced by His, Asn, Thr, Ser, or even Ala in actinobacterialType A SQORs, however it is not clear that these complexes have a proximal Q site. If so, this may be related to different endogenous quinone present in these bacteria

This common feature has at least one exception: it has been reported that the inhibitor thiabendazole, in porcine SQR, binds with a water molecule in the position of this universal oxygen, bridging between a nitrogen atom of the inhibitor’s benzimidazol ring and the D:Tyr58 and B:Trp173 residues [107]. Even in the structures with no inhibitor, there is density in this position, modeled as a water or part of an unidentified small molecule (Sections S2.4.7-8).

It is presumed that one of the carbonyl oxygens of the substrate ubiquinone binds in this position, as modeled in the structures PDB ID: 1NEK, 1ZOY, 3VR8 and 5C3J and discussed in section 2.2.8. D:Tyr58 and B:Trp173 are surface residues. Looking from outside as in Figure 4b and 4d the inhibitor is behind them, and the oxygen making the H-bonds is on the outward-facing edge of the inhibitor.

2.2.2. Possible H-bonds to SdhC from the other (inward-facing) edge of the Inhibitor

Two of the inhibitors have an electronegative atom on the other side of the inhibitor head group that might fill the role (if any) of the other carbonyl oxygen of quinone: the nitrile N of thiapronil, and either the ring N or OH group of AA5. Two other inhibitors, carboxin and flutolanil, bind with a water molecule in this position, bridging between the inhibitor’s amide N and the protein. In the superposition (Figure 5a) the two waters, nitrile N of thiapronil, and OH group of AA5 superimpose closely (region 2 in Figure 5). These 4 atoms are positioned within possible H-bonding distance of C:Ser39, and it appears that H-bonds are formed in some of the structures. The carbonyl O of C:Met36 is also within long H-bonding distance of this position, possibly accepting an H-bond from water or the AA5 OH group at this position. In the Flutolanil structure, the S^δ atom of C:Met36 may also accept an H-bond from the water (Figure 5a). With AA5 the ring N is also in H-bonding distance of C:Ser39, actually closer than the OH. Both putative H-bonds are drawn in Figure 5b. Detailed discussion of individual cases is presented in the Supplemental Materials (section S2.4) .

TTFA has no H-bond with SdhC, unless possibly to the thiophene ring sulfur from the amide nitrogen of C:Ile40 (3.68 Å), and ferulenol has no H-bonds with SdhC. On the other hand thiabendazole mentioned above H-bonds C:Ser39 directly via the other N of its benzimidazole ring, while bonding the universal ligands D:Tyr58 and B:Trp173 via a water [107], thus switching the water to the other side in comparison with carboxin or flutolanil.

2.2.3. Lining of the central pocket; interface with the Fe₃S₄ cluster.

Ile218 and Pro169 of SdhB form the roof of the pocket as oriented in Figure 4, with B:Ile218 toward the head and B:Pro169 toward the tail of the inhibitor. As seen also in Figure 6 A, B and D, these two residues, one on each of the two segments bearing the Fe₃S₄ cluster, stand between that cluster and the occupant of the Q site. B:Ile218 is in loose (<3.8Å) van-der-Waals (vdW) contact with the cluster (S2 and S4 atoms) and with the innermost (‘head’) ring of most inhibitors. Thus B:Ile218 forms an important part of the cluster 3 environment as well as the binding pocket and may serve to position the substrate ubiquinone to accept an electron from the cluster. For most of the inhibitors the closest approach of the inhibitor to the cluster is farther toward the head pocket, passing between B:Ile218 and B:His216, with a distance of about 7 Å between the head ring and a sulfur atom of the cluster

B:Pro169, on the other cluster-S₃-bearing loop of SdhB, forms the roof over the midsection or tail of the inhibitors. These two residues are highly conserved, even to proteobacteria and mycobacteria (Figure 2A). Nonetheless, conservative mutation of B:Ile218 to valine is tolerated in Mycosphaerella graminicola [64, 65], where it results in carboxin resistance. In Botrytis cinerea, mutation at the residue corresponding to B:Pro169 to Phe, Leu, or Thr is tolerated and results in resistance to Boscalid [120-123].

Superimposing the cluster S₂ ligands on the cluster S₃ ligands (B:155-170 on B:212-227, main chain rmsd ~0.9 A), or superimposing Peptostreptococcus asaccharolyticus ferredoxin on the second domain of SdhB, suggests that B:Ile218 takes the place of the fourth Cys ligand present in an ancestral, Fe₄S₄ Cluster S3. Replacing the corresponding residue with Cys in fact results in formation of a Fe₄S₄ cluster in E.coli QFR [124] but not in B. subtilis SQR [125].

The floor of the pocket is formed mainly by C:Ile40. C:Ile40 is mostly conserved in eukaryotes and in E. coli, but is replaced by Ser or Gly in yeast, Gly in nematodes and Val in Mycobacteria (Figure 2B). Mutation from glycine to glutamate results in reduced activity, increased ROS production, and shortened lifespan in C. elegans [126]. Mutation of the corresponding residue from Ala to Val in Mycosphaerella graminicola was the most frequent cause of resistance to fluopyram reported by the Syngenta group [64]. A genotype of Botryris cinerea in which this residue is mutated from Gly to Ala (together with three other SdhC mutations outside the Q site) conferred resistance to fluopyram and penthiopyrad, but increased sensitivity to boscalid, and partially overcame the resistance to boscalid caused by the C: H216R or H216Y mutations [127].

The universal ligands D:Tyr58 and B:Trp173 described in section 2.2.1, enclosing the inhibitor from the outside, and C:Ser39 on the inner wall, complete the lining of the central part of the inhibitor pocket. These six residues can be seen circling the inhibitor pocket in cross-section in Figure 6D.

Residue C:Ile40 in SdhC takes on different conformations to accommodate the different inhibitors, as can be seen comparing Figure 5A and B. With the inhibitors carboxin, flutolanil, or thiapronil C:Ile40 is in rotamer^* 1 (Figures 5A), which puts the two terminal atoms (C^γ1 and C^δ1) in loose vdW contact (3.6A) with, and running parallel to the plane of, the inhibitor at the level of the aniline rings of carboxin and flutolanil and the thiazole ring of thiapronil. These three rings closely superimpose when the protein structures are aligned, and provide a flat surface against which the terminal atoms of C:Ile40 stack. The other three inhibitors would clash with C:Ile40 in rotamer 1, and the residue is found instead in rotamer 5. This moves C^γ1 and C^δ1 away from the inhibitor, and, reaching across the inhibitor, closer to D:Tyr58 (Figures 5B). The two residues approach within four vdW radii and thus shield more of the bound inhibitor from the solvent.

2.2.4. Head pocket

This area is a pocket surrounded by His216 of SdhB, Ser39 and Arg43 of SdhC, and the heme propionate groups. With the exception of AA5 and ferulenol, each of the inhibitors described here inserts its head ring into this pocket. AA5, and perhaps ubiquinone, project the ring methoxy groups into this area. The aromatic head rings of TTFA, carboxin, flutolanil, and thiapronil interact with the guanidino group of C:Arg43 in an apparent cation-π stacking interaction, as noted by the Syngenta group [64] and Zhu et al. [66]. Inaoka et al. [92] also noted the interaction between the corresponding Arg72 in the A. suum enzyme and flutolanil, although their interpretation was different. This arginine is likely to be protonated, the positive charge being stabilized by ion-pairs with D:Asp57 and a heme propionate, and by the relatively polar and well-hydrated nature of the region. Stacking with C:Arg43 tilts the head ring out of plane of the rest of the molecule, contributing to the left-hand twist mentioned above.

In the chicken and E. coli SQR structures, and in the later A. suum structures (pdb ID 4ysx, 4ysy, 4ysz, 4yt0, 4ytm, 4ytn, 5c2t, 5c3j), the residue corresponding to C:Arg43 is in a conformation close to rotamer 30 (ttm180 of ref [128]) but with the χ² angle rotated significantly, placing the guanidino group in a position parallel to the inhibitor. This off-rotameric position is probably stabilized by the polar interactions with D:Asp57 and D:Tyr58 of SdhD and a heme propionate, rather than by interaction with the inhibitor, since it did not vary significantly depending on the Q-site occupant.

Mutation of the residue in E. coli SQR corresponding to C:Arg43 to Ala, Lys, or His [79], or Leu [118] resulted in loss of aerobic growth. However in M. graminicola the corresponding residue is found mutated to Cys in one carboxin-resistant mutant [64]. The cation-π interaction is not likely to contribute to ubiquinone binding, since even if quinone binds in the “Q₂–site” position [105] like AA5 as discussed below (section 2.2.8), only the methoxy groups would be stacking on C:Arg43. This residue is involved in holding together the SdhC and D subunits via interaction of its guanidino group with D:Asp57; and may modulate the H-bonding properties of quinone-ligand D:Tyr58 by an H-bond to that residue, making it a better H-bond donor [8]. Its positive charge may be important for stabilizing semiquinone anion.

The head pocket is terminated on the deep end by B:His216 and the heme propionates. In this end it connects with a cavity between SdhB and SdhC containing a chain of ordered water molecules (labeled 1 – 6 in Figure 5; region labeled “2” in Figure 6c), starting with a water H-bonding between the two heme propionates.

2.2.5. Heme propionates.

The first two transmembrane helices in each of SdhC and SdhD come together to form a 4-helix bundle, with heme in the center, its iron coordinated by C:His98 and D:His46 (each in the second TMH of their respective subunits). The helix bundle spirals significantly, but at the level of the heme the two C helices are on one side of the heme macrocycle and the D helices on the other.

Interestingly in Saccharomyces cerevisiae the residue corresponding to heme ligand D:His46 is mutated to tyrosine (Figure 2c). Demonstration of rapidly fumarate-oxidizable cytochrome b in yeast membranes suggested that nonetheless it has heme [129]. Substitution of the corresponding residue to Tyr in the E. coli enzyme resulted in a heme-free Complex II that was still functional but less stable [18], proving that heme per se is not required for function. It was suggested that tyrosine H-bonds the His heme ligand in SdhC, cross-linking SdhC and SdhD in much the way that heme does, thus providing the structural role of heme. The same group later showed that in yeast membranes fumarate rapidly oxidizes cytochromes b of Complex III, presumably via the Q pool, further calling into question the evidence for heme in yeast Complex II [20]. Selection for carboxin resistance in Mycosphaerella graminicola [64] gave rise to a mutation in the His ligand in SdhC (H145R; corresponding to C:H98), suggesting function without heme or with pentavalent heme; or an Arg-His H-bond cross-linking the subunits.

Note that C:Gly46 in the first TMH is completely conserved in the sequences of Figure 2b except in S. cerevisiae, and is always preceded by Ser or Thr. Examination of the structure shows that these residues correspond to a common feature in heme-bearing 4-helix bundles. The glycine makes a “notch” in TMH1 where the edge of the heme ring approaches [130-132], and the serine or threonine hydrogen-bonds the heme-ligand histidine in TMH2, enforcing the orientation of its imidazole ring [133]. The His ligands have their imidazole planes approximately orthogonal, accounting for the “highly anisotropic low spin” heme epr spectrum [134, 135] observed [136-139]. The C:Gly46 preceded by Ser or Thr is conserved in SDH2 of Actinobacteria (M. tuberculosis in Figure 2c), which have the proximal heme positioned as that of Complex II.

As usual in such transmembrane heme-ligating helix bundles, the heme is located with its propionates directed toward the membrane surface. This brings them also to the surface of the Q-site pocket. This is confirmed by rolling-water surfaces which show the heme propionates form part of the surface of the head pocket. The closest distance between heme and Flutolanil-type inhibitors is 4.1 Å between the head ring and a carboxyl O of a heme propionate. This propionate H-bonds/ion-pairs with C:Arg43 (Figure 5). As discussed in section 2.3, the other heme propionate makes an H-bond with B:His216, indirectly via a water molecule in the vertebrate structures and directly in the E. coli structures. If the heme is removed before generating the surface, the head pocket connects with the heme pocket through a tunnel passing between C:Arg43 (main chain and side chain) on one side and B:His216 side chain on the other. This also connects to another cavity containing ordered water (labeled “3” in Figure 6c), including the water linking B:His216 and heme propionate just mentioned, and leading into the space between SdhB and SdhD. As a result of these two water-filled cavities and the ionic interactions noted above, the heme propionates are in a rather polar environment.

The orientation of the heme about its pseudosymmetry axis is ambiguous, because the density does not show a clear distinction between methyl and vinyl groups on pyrrole rings B and C^* . The orientation may in fact be random, which would result in low occupancy of the vinyl C^β atoms at both positions, or the C^β atoms may be rotationally disordered, or both. In the orientation we have chosen, the methyl group on the B ring is in loose vdW contact (3.5 A) with two atoms of C:Ser50. It could be argued that a vinyl group here would be sterically restrained by the contact and so should be visible if present, so the absence of density for C^β favors the assignment we have, in which this is a methyl group. In any case we have omitted the vinyl C^β’s from the deposited models, resulting in a symmetric heme model.

2.2.6. Cavity for Ortho-substituent of head ring

Three of the inhibitors have ortho-substituents on the head ring that project into the space behind D:Tyr58 and B:Trp173, (region 5 in Figure 5). Carboxin has a methyl group, thiapronil has Cl, and flutolanil has trifluoromethyl. One methoxy group of AA5 also extends into this area.

Structure-activity studies of benzanilide inhibitors [104] show that such an ortho-substituent on the benzoate moiety is required for significant inhibition. The potency increases with size of the substituent at least up to iodide, and 2-ethyl is almost 3-fold more potent than 2-methyl benzanilide [104]. This is consistent with the spacious cavity into which this substituent projects, on the opposite side of the head pocket from C:Ser39 and the water-filled cavity; surrounded by Pro169, Ser170, Trp173, His216, and Ile218 from SdhB, Arg43 from SdhC, and Asp57 and Tyr58 from SdhD. The trifluoromethyl group of flutolanil exploits this cavity, making contacts closer than 3.6 Å with all of these 8 residues. Inaoka et al. [92] explained the small difference between the F₃Me- and I- substituted compounds as due to electron-withdrawing properties, but more favorable non-bonded interactions between F₃Me- and the lining of this pocket may be another explanation.

The surface of this cavity can clearly be seen in Figure 6: Directly behind the leftmost (“head”) end of the inhibitor in Figures 6A and B, and projecting downward from the main pocket into the region labeled “1” in 6C. The electrostatically-colored surface shows the end of the cavity is negatively charged due to the carboxylate of D:Asp57.

The cavity is wide in the area of the MeF₃ group, encircled by the residues listed above excepting B:Ser170 and B:Trp173. At the end it is partially blocked by these two residues, but a narrow spur continues between these residues and D:Asp57, until it too is blocked by B:Asn174. However there is a water in this narrow end of the cavity in all of the structures, with H-bonds to B:Asn174 as well as D:Asp57 and B:Ser170. This water is visible in Figure 5 (labeled “7”) and Figure 6b. It is likely to be a structural water, in which case the part of the cavity available to ortho- substituents would not include this narrow volume: the wide, accessible volume would be terminated by B:Trp173, B:Ser170, and the water. These are in loose contact (<3.6 A) with the MeF₃ of flutolanil, so trifluoromethyl is probably the largest ortho substituent that could be accommodated without displacement of the water or some side-chains. The eight residues surrounding the cavity, including B:Ser170 and B:Asn174 which have not been mentioned in previous sections, are conserved in all of the Complex II sequences of Figure 2a, and apparently through mitochondrial and α-, β-, and λ-proteobacterial Complex II. Mutation of B:Asn174 to Ile, which would remove the H-bond stabilizing this water, results in resistance to Boscalid in Botrytis cinerea [121, 123].

In the structure with TTFA (which has no ortho-substituent), there is a second water near the position occupied by the trifluoromethyl carbon of Flutolanil. This second water is also seen in one of the structures without inhibitor (PDB ID 2H88). In the TTFA structure, the two waters make a chain linking B:Asn174 at the end of the cavity to the thenoyl carbonyl of TTFA (in the universal O position), possibly displacing B:Trp173 from its role as universal ligand. The two waters are positioned to also H-bond the carboxylate oxygens of D:Asp57.

2.2.7. Tail pocket:

As mentioned earlier, this is an area in the angle between the start of SdhC TMH1 and its N-terminal extension, into which the tails of Flutolanil and Thiapronil extend (Region 3 in Figure 5; Figure 6). It is enclosed by the SdhC backbone between C:Ile27 and C:Trp32 and the side chains of these two residues, C:Met36, and B:Trp172. The surrounding residues are hydrophobic, and no water has been found here in any of the structures. In the structures with flutolanil or thiapronil, the tail of the inhibitor inserts between the three “tail gate” residues, C:Ile27, C:Trp32, and C:Met36; displacing the side chains of these residues.

The shape of the pocket, and changes induced by inhibitor tail insertion, are illustrated with surface drawings in Figure 6. Panels a and b compare the structure with AA5, which does not use the tail pocket, with that with flutolanil which does. In structures with no inhibitor or with inhibitors that do not contact these three residues, the tail gate residues fold compactly as illustrated in Figure 5b, and surface representation shows no cavity between them (2H88 or the ferulenol structure, not shown) or a small isolated cavity in the structure with AA5, shown in Figure 6a. As Figure 6a shows, residues C:Ile27 and C:Met36 approach each other, cutting off the tail pocket from the main binding cleft. C:Trp32 is also involved in this, but not visible in the figure.

In the structures with AA5 and TTFA (not shown) the cavity is larger but still cut off from the main binding cleft. These inhibitors make vdW contacts with C:Met36 and/or C:Ile27 but do not insert. The side chains of these residues maintain the same rotameric state but move slightly apart under the influence of contacts with the inhibitor.

In the structure with carboxin, C:Ile27 undergoes conformational change (rotamer 5 to 3) avoiding a closer-than vdW contact in the original position. That change moves the C^γ1 and C^δ1 atoms out of the way, opening a path between the tail cavity and the main binding pocket. The conformation of C:Met36 also changes slightly, rotating the terminal atom C^ε away from the approaching tail of the inhibitor.

Flutolanil and thiapronil induce a larger change in C:Ile27 (rotamer 5 to 1, Figure 5a) and insert through the resulting path. These two inhibitors also induce conformational changes in C:Trp32 and C:Met36. C:Trp32 swings away as in Figure 5a, opening the tail pocket to the outside (Figure 6b,c ). C:Met36 takes different new conformations with the two inhibitors (section S2.4.3).

A similar situation was described by Inaoka et al. [92] for binding of flutolanil derivatives in the A. suum QFR. In A. suum the SdhC tail gate residues C:Ile27 and C:Met36 are replaced by Leu(60) and Trp(69). C:Trp32 is replaced by Pro(65) which does not closely approach the tail of Flutolanil. It was noted that in the structure with flutolanil the inhibitor tail inserts between Trp69 and Leu60 while in the absence of inhibitor, these two residues are in close contact [92]. It was also shown [92] that upon replacing the isopropoxy group of flutolanil with phenyl (similar to the tail of thiapronil), the tail still resided between the tail gate residues in the A. suum (4YTM) or porcine(3ABV) structures. However when replaced with pentafluorophenoxy, which is longer, in both the porcine (3AE9) and A. suum (4YTN) structures the tail extended out of the protein (like AA5 or ferulenol) instead of occupying the tail pocket.

The IC₅₀ values for this last inhibitor (N-[3-(pentafluorophenoxy)phenyl]-2-(trifluoromethyl)benzamide) were higher than for the previous (N-biphenyl-3-yl-2-(trifluoromethyl)benzamide) or for flutolanil, suggesting that insertion into the tail pocket contributes to binding energy. In the previously mentioned study of benzanilide inhibitors, White [104] mentions that for alkoxy substituents at the 3’ position (which has isopropoxy in flutolanil), potency reached a maximum around C5 and diminished thereafter. This may also be due to inability of the tail pocket to accommodate the longer inhibitors. In the highest resolution A. suum structure (4YSX), the (N-[3-t-butyl-benzyl]-2-(trifluoromethyl)benzamide) inhibitor also has the tail extending outside.

2.2.8. How does the substrate ubiquinone bind?

In chicken Complex II crystals that have not been treated with Q-site inhibitors or that have been soaked with ubiquinone analogs there is still some density in the Q-site. This was modeled as quinone in the first structure (1YQ3), however the density was not well defined and would not support an unambiguous assignment. In the 1.8 A structure 2H88 [13] the density was considerably clearer but did not seem consistent with a ubiquinone molecule, and so was originally modeled as a cluster of waters. The density could be accounted for by poorly ordered waters in several different overlapping configurations, or by an unknown ligand co-purified or from the crystallization droplet. Both structures 1YQ3 and 2H88 are being updated with the latter option. In this report we provide a third structure with an unknown ligand in the Q-site, from a crystal that had been crystallized in the presence of HQNO. These structures are discussed further in section S2.4.7-8.

It is likely that endogenous quinone is lost in the exhaustive purification procedure. In an unpublished experiment (Sergei Dikanov and Rimma Samoilova), the semiquinone epr signal was undetectable in samples of our purified chicken Complex II poised with succinate:fumarate at different ratios. Miki et al. [141] concluded that ubiquinone was depleted in bovine SQR isolated by a different method, based on lack of a semiquinone signal in the epr spectrum. In their case incubation with five equivalents of Q₂ restored the semiquinone signal [141]. Reconstitution would seem to be an option, but the structure 2H88 is from a crystal soaked with Coenzyme Q₂, and as mentioned, density in the Q site was not consistent with ubiquinone.

Ubiquinone or a short-chain analog is present in structures PDB ID:1NEK (E. coli), 1ZOY (porcine), and 5C3J (A. suum). Rhodoquinone analogs are present in A. suum structures 3VR8 and 5C2T. In all of these structures, quinone is modeled with one O atom (O1) in H-bonding distance of the universal ligands D:Tyr58 and B:Trp173. It might be expected that the other carbonyl oxygen (O4) binds to C:Ser39, perhaps from region 2 of Figure 5, as discussed above in section 2.2.2.

Comparing UQ binding in the structure 1NEK, and AA5 binding in 2ACZ; and noting structural similarities between UQ and AA5, Horsefield et al. [105] proposed that UQ might have an alternate binding position similar to that of AA5, which they called the “Q₂-site”. The change would involve rotation, pivoting about the O1 atom bound in the universal position, so that the C4 atom would be in the position of the ring N of AA5, and the ring and the methoxy groups would superimpose roughly with those of AA5. This would put the O₄ atom near position 2 of Figure 5, directed toward C:Ser39.

Four later structures with UQ or RQ analogs do in fact show partial rotation toward the AA5 position. Of these, The closest to AA5 position is the RQ in the A. suum structure 3VR8. It is in a shallower position (farther from B:His216) than AA5 (Figure S1a), but close enough for a H-bond between O₄ and C:Ser39 (2.9 Å). The other two Ascaris structures (5C2T, 5C3J) and the porcine structure (1ZOY), the quinone is rotated to nearly the same extent as 3VR8 but is translated farther from the His216 end (Figure S1b).. This puts the O4 atom out of reach of C:Ser39, and closer to the carbonyl O of C:Met36; which however could not provide a hydrogen bond to the quinone carbonyl or donate an H⁺ when quinone gets reduced to quinol.

C:Ser39 is highly or completely conserved in mitochondrial and proteobacterial SdhC. Mutation of the equivalent Ser residue in E. coli SQR to Ala, Cys, or even Thr [79]; or in yeast to Ala [118], results in cells unable to grow on non-fermentable substrates; but mutation to Gly has been observed in M. graminicola upon selection for Fluopyram resistance [64]. Thus an important, but perhaps not obligatory, role for C:Ser39 is indicated.

As suggested in the introduction, it is likely that the mitochondrial ubiquinone-binding site can also accommodate naphthoquinones. Comparing menaquinone and ubiquinone, it might be expected that the second ring of menaquinone would occupy roughly the same area as the methoxy groups of ubiquinone. However vdW forces will force the two methoxy methyl groups apart, as can be seen in the structures with Atpenin. In our structure (section S2.4.5) the methoxy groups diverge, one into the ortho-substituent cavity and one into the connected water-filled cavity mentioned above. The second ring of naphthoquinone might clash with residues at the end of the head pocket. This was tested by positioning menadione with the di-oxo ring in possible ubiquinone positions. Superimposing on UQ in the porcine structure (PDB ID: 1ZOY), there were no close contacts with the second ring. Superimposing on RQ in 3VR8 there were mild clashes with the side chains of B:His216, D:Asp57, and C:Arg43, which could probably be accommodated by slight movement of these side chains. Superimposing on AA5 in the chicken structure presented here, there were more severe clashes.

2.2.9. H-bond with B:His216?

It has been proposed [105] that the ring of B:His216 may re-orient by rotation around its χ² bond to position its N^δ1 atom to H-bond the methoxy O atoms of AA5 (and, by extension, ubiquinone in the Q₂-site position). Unfortunately the orientation of the imidazole ring cannot be reliably determined from sub-atomic resolution crystal structures, so the ring is oriented based on chemical plausibility, to make the most favorable H-bonds. All of the chicken, porcine and A. suum structures, and the E. coli structures other than 2ACZ, have B:His216 oriented close to rotamer 1. This allows H-bonds involving both ring N's, one to D:Asp57 or heme propionate (see next section) and the other to a water molecule. These bonds would have to be broken to make the bond to the inhibitor. The proposal for reorientation was based in part on the first structure with Atpenin A5 (E. coli structure 2ACZ) [105]. In this structure waters were not modeled (appropriate given the resolution), and this histidine was rotated around χ² to direct the N^δ1 atom toward the inhibitor (rotamer 6). This model was in part the basis for proposing the Q₂-site for ubiquinone mentioned in the previous section. While the position of AA5 in this model has been confirmed by other structures included that presented here, no other structure has supported this rotamer of B:His216.

An H-bond with B:H216 cannot be a required part of ubiquinone binding, since functional SQRs with B:H216 replaced by Leu, Asn, Gln, Tyr, or Arg are found in inhibitor-resistant fungi [64, 65, 81, 122, 142] and Pc. denitrificans [74, 83]. Furthermore this residue has been replaced with Thr by site-directed mutagenesis in E. coli SQR with little effect on activity [143]. Interestingly, the mutated residue (B:Thr207) does make a long (3.4 Å) H-bond with the inhibitor carboxin, which was co-crystallized and binds exactly as in wild-type. Despite this extra H-bond and very subtle differences in the rest of the structure, the K_I for the inhibitor in the mutant enzyme (160 μM) is five times higher than the already high wild-type K_I of 30 μM [75].

Arg is found at this position (Figure 2a) in one isoform of plant SDH2 (e.g. A. thaliana SDH2-3, NCBI accession AT5G65165.1). The His216 inhibitor-resistant mutants can incur a fitness penalty, with the Asn, Tyr, and Leu mutations giving 40%, 35%, and 18% of the succinate-cytochrome c reductase specific activity in Aspergillus oryzae mitochondria [144].

On the other hand indirect evidence for an H-bond between His216 and inhibitors is available from comparing the effect of His216 mutation on affinity of inhibitors that can or cannot accept H-bonds to the head ring. The B:His216Y and B:His216L mutants were resistant to Fluopyram, which has no H-bond acceptor in the head ring, by a much smaller factor than to carboxin, Isopyrazam, and Boscalid, which have [64]. This would be consistent with an H-bond from B:His216 to the inhibitor making a significant contribution to the binding energy for those inhibitors that can accept it.

A similar correlation is seen from the relative sensitivity to carboxin and o-toluanilide, which differ only by replacing the methyl-oxathiin ring of carboxin (capable of accepting H-bonds) with a phenyl ring having a methyl group in the same (ortho) position. For controlling mycelial growth in wild-type A. oryzae, carboxin is effective at 10-fold lower concentrations than o-toluanilide. Mutation of B:His216 to L, Y, or N has little effect or slightly increases sensitivity to o-toluanilide, while decreasing sensitivity to carboxin by about 10-fold, to about the same level as toluanilide [142]. Put another way, the 10-fold greater sensitivity to carboxin depends on the presence of B:His216. Mutation of D:Asp58 (Which ion-pairs with His216) to Glu, or C:Thr45 to Ile also reduced the differential efficacy of these two inhibitors, but carboxin remained significantly more potent [142]. However suggestive these observations may be, as mentioned above there is no convincing evidence from the structures for an H-bond between His216 and any of the inhibitors.

2.2.10. Substrate and proton access to the Q-site.

Based on its position, near the matrix-side end of the transmembrane helices, the mouth of the Q-site pocket is likely to be near the surface of the membrane. There is controversy regarding the position of free ubiquinone in a bilayer, with some studies suggesting that the entire molecule is in the middle between the two leaflets, and others saying the head is at the surface at about the level of glycerol in the lipid headgroups. In any case it must visit the surfaces, and be able to “flip-flop” from one surface to the other. This is demonstrated by its ability to carry out electron transfer between water-soluble reagents across vesicle membranes [145] , as well as it’s natural chemiosmotic role as an H-carrier: In vertebrate mitochondria the quinone reduction sites, where protons are consumed, are all on the “N” side of the membrane, while the oxidation site (The Qo site of Complex III) is on the P side (Figure 7, ref [146]).

Figure 7. — The ubiquinone reduction sites (blue) of Complex I and II, and the Qi site of Complex III, are near the N side of the membrane. Ubiquinol oxidation (Red) takes place at the Qo site of complex III, near the P side of the membrane. This allows protonic equilibrium with the chemiosmotically appropriate phase. The presumed ubiquinone-binding ligands of Complex II are surface residues, obviating the need for a proton wire to connect with the bulk phase water. (Reproduced from ref [146]: Teixeira and Arantes, RSC Adv., 2019,9, 16892-16899 (published by the Royal Society of Chemistry, licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License))

We have described above two pockets of water molecules in the space between the extrinsic subunits and the membrane anchor. Neither of these corresponds to the water chains described for the E. coli enzyme [15, 105]. However the universal ligands D:Tyr58 and B:Trp173 are both highly exposed surface residues, and it is likely that they are at the level of the polar lipid headgroup layer. Proximity to the membrane surface could provide a proton source for the redox reaction, allowing re-protonation after the quinol leaves. There does not seem to be any need to propose a pathway through the protein for proton uptake and release, as was initially suggested [105, 147]. It is not unusual to have water molecules in the interface between protein subunits, where they likely compensate for imperfect surface complementarity and provide water-bridged H-bonds where the surfaces are too far apart for direct H-bonds or vdW contacts.

2.3. Differences between the E. coli and mitochondrial Q-sites, resulting from different binding of the extrinsic subunits to the membrane anchor domain.

The E. coli Complex II is in many ways an excellent model for the mitochondrial enzyme [87, 97]. Unlike fumarate reductase [3, 86, 91, 148], the E. coli SQR enzyme has a quinone binding site very similar to that of mitochondrial SQR. Most of the Q-site residues discussed above and highlighted in yellow in Figure 2 are conserved in the E. coli enzyme, and the differences are all in the “tail gate” residues and so unlikely to affect binding of TTFA and carboxin. However major differences have been reported in the sensitivity of the E. coli and mitochondrial SQR proteins to Q-site inhibitors. Carboxin and TTFA inhibit with K_Is of 1 and 10 μM in beef heart mitochondria [111], but about 30 [75] or 100 [149] fold greater in E. coli . Atpenin A5 was reported to inhibit Complex II with IC₅₀ 3.6 nM for the bovine enzyme, but 5 μM for E. coli. [116].

This difference in inhibitor sensitivity might seem puzzling given the high degree of conservation of the key residues in the Q-site between the E. coli and mitochondrial enzymes. Carboxin does in fact bind to the E. coli complex at sufficiently high concentration, and the crystal structure [15] shows that the binding mode is basically the same as in vertebrates. Comparing the structures, however, shows significant differences in the residue interactions due to a rotation of the small subunits relative to SdhB between the two structures. Figure 8 illustrates this with the carboxin-bound structures from chicken (PDB ID: 2FBW) and E. coli (2WDQ).

Figure 8. — The view is from within the membrane up toward the extrinsic subunits. The two structures (PDB ID: 2FBW chains O, P, Q and 2WDQ chains F, G, H) have been superimposed based on the segments of SdhC (salmon) and SdhD (blue) depicted. Rotation of SdhB (green) relative to the anchor subunits, as compared to the vertebrate structure, separates residues corresponding to SdhB His216 and SdhD Asp57 in the *E. coli* structure while bringing His216 closer to a heme propionate. As a result the direct H-bond between His216 and D57 in the vertebrate structure is absent and an H-bond to heme is formed in the *E. coli* structure.

Although the C and D subunits of E. coli and vertebrate SQRs are significantly different in their fold, and cannot be superimposed overall very well, the backbone shape and the relative position of the two subunits has been conserved in the region making up the Q site so that selected regions (SdhC:29-50 and SdhD:38-60, chicken mature sequence) can be simultaneously superimposed quite accurately (C^α rmsd 0.71 Å) between the chicken and E. coli structures. Likewise the second domain of SdhB, which participates in the Q site, can be superimposed with RMSD 0.55 (residues 141-243). However this is achieved with a further rotation of ~7° after the SdhC, D segments have been superimposed.

The rotation is species specific and not significantly dependent on crystal packing, or presence or type of inhibitor bound: Comparing the two molecules in the chicken carboxin structure with three in the E. coli structure gives six measurements of this rotation, average 6.79° ±0.25°. Comparing with E. coli in a different crystal form (PDB ID:1NEK) gave rotations of 6.62° and 6.96°. Comparing our type 1 and type 2 crystals (with different inhibitors bound) gives rotation values around 1° or less, and comparing chicken with the porcine structures 1ZOY and 3SFD gave rotations of less than 1°. Comparing E. coli structures 2WDQ with 1NEK gives 0.71°, −1.06°, and 0.67° for the 3 tetramers in 2WDQ.

Thus SdhB in E. coli is twisted 7° relative to the membrane subunits as compared to the situation in vertebrate mitochondrial Complex II. The axis is about 26° from the presumed normal to the membrane, and displaced to one side of the interface. This results in slight shifting of Q-site residues contributed from SdhB relative to those from the anchor peptides, thus subtly changing the geometry of the site, and may be responsible for the differences in inhibitor affinity. In Figure 8 the structures PDB ID: 2FBW (chicken) and 2WDQ (E. coli) are aligned based on the anchor subunits as described above, and the displacement in SdhB is apparent.

The most significant difference involves His216 of SdhB (His207 in E. coli), which in both structures is located between Asp57 in SdhD and a heme propionate carboxyl. As the latter two are in the anchor domain, the relative rotation of SdhB to the anchor domain moves B:His216 closer to D:Asp57 in the vertebrate enzymes, making an ion pair (~2.7 Å, range 2.40-2.97 Å) with that residue, while in the E. coli complex it is closer to the heme propionate and forms an ion pair (~2.55 Å) with it. This structural difference might be expected to contribute to the difference in midpoint potential of the hemes (−185 mV in vertebrates [136] vs +35 mV in E. coli [8]), as the polar interaction with a protonated His in E. coli would tend to stabilize the reduced form, but mutation of B:His216 to Thr had little effect on the heme midpoint potential of the E. coli enzyme [143].

The other heme propionate is ion-paired with C:Arg43 of SdhC in both structures, unaffected by rotation of SdhB. And Arg214 of chain SdhB maintains similar interactions with Asp57 and Gln53 in SdhD, due to flexibility of the arginine side chain.

The two universal inhibitor ligands, Tyr58 in SdhD and Trp173 in the SdhB, are affected by this, but the distance between them hardly changes: the difference in separation between C^αs being 0.3 Å and between the liganding atoms N^ε1 and OH only 0.1 Å. This is because the relative movement is nearly perpendicular to the distance between these residues, i.e. sideways instead of closer or farther. (Figure 8). Meanwhile the ligand carboxin takes an intermediate position and so is positioned a little differently with respect to either SdhB or the anchor, in the E. coli as compared to the mitochondrial complex. Oddly, the water bridging between carboxin and SdhC seems to move relative to SdhC even more than the carboxin does, putting it nearly equidistant from the amide N and ring S of the inhibitor in the E. coli structure, and closer to the amide N in the avian. Whether or not this is a significant difference is unclear.

B:His216 and D:Asp57 are the most frequently mutated residues found in mutants resistant to carboxin or related fungicides. Putting this together with the fact that the interaction between these two residues is different in E. coli which is resistant to carboxin compared to the mitochondria, which are sensitive; it is tempting to speculate that this salt bridge cross-linking residues in SdhB and D is critical for binding of carboxin. Is this salt bridge fixing the distance between these two parts of the Q site, and in doing so modulating the affinity for carboxin and TTFA? Carboxin sensitivity seems to correlate with presence of a short, direct ion pair between these two residues, where the B:H216Y, H216R and the D57E mutations would make a longer linker like the water-mediated E. coli situation, and H216L none at all. This would be an alternative to the idea discussed in section 2.2.9 that His216 is important for providing an H-bond with the head ring of inhibitors like carboxin.

But do the mutations of B:His216 or D:Asp57 significantly affect the geometry of the active site? Does the H-bonding arrangement of B:His216 fix the otherwise rotatable orientation of SdhB on the anchor, rather than being determined by that rotation? This is unlikely, at least in E. coli, because this angle is essentially the same in the structure PDB ID: 3WP9, in which B:His216 (207) is mutated to Thr [143]. The mutated residue makes a long H-bond to carboxin, but no H-bond to the aspartate or heme propionate; nor to any water that was modeled. After superposing the mutated structure 3WP9 with wild-type 3WDQ, based on SdhC and D (rmsd 0.211 Å), superimposition of the SdhB subunits requires a reorientation of less than 1 degree. The C^α atoms of the wild-type and mutant SdhB subunits can be aligned over the full length with rmsd 0.147 Å. The largest C^α deviation, only 0.52 Å, is at the mutated residue 207. Thus there is not a major rearrangement in the structure of SdhB, or its orientation with respect to the anchor peptides, on mutation of B:His216 and loss of its interaction with the anchor subunits. The His216Ser mutation in E. coli had no significant effect on activity [143]. On the other hand mutation of D:Asp57 to Leu in E. coli resulted in an inactive enzyme. [118].

In any case, B:His216 or D:Asp57 are the same in E. coli and in mitochondria, and so cannot be responsible for the difference. Something else must be enforcing the slightly different orientation that results in the different H-bonding pattern of D:His216. Inter-protomer contacts in the trimeric bacterial complex may affect the structure.

Alternatively, the different orientation may be due to differences in other interface residues. Residues important in binding the chicken SdhB to the anchor but not conserved in E. coli include B:Gln207, B:Arg223, B:Lys240, C:Lys31, C:Glu120; D:Lys4 and D:Ser7. In fact, comparing polar interactions between SdhB and the anchor peptides, there is little conservation between vertebrate and E. coli. Excluding the N-terminal extension of SdhC before the first TMH, which is long and flexible (and so not likely to fix the orientation), 21 polar interactions can be identified in the chicken structure between SdhB and the membrane domain proper of the anchor subunits (Table S3). Only one of these is conserved in the E. coli structure: the above-mentioned ion pair between B:Arg214 and D:Asp57 is maintained, allowed by the flexibility of the Arg side chain. Some other contacts are preserved in altered form. In the chicken structure Asn165 of SdhB H-bonds with the carbonyl O of D:Asp57, while in E. coli the Asn is conserved, but it bonds with the carbonyl of the residue corresponding to D:56 instead of 57. In both structures the residue corresponding to SdhD:His60 H-bonds with O^δ1 of that conserved Asn165 in SdhB. But in E. coli His60 is mutated to Lys, the extra length of the latter making up for the different relative position of the two subunits.

While both E. coli and vertebrate SQR can be reconstituted from resolved SDH and anchor proteins, attempts to make an active hybrid enzyme were unsuccessful [150], which could be due to differences in the interactions holding the parts together and/or a different angle required in the trimeric bacterial complex. On the other hand a hybrid consisting of bovine and Rhodospirillum rubrum was active [48]. It might be expected from their evolutionary origin that mitochondrial Complex II is more similar to α-proteobacterial Complex II than it is to the E. coli complex. The Pc. denitrificans complex, unlike that of E. coli, is inhibited by carboxin with a K_I close to that of the vertebrate enzyme [83] [89]. The heme has a midpoint potential (−176 mv) essentially the same as vertebrate, and is not reducible by succinate [89]. On the other hand sequence alignment of SdhD suggests that Pc. denitrificans, like E. coli, has an amphipathic transverse helix between TMH 1 and 2 rather than after TMH3 as in mitochondrial complex II. Also Pc. denitrificans Complex II is believed to be trimeric, like the E. coli complex (Figure 2 of [151]), unlike monomeric mitochondrial Complex II. However these structural differences may not affect the Q-site or the heme environment..

2.4. Details of the individual inhibitors.

Figure 9 shows electron density of the inhibitor and interactions with surrounding residues for each structure. Detailed descriptions and stereo figures are available in the supplementary materials available at the publisher’s web site.

2.5. Conclusion

Seven new structures of chicken Complex II have been provided, with five different inhibitors at the Q-site. An earlier structure with carboxin and a high resolution structure with no added inhibitor have been further refined. Using these structures as well as structures from other research groups, we have characterized binding of the different inhibitors, noting similarities and differences in their mode of binding. We have compared the Q-site and carboxin binding of the vertebrate mitochondrial enzyme with that in the E. coli enzyme, finding a difference in the orientation of the extrinsic subunits on the membrane anchor that may be responsible for the difference in inhibitor sensitivity. We hope the information will be useful in explaining the relative affinity of the inhibitors and the mechanism by which resistance arises as a result of certain mutations, and that this improved understanding will contribute to the design of higher affinity, more specific inhibitors and ones that are effective against the current inhibitor-resistant mutant enzymes. Reason for optimism is provided by the 200-fold greater sensitivity of the A. suum vs vertebrate Complex II to flutolanil [63], and the fact that mutations conferring resistance to some inhibitors actually increase the sensitivity to others [115, 121] (“negative cross-resistance”).

3. Methods

Thiapronil was provided by Bayer Crop Sciences (Monheim). Carboxin was purchased from Sigma-Aldrich/Supelco, TTFA and Flutolanil from Sigma-Aldrich, and ferulenol and atpenin A5 from Alexis (now Enzo Life Sciences).

Purification and crystallization were performed essentially as described [11]. Briefly, Complex II was extracted from chicken heart mitochondria using Thesit (C₁₂E₉) detergent and exchanged to dodecyl maltoside during the purification.

The final product was exchanged by repeated ultrafiltration and dilution, first into 20 mM Tris HCl pH 7.5, 0.5 mM EDTA containing 0.3 g/L undecyl maltoside and then into the same buffer containing 20 g/L octyl glucoside. Exchange directly into the octyl glucoside buffer tended to result in a more viscous solution of the protein and failure to crystallize.

No effort was made to remove endogenous oxaloacetate (OAA), and it seems likely that OAA stabilizes a conformation that is preferentially incorporated into the crystal, since the structures contain OAA at high occupancy [13].

For crystallization, protein at ~70 g/L was mixed with an equal volume of a precipitant consisting of 0.1 M HEPES pH 7.5, 50 ml/L 2-propanol, 100 g/L PEG 3350, 1.33 mM MgCl₂, 3 mM NaN₃ and various additives (MnCl₂, PEG 400; detailed in the individual PDB depositions). Crystals were set up at room temperature but incubated and stored at ~4°C. In the case of TTFA, (and HQNO) the inhibitor was added to the protein at 2-fold molar ratio to protein before the final concentration step, for co-crystallization. The other inhibitors were added to droplets after crystal growth was complete (“soaking”). Crystals were soaked with the inhibitor at least two days before freezing for data collection, at 2-fold or greater molar ratio to the total protein in the well. Details for the carboxin-soaked crystal were described previously [12] ; other compounds were soaked similarly. As described [11] two different crystal forms were obtained: type 1 crystals are orthorhombic (P2₁2₁2₁) and have a monomer in the asymmetric unit. Type 2 crystals are monoclinic (P2₁) with a dimer in the asymmetric unit.

Before flash freezing for data collection, the crystals were transferred briefly to a cryoprotectant solution consisting of glycerol diluted to 300 g/L in the precipitant. Data were collected at the ALS and SSRL synchrotron sources, and processed and reduced with denzo and scalepack (HKL package [152]). The merged scalepack files were used to start refinement in Phenix.refine (Phenix crystallography suite [153, 154]), applying the French and Wilson procedure in converting measured intensities to amplitudes. In some but not all cases the resulting amplitudes and final map coefficients were put on an absolute scale by scaling against Fc. A single complete set of free-R flags to high resolution was generated by random selection in CNS for each of the two crystal forms, and applied to the experimental datasets to ensure free reflections were not used in refinement in any of the structures. The deposited structure factor files include the merged intensities from scalepack and resulting amplitudes together with their sigma values, the map coefficients (model plus solvent) from refinement, and the free-R flags for the observed reflections.

The native and carboxin-bound structures described here are further refinements of previous structures (PDB ID 2H88 and 2FBW), while another native (6MYT) and the structures with TTFA, flutolanil, thiapronil, ferulenol, and Atpenin A5 are based on previously unpublished data. The first chicken Complex II structures [11] were phased by molecular replacement using the E. coli structure [8] (kindly provided by S. Iwata prior to its publication). Subsequent structures were solved with Phenix by rigid-body rotation of previous structures to account for slight non-isomorphicity. In the case of the type 2 (P2₁, pseudo-orthorhombic) crystals, it was necessary to make a consistent choice of two non-equivalent possibilities for indexing in order for rigid-body refinement to succeed. Then the structure was refined by individual ADP, positional, and occupancy refinement with the maximal likelihood target in Phenix, interspersed with examination and manual rebuilding using Coot. New ligands were introduced and fit into the density manually using the graphics program O [155] or Coot [156]. Ligand models for ligands existing in the PDB were obtained by Coot. Novel ligands were constructed using the Dundee ProDRG2 [157] server or Lidia in Coot. Water molecules were added by phenix.refine, with additional waters and unidentified solvent ligands added manually. In the final rounds of refinement TLS as implemented by Phenix was used, with a single TLS group.

In the deposited structure factor files, the raw data (I-obs) are on an arbitrary scale (as output by scalepack). In most of the structures “F-obs-filtered” and map coefficients have been put on an approximate absolute scale by scaling against F-model. This was not done with the structures containing TTFA or ferulenol, or that soaked with HQNO (PDB ID: 6MYP, 6MYQ, and 6MYU). Maps based on these coefficients should be contoured by RMSD, or the density levels should be multiplied by 5.95, 8.44, or 8.14; respectively, to approximate absolute scale.

Figures 1, 4, 5, 8 and 9 were made using Molscript [158] and rendered with Raster3D [159]. Maps in Figure 9 and S9 are 2mFo - DFc maps calculated in phenix, "filling-in" missing Fo with Fc. The "rmsd" levels indicated are the RMS deviation from average electron density calculated over one asymmetric unit. Maps from these coefficients were calculated by CCP4 “fft” and converted to DSN6 maps and contoured in O. The contour renderings were exported as object files for molscript figures. The cross-sectional surface diagrams of Figure 6 were calculated and rendered with Coot. The surfaces represent the closest position the center of a water molecule could occupy without clashing with the protein, i.e. sum of vdW radii of the water and the protein atom. Sequence alignments (Figure 2) were made using ClustalW implemented at the NPS@ site [160] and edited with Mozilla HTML editor and Microsoft Word.

Supplementary Material

Supplement

Table S1 - Data Reduction Statistics

Table S2 - Refinement Statisitics

Table S3 - Polar interactions at anchor interface

Figure S1- Position of UQ and RQ from other group’s structures

Section S2.4: Detailed descriptions of binding in each structure

NIHMS1742189-supplement-Supplement.pdf^{(258.6KB, pdf)}

Stereo Figures x-eye

Figures-sx.pdf: Stereo Figures for convergent (“cross-eyed”) viewing Convergent Stereo views of Figures 1, 4, 5, 8, and 9

NIHMS1742189-supplement-Stereo_Figures_x-eye.pdf^{(1.8MB, pdf)}

Stereo Figure wall-eye

Figures-sw.pdf: Stereo Figures for divergent (“wall-eyed”) viewing Divergent Stereo views of Figures 1, 4, 5, 8, and 9

NIHMS1742189-supplement-Stereo_Figure_wall-eye.pdf^{(1.8MB, pdf)}

Rocking Gifs (ppt)

Figures.ppt has figures with the stereo drawings in motion, rocking back and forth to provide a sense of depth perception.

NIHMS1742189-supplement-Rocking_Gifs__ppt_.ppt^{(15.5MB, ppt)}

4. Acknowledgements

We would like to thank staff at the Advanced Light Source (ALS) and Stanford Synchrotron Radiation Lab (SSRL) for help with data collection, and Dieter Berg and Michael Schindler of Bayer Crop Sciences for providing the sample of thiapronil. Initial stages of the project were funded by National Institutes of Health (NIH) NIGMS grant GM62563 to EAB. During the final analysis and manuscript preparation, EAB and LSH were supported by startup funds to EAB from the State University of New York. This work is based in part on research conducted at the Lawrence Berkeley National Laboratory, which is operated by the Department of Energy (DOE), contract DE-AC03-76F00098 to the University of California. The Advanced Light Source is supported by the DOE, Office of Basic Energy Sciences, under Contract No. DE-AC02-05CH11231. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. SSRL is operated by the DOE, Office of Basic Energy Sciences. The SSRL Biotechnology Program was supported by the NIH National Center for Research Resources, Biomedical Technology Program, and by the DOE Office of Biological and Environmental Research.

Footnotes

^{(1) *}

Abbreviations used: Q-site, quinone binding site. SDH, succinate dehydrogenase. FRD, fumarate reductase. SQR, succinate-quinone reductase. QFR, quinol-fumarate reductase. SQOR, succinate-quinone oxido-reductase family. RQ, rhodaquinone. MQ₁, menaquinone-1. UQ, ubiquinone. Q₁, Q₂: ubiquinone with one or two isoprenoid units; except “Q₂-site” refers to a proposed alternate binding mode of UQ. TTFA, 2-thenoyltrifluoroacetone. AA5, Atpenin A5. vdW, van der Waals. RMS, root-mean-square. RMSD, RMS deviation (between two structures, or from the mean of electron density. Specific amino acid residues are designated by the chain letter followed by 3-letter representation of residue type and sequence number, as B:His216. Sequence numbers used for the porcine and chicken structures correspond with the numbering used in the pdb coordinates files, which mainly use the mature sequence as far as it is known. The A. suum structures use the gene product (pre-protein) sequence The sequence numbers of corresponding Q-site residues in different species are listed in the Table. Mass concentrations are expressed in units of g/L; equivalent to the values in standard SI units of kg/m³.

^{(2) *}

Menaquinone molecules are modeled in a recent cryo-EM structure of M. smegmatis SDH2 [161], but this is a different type of SQOR (Type A; with two hemes in the membrane sector), and the menaquinones are modeled near the distal side of the membrane. The proximal site that could be analogous to the Complex II Q-site is empty in the structure and is suggested to be inactive in this enzyme.

^{(3) *}

Amino acid rotamer numbers cited here refer to the system used in the molecular graphics program “coot”, based on the Richardson classification [128].

^{(4) *}

For convenience in describing structures, the nomenclature of the heme atoms defined for the protein database ligand “HEM” is used for describing heme atoms. Pyrrole rings B, C, D, and A correspond to rings I,II, III, IV or A, B, C, D of the Fischer system [140].

Accession Numbers (PDB coordinates files)

Chicken

PDB ID: 6MYO Avian Mitochondrial Complex II With Flutolanil Bound

PDB ID: 6MYP Avian Mitochondrial Complex II With TTFA (Thenoyltrifluoroacetone) Bound

PDB ID: 6MYQ Avian Mitochondrial Complex II With Ferulenol Bound

PDB ID: 6MYR Avian Mitochondrial Complex II With Thiapronil Bound

PDB ID: 6MYS Avian Mitochondrial Complex II With Atpenin A5 Bound, Sidechain Outside

PDB ID: 6MYT Avian Mitochondrial Complex II With Atpenin A5 Bound, Sidechain In Pocket

PDB ID: 6MYU Avian Mitochondrial Complex II Crystallized In The Presence Of HQNO

PDB ID: 2H88 Avian Mitochondrial Respiratory Complex II At 1.8 Angstrom Resolution

PDB ID: 2FBW Avian Respiratory Complex II With Carboxin Bound

PDB ID: 2H89 Avian Respiratory Complex II With Malonate Bound

PDB ID: 1YQ3 Avian Respiratory Complex II With Oxaloacetate And Ubiquinone

PDB ID: 1YQ4 Avian Respiratory Complex II With 3-Nitropropionate And Ubiquinone

PDB ID: 2WQY (Remodeling Of Carboxin In 2BFW)

Other structures cited:

Sus scrofa: PDB ID: 1ZOY 1ZP0 3ABV 3AE1 3AE2 3AE3 3AE4 3AE5 3AE6 3AE7 3AE8 3AE9 3AEA 3AEB 3AEC 3AED 3AEE 3AEF 3AEG 3SFD 3SFE 4YTP 4YXD

A. suum: PDB ID: 3VR8 3VR9 3VRA 3VRB 4YSX 4YSY 4YSZ 4YT0 4YTM 4YTN 5C2T 5C3J

Escherichia coli: PDB ID: 1NEK 1NEN 2ACZ 2WDQ 2WDR 2WDV 2WP9 2WS3 2WU2 2WU5

5. References

[1].Cecchini G Function and structure of complex II of the respiratory chain. Annu Rev Biochem. 2003;72:77–109. [DOI] [PubMed] [Google Scholar]
[2].Rutter J, Winge DR, Schiffman JD. Succinate dehydrogenase - Assembly, regulation and role in human disease. Mitochondrion. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
[3].Iverson TM. Catalytic mechanisms of complex II enzymes: a structural perspective. Biochim Biophys Acta. 2013;1827:648–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
[4].Hägerhäll C Succinate: quinone oxidoreductases. Variations on a conserved theme. Biochim Biophys Acta. 1997;1320:107–41. [DOI] [PubMed] [Google Scholar]
[5].Westenberg DJ, Guerinot ML. Succinate dehydrogenase (Sdh) from Bradyrhizobium japonicum is closely related to mitochondrial Sdh. J Bacteriol. 1999;181:4676–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
[6].Schäfer G, Anemüller S, Moll R. Archaeal complex II: 'classical' and 'non-classical' succinate:quinone reductases with unusual features. Biochim Biophys Acta. 2002;1553:57–73. [DOI] [PubMed] [Google Scholar]
[7].Lemos RS, Fernandes AS, Pereira MM, Gomes CM, Teixeira M. Quinol:fumarate oxidoreductases and succinate:quinone oxidoreductases: phylogenetic relationships, metal centres and membrane attachment. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2002;1553:158–70. [DOI] [PubMed] [Google Scholar]
[8].Yankovskaya V, Horsefield R, Tornroth S, Luna-Chavez C, Miyoshi H, Leger C, et al. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science. 2003;299:700–4. [DOI] [PubMed] [Google Scholar]
[9].Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, et al. Crystal Structure of Mitochondrial Respiratory Membrane Protein Complex II. Cell. 2005;121:1043–57. [DOI] [PubMed] [Google Scholar]
[10].Huo X, Su D, Wang A, Zhai Y, Xu J, Li X, et al. Preliminary molecular characterization and crystallization of mitochondrial respiratory complex II from porcine heart. Febs J. 2007;274:1524–9. [DOI] [PubMed] [Google Scholar]
[11].Huang LS, Borders TM, Shen JT, Wang CJ, Berry EA. Crystallization of mitochondrial respiratory complex II from chicken heart: a membrane-protein complex diffracting to 2.0 A. Acta Crystallogr D Biol Crystallogr. 2005;61:380–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
[12].Huang L-S, Sun G, Cobessi D, Wang AC, Shen JT, Tung EY, et al. 3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic-base arginine in the active site of the enzyme. J Biol Chem. 2006;281:5965–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
[13].Huang LS, Shen JT, Wang AC, Berry EA. Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state. Biochim Biophys Acta. 2006;1757:1073–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
[14].Shimizu H, Osanai A, Sakamoto K, Inaoka DK, Shiba T, Harada S, et al. Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum. J Biochem. 2012;151:589–92. [DOI] [PubMed] [Google Scholar]
[15].Ruprecht J, Yankovskaya V, Maklashina E, Iwata S, Cecchini G. Structure of Escherichia coli succinate:quinone oxidoreductase with an occupied and empty quinone-binding site. J Biol Chem. 2009;284:29836–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
[16].Moosavi B, Berry EA, Zhu XL, Yang WC, Yang GF. The assembly of succinate dehydrogenase: a key enzyme in bioenergetics. Cell Mol Life Sci. 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
[17].Sharma P, Maklashina E, Cecchini G, Iverson TM. Maturation of the respiratory complex II flavoprotein. Curr Opin Struct Biol. 2019;59:38–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
[18].Tran QM, Rothery RA, Maklashina E, Cecchini G, Weiner JH. Escherichia coli succinate dehydrogenase variant lacking the heme b. Proc Natl Acad Sci U S A. 2007;104:18007–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
[19].Oyedotun KS, Sit CS, Lemire BD. The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction. Biochim Biophys Acta. 2007;1767:1436–45. [DOI] [PubMed] [Google Scholar]
[20].Maklashina E, Rajagukguk S, McIntire WS, Cecchini G. Mutation of the heme axial ligand of Escherichia coli succinate-quinone reductase: Implications for heme ligation in mitochondrial complex II from yeast. Biochim Biophys Acta. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
[21].Ackrell BA. Cytopathies involving mitochondrial complex II. Mol Aspects Med. 2002;23:369–84. [DOI] [PubMed] [Google Scholar]
[22].Iverson TM, Maklashina E, Cecchini G. Structural basis for malfunction in complex II. J Biol Chem. 2012;287:35430–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
[23].Bezawork-Geleta A, Rohlena J, Dong L, Pacak K, Neuzil J. Mitochondrial Complex II: At the Crossroads. Trends Biochem Sci. 2017;42:312–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
[24].Moosavi B, Zhu XL, Yang WC, Yang GF. Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect. Eur J Cell Biol. 2020;99:151057. [DOI] [PubMed] [Google Scholar]
[25].Burnichon N, Briere JJ, Libe R, Vescovo L, Riviere J, Tissier F, et al. SDHA is a tumor suppressor gene causing paraganglioma. Hum Mol Genet. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
[26].Korpershoek E, Favier J, Gaal J, Burnichon N, van Gessel B, Oudijk L, et al. SDHA immunohistochemistry detects germline SDHA gene mutations in apparently sporadic paragangliomas and pheochromocytomas. J Clin Endocrinol Metab. 2011;96:E1472–6. [DOI] [PubMed] [Google Scholar]
[27].Dwight T, Mann K, Benn DE, Robinson BG, McKelvie P, Gill AJ, et al. Familial SDHA mutation associated with pituitary adenoma and pheochromocytoma/paraganglioma. J Clin Endocrinol Metab. 2013;98:E1103–8. [DOI] [PubMed] [Google Scholar]
[28].Dwight T, Benn DE, Clarkson A, Vilain R, Lipton L, Robinson BG, et al. Loss of SDHA expression identifies SDHA mutations in succinate dehydrogenase-deficient gastrointestinal stromal tumors. Am J Surg Pathol. 2013;37:226–33. [DOI] [PubMed] [Google Scholar]
[29].Hao HX, Khalimonchuk O, Schraders M, Dephoure N, Bayley JP, Kunst H, et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science. 2009;325:1139–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
[30].McNeil MB, Clulow JS, Wilf NM, Salmond GP, Fineran PC. SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria. J Biol Chem. 2012;287:18418–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
[31].Eletsky A, Jeong MY, Kim H, Lee HW, Xiao R, Pagliarini DJ, et al. Solution NMR Structure of Yeast Succinate Dehydrogenase Flavinylation Factor Sdh5 Reveals a Putative Sdh1 Binding Site. Biochemistry. 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
[32].Ziegler DM, Doeg KA. The isolation of a functionally intact succinic dehydrogenase-cytochrome b complex from beef heart mitochondria. Arch Biochem Biophys. 1959;85:282–4. [DOI] [PubMed] [Google Scholar]
[33].Hatefi Y, Haavik AG, Fowler LR, Griffiths DE. Studies on the electron transfer system. XLII. Reconstitution of the electron transfer system. J Biol Chem. 1962;237:2661–9. [PubMed] [Google Scholar]
[34].Capaldi RA, Sweetland J, Merli A. Polypeptides in the succinate-coenzyme Q reductase segment of the respiratory chain. Biochemistry. 1977;16:5707–10. [DOI] [PubMed] [Google Scholar]
[35].Wang TY, Tsou CL, Wang YL. Scientia Sinica. 1956;5:73–90. [Google Scholar]
[36].Singer TP, Kearney EB, Bernath P. Studies on succinic dehydrogenase. II. Isolation and properties of the dehydrogenase from beef heart. J Biol Chem. 1956;223:599–613. [PubMed] [Google Scholar]
[37].Basford RE, Tisdale HD, Green DE. Studies on the terminal electron transport system. VIII. Conversion of succinic dehydrogenase complex to soluble succinic dehydrogenase. Biochim Biophys Acta. 1957;24:290–4. [DOI] [PubMed] [Google Scholar]
[38].Davis KA, Hatefi Y. Succinate dehydrogenase. I. Purification, molecular properties, and substructure. Biochemistry. 1971;10:2509–16. [DOI] [PubMed] [Google Scholar]
[39].Ackrell BA, Kearney EB, Coles CJ. Isolation of reconstitutively active succinate dehydrogenase in highly purified state. J Biol Chem. 1977;252:6963–5. [PubMed] [Google Scholar]
[40].Yu CA, Yu L. Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase. Biochim Biophys Acta. 1980;591:409–20. [DOI] [PubMed] [Google Scholar]
[41].King TE. Reconstitution of Respiratory Chain Enzyme Systems. Xii. Some Observations on the Reconstitution of the Succinate Oxidase System from Heart Muscle. J Biol Chem. 1963;238:4037–51. [PubMed] [Google Scholar]
[42].Ohnishi T, Lim J, Winter DB, King TE. Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain. J Biol Chem. 1976;251:2105–9. [PubMed] [Google Scholar]
[43].Keilin D, King TE. Reconstitution of the succinic oxidase system from soluble succinic dehydrogenase and a particulate cytochrome system preparation. Nature. 1958;181:1520–2. [DOI] [PubMed] [Google Scholar]
[44].King TE, Takemori S. Reconstitution of Respiratory Chain Enzyme Systems. Xiv. Reconstitution of Succinate-Cytochrome C Reductase from Soluble Succinate Dehydrogenase and the Cytochrome B-C1 Particle. J Biol Chem. 1964;239:3559–69. [PubMed] [Google Scholar]
[45].Yu CA, Yu L, King TE. Soluble cytochrome b-c1 complex and the reconstitution of succinate- cytochrome c reductase. J Biol Chem. 1974;249:4905–10. [PubMed] [Google Scholar]
[46].Yu CA, Yu L, King TE. Reconstitution of succinate-Q reductase. Biochem Biophys Res Commun. 1977;79:939–46. [DOI] [PubMed] [Google Scholar]
[47].Ackrell BA, Ball MB, Kearney EB. Peptides from complex II active in reconstitution of succinate-ubiquinone reductase. J Biol Chem. 1980;255:2761–9. [PubMed] [Google Scholar]
[48].Hatefi Y, Galante YM. Isolation of cytochrome b560 from complex II (succinateubiquinone oxidoreductase) and its reconstitution with succinate dehydrogenase. J Biol Chem. 1980;255:5530–7. [PubMed] [Google Scholar]
[49].Yu CA, Yu L. Isolation and properties of a mitochondrial protein that converts succinate dehydrogenase into succinate-ubiquinone oxidoreductase. Biochemistry. 1980;19:3579–85. [DOI] [PubMed] [Google Scholar]
[50].Vinogradov AD, Gavrikov VG, Gavrikova EV. Studies on the succinate dehydrogenating system. II. Reconstitution of succinate-ubiquinone reductase from the soluble components. Biochim Biophys Acta. 1980;592:13–27. [DOI] [PubMed] [Google Scholar]
[51].Davis KA, Hatefi Y. Resolution and reconstitution of complex II (succinate-ubiquinone reductase) by salts. Arch Biochem Biophys. 1972;149:505–12. [DOI] [PubMed] [Google Scholar]
[52].Lancaster CR. Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase. FEBS Lett. 2003;555:21–8. [DOI] [PubMed] [Google Scholar]
[53].Hards K, Rodriguez SM, Cairns C, Cook GM. Alternate quinone coupling in a new class of succinate dehydrogenase may potentiate mycobacterial respiratory control. FEBS Lett. 2019;593:475–86. [DOI] [PubMed] [Google Scholar]
[54].Schirawski J, Unden G. Menaquinone-dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential. Eur J Biochem. 1998;257:210–5. [DOI] [PubMed] [Google Scholar]
[55].Matsson M, Tolstoy D, Aasa R, Hederstedt L. The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone. Biochemistry. 2000;39:8617–24. [DOI] [PubMed] [Google Scholar]
[56].Ohnishi T, Moser CC, Page CC, Dutton PL, Yano T. Simple redox-linked proton-transfer design: new insights from structures of quinol-fumarate reductase. Structure Fold Des. 2000;8:R23–32. [DOI] [PubMed] [Google Scholar]
[57].Lancaster CR, Herzog E, Juhnke HD, Madej MG, Muller FG, Paul R, et al. Electroneutral and electrogenic catalysis by dihaem-containing succinate:quinone oxidoreductases. Biochem Soc Trans. 2008;36:996–1000. [DOI] [PubMed] [Google Scholar]
[58].Lancaster CR. The di-heme family of respiratory complex II enzymes. Biochim Biophys Acta. 2013;1827:679–87. [DOI] [PubMed] [Google Scholar]
[59].Hinkle PC, Kumar MA, Resetar A, Harris DL. Mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Biochemistry. 1991;30:3576–82. [DOI] [PubMed] [Google Scholar]
[60].Ernster L P/O ratios--the first fifty years. Faseb J. 1993;7:1520–4. [DOI] [PubMed] [Google Scholar]
[61].Mowery PC, Steenkamp DJ, Ackrell AC, Singer TP, White GA. Inhibition of mammalian succinate dehydrogenase by carboxins. Arch Biochem Biophys. 1977;178:495–506. [DOI] [PubMed] [Google Scholar]
[62].Lisse D, Huang L-s, Gasteiger T, Berry EA, Lümmen P. Kinetic and structural analysis of succinate:ubiquinone oxidoreductase (complex II) inhibition by thiapronil. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2010;1797, Supplement:18–9. [Google Scholar]
[63].Osanai A, Harada S, Sakamoto K, Shimizu H, Inaoka DK, Kita K. Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009;65:941–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
[64].Scalliet G, Bowler J, Luksch T, Kirchhofer-Allan L, Steinhauer D, Ward K, et al. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola. PLoS One. 2012;7:e35429. [DOI] [PMC free article] [PubMed] [Google Scholar]
[65].Fraaije BA, Bayon C, Atkins S, Cools HJ, Lucas JA, Fraaije MW. Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat. Mol Plant Pathol. 2012;13:263–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
[66].Zhu XL, Xiong L, Li H, Song XY, Liu JJ, Yang GF. Computational and experimental insight into the molecular mechanism of carboxamide inhibitors of succinate-ubquinone oxidoreductase. ChemMedChem. 2014;9:1512–21. [DOI] [PubMed] [Google Scholar]
[67].Xiong L, Zhu XL, Shen YQ, Wishwajith WK, Li K, Yang GF. Discovery of N-benzoxazol-5-yl-pyrazole-4-carboxamides as nanomolar SQR inhibitors. Eur J Med Chem. 2015;95:424–34. [DOI] [PubMed] [Google Scholar]
[68].Xiong L, Zhu XL, Gao HW, Fu Y, Hu SQ, Jiang LN, et al. Discovery of Potent Succinate-Ubiquinone Oxidoreductase Inhibitors via Pharmacophore-linked Fragment Virtual Screening Approach. J Agric Food Chem. 2016;64:4830–7. [DOI] [PubMed] [Google Scholar]
[69].Xiong L, Li H, Jiang LN, Ge JM, Yang WC, Zhu XL, et al. Structure-Based Discovery of Potential Fungicides as Succinate Ubiquinone Oxidoreductase Inhibitors. J Agric Food Chem. 2017;65:1021–9. [DOI] [PubMed] [Google Scholar]
[70].Grivennikova VG, Vinogradov AD. [Interaction of mitochondrial succinate:ubiquinone reductase with thenoyltrifluoroacetone and carboxin]. Biokhimiia. 1985;50:375–83. [PubMed] [Google Scholar]
[71].Tushurashvili PR, Gavrikova EV, Ledenev AN, Vinogradov AD. Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. Biochim Biophys Acta. 1985;809:145–59. [DOI] [PubMed] [Google Scholar]
[72].Xu JX, King TE. Two-site property of thenoyltrifluoroacetone inhibiting succinate-ubiquinone reductase. Sci China B. 1992;35:162–8. [PubMed] [Google Scholar]
[73].Yankovskaya V, Sablin SO, Ramsay RR, Singer TP, Ackrell BA, Cecchini G, et al. Inhibitor probes of the quinone binding sites of mammalian complex II and Escherichia coli fumarate reductase. J Biol Chem. 1996;271:21020–4. [DOI] [PubMed] [Google Scholar]
[74].Matsson M, Ackrell BA, Cochran B, Hederstedt L. Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase. Arch Microbiol. 1998;170:27–37. [DOI] [PubMed] [Google Scholar]
[75].Maklashina E, Cecchini G. Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli. Arch Biochem Biophys. 1999;369:223–32. [DOI] [PubMed] [Google Scholar]
[76].Oyedotun KS, Lemire BD. The Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. Identification of Sdh3p amino acid residues involved in ubiquinone binding. J Biol Chem. 1999;274:23956–62. [DOI] [PubMed] [Google Scholar]
[77].Grigolava IV, Konstantinov AA, Ksenzenko M, Ruuge EK, Tikhonov AN. [Interaction of ubisemiquinone with succinate dehydrogenase and the cytochrome chain of mitochondria]. Biokhimiia. 1982;47:1970–82. [PubMed] [Google Scholar]
[78].Westenberg DJ, Gunsalus RP, Ackrell BA, Sices H, Cecchini G. Escherichia coli fumarate reductase frdC and frdD mutants. Identification of amino acid residues involved in catalytic activity with quinones. J Biol Chem. 1993;268:815–22. [PubMed] [Google Scholar]
[79].Yang X, Yu L, He D, Yu CA. The quinone-binding site in succinate-ubiquinone reductase from Escherichia coli. Quinone-binding domain and amino acid residues involved in quinone binding. J Biol Chem. 1998;273:31916–23. [DOI] [PubMed] [Google Scholar]
[80].Oyedotun KS, Lemire BD. The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. J Biol Chem. 2001;276:16936–43. [DOI] [PubMed] [Google Scholar]
[81].Broomfield PL, Hargreaves JA. A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Curr Genet. 1992;22:117–21. [DOI] [PubMed] [Google Scholar]
[82].Skinner W, Bailey A, Renwick A, Keon J, Gurr S, Hargreaves J. A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Curr Genet. 1998;34:393–8. [DOI] [PubMed] [Google Scholar]
[83].Matsson M, Hederstedt L. The carboxin-binding site on Paracoccus denitrificans succinate:quinone reductase identified by mutations. J Bioenerg Biomembr. 2001;33:99–105. [DOI] [PubMed] [Google Scholar]
[84].Lee GY, He DY, Yu L, Yu CA. Identification of the ubiquinone-binding domain in QPs1 of succinate-ubiquinone reductase. J Biol Chem. 1995;270:6193–8. [DOI] [PubMed] [Google Scholar]
[85].Shenoy SK, Yu L, Yu C. Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase. J Biol Chem. 1999;274:8717–22. [DOI] [PubMed] [Google Scholar]
[86].Cecchini G, Maklashina E, Yankovskaya V, Iverson TM, Iwata S. Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases. FEBS Lett. 2003;545:31–8. [DOI] [PubMed] [Google Scholar]
[87].Maklashina E, Cecchini G. The quinone-binding and catalytic site of complex II. Biochim Biophys Acta. 2010;1797:1877–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
[88].Nasiri HR, Madej MG, Panisch R, Lafontaine M, Bats JW, Lancaster CR, et al. Design, synthesis, and biological testing of novel naphthoquinones as substrate-based inhibitors of the quinol/fumarate reductase from Wolinella succinogenes. J Med Chem. 2013;56:9530–41. [DOI] [PubMed] [Google Scholar]
[89].Hederstedt L Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis. Biochim Biophys Acta. 2002;1553:74–83. [DOI] [PubMed] [Google Scholar]
[90].Tappel AL. Inhibition of electron transport by antimycin A, alkyl hydroxy naphthoquinones and metal coordination compounds. Biochem Pharmacol. 1960;3:289–96. [DOI] [PubMed] [Google Scholar]
[91].Maklashina E, Cecchini G. The quinone-binding and catalytic site of complex II. Biochim Biophys Acta. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
[92].Inaoka DK, Shiba T, Sato D, Balogun EO, Sasaki T, Nagahama M, et al. Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria. Int J Mol Sci. 2015;16:15287–308. [DOI] [PMC free article] [PubMed] [Google Scholar]
[93].Miyadera H, Hiraishi A, Miyoshi H, Sakamoto K, Mineki R, Murayama K, et al. Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity. Eur J Biochem. 2003;270:1863–74. [DOI] [PubMed] [Google Scholar]
[94].Amino H, Wang H, Hirawake H, Saruta F, Mizuchi D, Mineki R, et al. Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit. Mol Biochem Parasitol. 2000;106:63–76. [DOI] [PubMed] [Google Scholar]
[95].Iwata F, Shinjyo N, Amino H, Sakamoto K, Islam MK, Tsuji N, et al. Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host. Parasitol Int. 2008;57:54–61. [DOI] [PubMed] [Google Scholar]
[96].Grivennikova VG, Gavrikova EV, Timoshin AA, Vinogradov AD. Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction. Biochim Biophys Acta. 1993;1140:282–92. [DOI] [PubMed] [Google Scholar]
[97].Cecchini G, Schroder I, Gunsalus RP, Maklashina E. Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta. 2002;1553:140–57. [DOI] [PubMed] [Google Scholar]
[98].Cheng VW, Tran QM, Boroumand N, Rothery RA, Maklashina E, Cecchini G, et al. A conserved lysine residue controls iron-sulfur cluster redox chemistry in Escherichia coli fumarate reductase. Biochim Biophys Acta. 2013;1827:1141–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
[99].Maklashina E, Berthold DA, Cecchini G. Anaerobic expression of Escherichia coli succinate dehydrogenase: functional replacement of fumarate reductase in the respiratory chain during anaerobic growth. J Bacteriol. 1998;180:5989–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
[100].Ball EG, Anfinsen CB, Cooper O. The inhibitory action of naphthoquinones on respiratory processes. J Biol Chem. 1947;168:257–70. [PubMed] [Google Scholar]
[101].Takemori S, King TE. Reconstitution of Respiratory Chain Enzyme Systems. 13. Sequential Fragmentation of Succinate Oxidase: Preparation and Properties of Succinate-Cytochrome C Reductase and the Cytochrome B-C1 Particle. J Biol Chem. 1964;239:3546–58. [PubMed] [Google Scholar]
[102].Fry M, Pudney M. Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem Pharmacol. 1992;43:1545–53. [DOI] [PubMed] [Google Scholar]
[103].Kessl JJ, Moskalev NV, Gribble GW, Nasr M, Meshnick SR, Trumpower BL. Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex. Biochim Biophys Acta. 2007;1767:319–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
[104].White GA. Substituted Benzanilides: Structural Variation and Inhibition of Complex II Activity in Mitochondria from a Wild-Type Strain and a Carboxin-Selected Mutant Strain of Ustilago maydis. Pestic Biochem Physiol. 1987;27:249–60. [Google Scholar]
[105].Horsefield R, Yankovskaya V, Sexton G, Whittingham W, Shiomi K, Omura S, et al. Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction. J Biol Chem. 2006;281:7309–16. [DOI] [PubMed] [Google Scholar]
[106].Berman HM, Kleywegt GJ, Nakamura H, Markley JL. The future of the protein data bank. Biopolymers. 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
[107].Zhou Q, Zhai Y, Lou J, Liu M, Pang X, Sun F. Thiabendazole inhibits ubiquinone reduction activity of mitochondrial respiratory complex II via a water molecule mediated binding feature. Protein Cell. 2011;2:531–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
[108].Huang L-S, Borders TM, Shen JT, Wang C-J, Berry EA. Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å. Acta Cryst D. 2005;D61. [DOI] [PMC free article] [PubMed] [Google Scholar]
[109].Tomasiak TM, Archuleta TL, Andrell J, Luna-Chavez C, Davis TA, Sarwar M, et al. Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase. J Biol Chem. 2011;286:3047–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
[110].White GA, Thorn GD. Structure-Activity Relationships of Carboxamide Fungicides and the Succinic Dehydrogenase Complex of Cryptococcus laurentii and Ustilago maydis’. Pestic Biochem Physiol. 1975;5:380–95. [Google Scholar]
[111].Mowery PC, Ackrell BA, Singer TP, Thorn GD, White GA. Carboxins: powerful selective inhibitors of succinate oxidation in animal tissues. Biochem Biophys Res Commun. 1976;71:354–61. [DOI] [PubMed] [Google Scholar]
[112].Coles CJ, Singer TP, White GA, Thorn GD. Studies on the binding of carboxin analogs to succinate dehydrogenase. J Biol Chem. 1978;253:5573–8. [PubMed] [Google Scholar]
[113].Ramsay RR, Ackrell BA, Coles CJ, Singer TP, White GA, Thorn GD. Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II. Proc Natl Acad Sci U S A. 1981;78:825–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
[114].White GA. Pyrazole Carboxanilide Fungicides. I. Correlation of Mitochondrial Electron Transport Inhibition and Anti-fungal Activity. Pestic Biochem Physiol. 1986;25:163–8. [Google Scholar]
[115].White GA. Substituted 2-methylbenzaniIides and Structurally Related Carboxamides: Inhibition of Complex II Activity in Mitochondria from a Wild-Type Strain and a Carboxin-Resistant Mutant Strain of Ustilago maydis. Pestic Biochem Physiol. 1989;34:255–76. [Google Scholar]
[116].Miyadera H, Shiomi K, Ui H, Yamaguchi Y, Masuma R, Tomoda H, et al. Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase). Proc Natl Acad Sci U S A. 2003;100:473–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
[117].Silkin Y, Oyedotun KS, Lemire BD. The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase. Biochim Biophys Acta. 2007;1767:143–50. [DOI] [PubMed] [Google Scholar]
[118].Tran QM, Rothery RA, Maklashina E, Cecchini G, Weiner JH. The quinone binding site in Escherichia coli succinate dehydrogenase is required for electron transfer to the heme b. J Biol Chem. 2006;281:32310–7. [DOI] [PubMed] [Google Scholar]
[119].Milunsky JM, Maher TA, Michels VV, Milunsky A. Novel mutations and the emergence of a common mutation in the SDHD gene causing familial paraganglioma. Am J Med Genet. 2001;100:311–4. [DOI] [PubMed] [Google Scholar]
[120].De Miccolis Angelini RM, Habib W, Rotolo C, Pollastro S, Faretra F. Selection, characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid. European Journal of Plant Pathology. 2010;128:185–99. [Google Scholar]
[121].Leroux P, Gredt M, Leroch M, Walker AS. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Appl Environ Microbiol. 2010;76:6615–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
[122].Yin YN, Kim YK, Xiao CL. Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple. Phytopathology. 2011;101:986–95. [DOI] [PubMed] [Google Scholar]
[123].Veloukas T, Leroch M, Hahn M, Karaoglanidis GS. Detection and Molecular Characterization of Boscalid-Resistant Botrytis cinerea Isolates from Strawberry. Plant Dis. 2011;95:1302–7. [DOI] [PubMed] [Google Scholar]
[124].Manodori A, Cecchini G, Schroder I, Gunsalus RP, Werth MT, Johnson MK. [3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis. Biochemistry. 1992;31:2703–12. [DOI] [PubMed] [Google Scholar]
[125].Hägerhäll C, Sled V, Hederstedt L, Ohnishi T. The trinuclear iron-sulfur cluster S3 in Bacillus subtilis succinate:menaquinone reductase; effects of a mutation in the putative cluster ligation motif on enzyme activity and EPR properties. Biochim Biophys Acta. 1995;1229:356–62. [DOI] [PubMed] [Google Scholar]
[126].Ishii N, Fujii M, Hartman PS, Tsuda M, Yasuda K, Senoo-Matsuda N, et al. A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes. Nature. 1998;394:694–7. [DOI] [PubMed] [Google Scholar]
[127].Amiri A, Zuniga AI, Peres NA. Mutations in the Membrane-Anchored SdhC Subunit Affect Fitness and Sensitivity to Succinate Dehydrogenase Inhibitors in Botrytis cinerea Populations from Multiple Hosts. Phytopathology. 2020;110:327–35. [DOI] [PubMed] [Google Scholar]
[128].Lovell SC, Word JM, Richardson JS, Richardson DC. The penultimate rotamer library. Proteins. 2000;40:389–408. [PubMed] [Google Scholar]
[129].Oyedotun KS, Lemire BD. The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562. FEBS Lett. 1999;442:203–7. [DOI] [PubMed] [Google Scholar]
[130].Tron T, Crimi M, Colson AM, Degli Esposti M. Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants. Eur J Biochem. 1991;199:753–60. [DOI] [PubMed] [Google Scholar]
[131].Yun CH, Wang Z, Crofts AR, Gennis RB. Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides. J Biol Chem. 1992;267:5901–9. [PubMed] [Google Scholar]
[132].Crofts A, Hacker B, Barquera B, Yun CH, Gennis R. Structure and function of the bc-complex of Rhodobacter sphaeroides. Biochim Biophys Acta. 1992;1101:162–5. [PubMed] [Google Scholar]
[133].Berry EA, Walker FA. Bis-histidine-coordinated hemes in four-helix bundles: how the geometry of the bundle controls the axial imidazole plane orientations in transmembrane cytochromes of mitochondrial complexes II and III and related proteins. J Biol Inorg Chem. 2008;13:481–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
[134].Scheidt WR, Kirner JF, Hoard JL, Reed CA. Unusual orientation of axial ligands in metalloporphyrins. Molecular structure of low-spin bis(2-methylimidazole)(meso-tetraphenylporphinato)iron(III) perchlorate. Journal of the American Chemical Society. 1987;109:1963–8. [Google Scholar]
[135].Walker FA, Huynh BH, Scheidt WR, Osvath SR. Models of the Cytochromes b. The Effect of Axial Ligand Plane Orientation on the EPR and Mössbauer Spectra of Low-Spin Ferrihemes. J Am Chem Soc. 1986;108:5288–97. [Google Scholar]
[136].Yu L, Xu JX, Haley PE, Yu CA. Properties of bovine heart mitochondrial cytochrome b560. J Biol Chem. 1987;262:1137–43. [PubMed] [Google Scholar]
[137].Peterson J, Vibat C, Gennis RB. Identification of the axial heme ligands of cytochrome b556 in succinate: ubiquinone oxidoreductase from Escherichia coli. FEBS Lett. 1994;355:155–6. [DOI] [PubMed] [Google Scholar]
[138].Crouse BR, Yu CA, Yu L, Johnson MK. Spectroscopic identification of the axial ligands of cytochrome b560 in bovine heart succinate-ubiquinone reductase. FEBS Lett. 1995;367:1–4. [DOI] [PubMed] [Google Scholar]
[139].Waldeck AR, Stowell MH, Lee HK, Hung SC, Matsson M, Hederstedt L, et al. Electron paramagnetic resonance studies of succinate:ubiquinone oxidoreductase from Paracoccus denitrificans. Evidence for a magnetic interaction between the 3Fe-4S cluster and cytochrome b. J Biol Chem. 1997;272:19373–82. [DOI] [PubMed] [Google Scholar]
[140].Moss GP. Nomenclature of tetrapyrroles. Recommendations 1986 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Eur J Biochem. 1988;178:277–328. [DOI] [PubMed] [Google Scholar]
[141].Miki T, Yu L, Yu CA. Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase. Arch Biochem Biophys. 1992;293:61–6. [DOI] [PubMed] [Google Scholar]
[142].Shima Y, Ito Y, Hatabayashi H, Koma A, Yabe K. Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides. Biosci Biotechnol Biochem. 2011;75:181–4. [DOI] [PubMed] [Google Scholar]
[143].Ruprecht J, Iwata S, Rothery RA, Weiner JH, Maklashina E, Cecchini G. Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster. J Biol Chem. 2011;286:12756–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
[144].Shima Y, Ito Y, Kaneko S, Hatabayashi H, Watanabe Y, Adachi Y, et al. Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae. Fungal Genet Biol. 2009;46:67–76. [DOI] [PubMed] [Google Scholar]
[145].Hauska G Plasto- and ubiquinone as translocators of electrons and protons through membranes: a facilitating role of the isoprenoid side chain. FEBS Lett. 1977;79:345–7. [DOI] [PubMed] [Google Scholar]
[146].Teixeira MH, Arantes GM. Effects of lipid composition on membrane distribution and permeability of natural quinones. RSC Adv. 2019;2019:16892–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
[147].Cheng VW, Johnson A, Rothery RA, Weiner JH. Alternative sites for proton entry from the cytoplasm to the quinone binding site in Escherichia coli succinate dehydrogenase. Biochemistry. 2008;47:9107–16. [DOI] [PubMed] [Google Scholar]
[148].Iverson TM, Luna-Chavez C, Schroder I, Cecchini G, Rees DC. Analyzing your complexes: structure of the quinol-fumarate reductase respiratory complex. Curr Opin Struct Biol. 2000;10:448–55. [DOI] [PubMed] [Google Scholar]
[149].Kita K, Vibat CR, Meinhardt S, Guest JR, Gennis RB. One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556. J Biol Chem. 1989;264:2672–7. [PubMed] [Google Scholar]
[150].Yang X, Yu L, Yu CA. Resolution and reconstitution of succinate-ubiquinone reductase from Escherichia coli. J Biol Chem. 1997;272:9683–9. [DOI] [PubMed] [Google Scholar]
[151].Stroh A, Anderka O, Pfeiffer K, Yagi T, Finel M, Ludwig B, et al. Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans. J Biol Chem. 2004;279:5000–7. [DOI] [PubMed] [Google Scholar]
[152].Otwinowski Z, Minor W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology. 1997;276:307–26. [DOI] [PubMed] [Google Scholar]
[153].Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68:352–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
[154].Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
[155].Jones TA, Zhou J-Y, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors on these models. Acta Crystallogr. 1991; A47,:110–9. [DOI] [PubMed] [Google Scholar]
[156].Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501. [DOI] [PMC free article] [PubMed] [Google Scholar]
[157].Schüttelkopf AW, van Aalten DM. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2004;60:1355–63. [DOI] [PubMed] [Google Scholar]
[158].Kraulis PJ. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr. 1991; 24:946–50. [Google Scholar]
[159].Merritt EA, Murphy ME. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994;50:869–73. [DOI] [PubMed] [Google Scholar]
[160].Combet C, Blanchet C, Geourjon C, Deleage G. NPS@: network protein sequence analysis. Trends Biochem Sci. 2000;25:147–50. [DOI] [PubMed] [Google Scholar]
[161].Gong H, Gao Y, Zhou X, Xiao Y, Wang W, Tang Y, et al. Cryo-EM structure of trimeric Mycobacterium smegmatis succinate dehydrogenase with a membrane-anchor SdhF. Nat Commun. 2020;11:4245. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement

Table S1 - Data Reduction Statistics

Table S2 - Refinement Statisitics

Table S3 - Polar interactions at anchor interface

Figure S1- Position of UQ and RQ from other group’s structures

Section S2.4: Detailed descriptions of binding in each structure

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[R1] [1].Cecchini G Function and structure of complex II of the respiratory chain. Annu Rev Biochem. 2003;72:77–109. [DOI] [PubMed] [Google Scholar]

[R2] [2].Rutter J, Winge DR, Schiffman JD. Succinate dehydrogenase - Assembly, regulation and role in human disease. Mitochondrion. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] [3].Iverson TM. Catalytic mechanisms of complex II enzymes: a structural perspective. Biochim Biophys Acta. 2013;1827:648–57. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] [4].Hägerhäll C Succinate: quinone oxidoreductases. Variations on a conserved theme. Biochim Biophys Acta. 1997;1320:107–41. [DOI] [PubMed] [Google Scholar]

[R5] [5].Westenberg DJ, Guerinot ML. Succinate dehydrogenase (Sdh) from Bradyrhizobium japonicum is closely related to mitochondrial Sdh. J Bacteriol. 1999;181:4676–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] [6].Schäfer G, Anemüller S, Moll R. Archaeal complex II: 'classical' and 'non-classical' succinate:quinone reductases with unusual features. Biochim Biophys Acta. 2002;1553:57–73. [DOI] [PubMed] [Google Scholar]

[R7] [7].Lemos RS, Fernandes AS, Pereira MM, Gomes CM, Teixeira M. Quinol:fumarate oxidoreductases and succinate:quinone oxidoreductases: phylogenetic relationships, metal centres and membrane attachment. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2002;1553:158–70. [DOI] [PubMed] [Google Scholar]

[R8] [8].Yankovskaya V, Horsefield R, Tornroth S, Luna-Chavez C, Miyoshi H, Leger C, et al. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science. 2003;299:700–4. [DOI] [PubMed] [Google Scholar]

[R9] [9].Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, et al. Crystal Structure of Mitochondrial Respiratory Membrane Protein Complex II. Cell. 2005;121:1043–57. [DOI] [PubMed] [Google Scholar]

[R10] [10].Huo X, Su D, Wang A, Zhai Y, Xu J, Li X, et al. Preliminary molecular characterization and crystallization of mitochondrial respiratory complex II from porcine heart. Febs J. 2007;274:1524–9. [DOI] [PubMed] [Google Scholar]

[R11] [11].Huang LS, Borders TM, Shen JT, Wang CJ, Berry EA. Crystallization of mitochondrial respiratory complex II from chicken heart: a membrane-protein complex diffracting to 2.0 A. Acta Crystallogr D Biol Crystallogr. 2005;61:380–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] [12].Huang L-S, Sun G, Cobessi D, Wang AC, Shen JT, Tung EY, et al. 3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic-base arginine in the active site of the enzyme. J Biol Chem. 2006;281:5965–72. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] [13].Huang LS, Shen JT, Wang AC, Berry EA. Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state. Biochim Biophys Acta. 2006;1757:1073–83. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] [14].Shimizu H, Osanai A, Sakamoto K, Inaoka DK, Shiba T, Harada S, et al. Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum. J Biochem. 2012;151:589–92. [DOI] [PubMed] [Google Scholar]

[R15] [15].Ruprecht J, Yankovskaya V, Maklashina E, Iwata S, Cecchini G. Structure of Escherichia coli succinate:quinone oxidoreductase with an occupied and empty quinone-binding site. J Biol Chem. 2009;284:29836–46. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] [16].Moosavi B, Berry EA, Zhu XL, Yang WC, Yang GF. The assembly of succinate dehydrogenase: a key enzyme in bioenergetics. Cell Mol Life Sci. 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] [17].Sharma P, Maklashina E, Cecchini G, Iverson TM. Maturation of the respiratory complex II flavoprotein. Curr Opin Struct Biol. 2019;59:38–46. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] [18].Tran QM, Rothery RA, Maklashina E, Cecchini G, Weiner JH. Escherichia coli succinate dehydrogenase variant lacking the heme b. Proc Natl Acad Sci U S A. 2007;104:18007–12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] [19].Oyedotun KS, Sit CS, Lemire BD. The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction. Biochim Biophys Acta. 2007;1767:1436–45. [DOI] [PubMed] [Google Scholar]

[R20] [20].Maklashina E, Rajagukguk S, McIntire WS, Cecchini G. Mutation of the heme axial ligand of Escherichia coli succinate-quinone reductase: Implications for heme ligation in mitochondrial complex II from yeast. Biochim Biophys Acta. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] [21].Ackrell BA. Cytopathies involving mitochondrial complex II. Mol Aspects Med. 2002;23:369–84. [DOI] [PubMed] [Google Scholar]

[R22] [22].Iverson TM, Maklashina E, Cecchini G. Structural basis for malfunction in complex II. J Biol Chem. 2012;287:35430–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] [23].Bezawork-Geleta A, Rohlena J, Dong L, Pacak K, Neuzil J. Mitochondrial Complex II: At the Crossroads. Trends Biochem Sci. 2017;42:312–25. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] [24].Moosavi B, Zhu XL, Yang WC, Yang GF. Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect. Eur J Cell Biol. 2020;99:151057. [DOI] [PubMed] [Google Scholar]

[R25] [25].Burnichon N, Briere JJ, Libe R, Vescovo L, Riviere J, Tissier F, et al. SDHA is a tumor suppressor gene causing paraganglioma. Hum Mol Genet. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] [26].Korpershoek E, Favier J, Gaal J, Burnichon N, van Gessel B, Oudijk L, et al. SDHA immunohistochemistry detects germline SDHA gene mutations in apparently sporadic paragangliomas and pheochromocytomas. J Clin Endocrinol Metab. 2011;96:E1472–6. [DOI] [PubMed] [Google Scholar]

[R27] [27].Dwight T, Mann K, Benn DE, Robinson BG, McKelvie P, Gill AJ, et al. Familial SDHA mutation associated with pituitary adenoma and pheochromocytoma/paraganglioma. J Clin Endocrinol Metab. 2013;98:E1103–8. [DOI] [PubMed] [Google Scholar]

[R28] [28].Dwight T, Benn DE, Clarkson A, Vilain R, Lipton L, Robinson BG, et al. Loss of SDHA expression identifies SDHA mutations in succinate dehydrogenase-deficient gastrointestinal stromal tumors. Am J Surg Pathol. 2013;37:226–33. [DOI] [PubMed] [Google Scholar]

[R29] [29].Hao HX, Khalimonchuk O, Schraders M, Dephoure N, Bayley JP, Kunst H, et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science. 2009;325:1139–42. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] [30].McNeil MB, Clulow JS, Wilf NM, Salmond GP, Fineran PC. SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria. J Biol Chem. 2012;287:18418–28. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] [31].Eletsky A, Jeong MY, Kim H, Lee HW, Xiao R, Pagliarini DJ, et al. Solution NMR Structure of Yeast Succinate Dehydrogenase Flavinylation Factor Sdh5 Reveals a Putative Sdh1 Binding Site. Biochemistry. 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] [32].Ziegler DM, Doeg KA. The isolation of a functionally intact succinic dehydrogenase-cytochrome b complex from beef heart mitochondria. Arch Biochem Biophys. 1959;85:282–4. [DOI] [PubMed] [Google Scholar]

[R33] [33].Hatefi Y, Haavik AG, Fowler LR, Griffiths DE. Studies on the electron transfer system. XLII. Reconstitution of the electron transfer system. J Biol Chem. 1962;237:2661–9. [PubMed] [Google Scholar]

[R34] [34].Capaldi RA, Sweetland J, Merli A. Polypeptides in the succinate-coenzyme Q reductase segment of the respiratory chain. Biochemistry. 1977;16:5707–10. [DOI] [PubMed] [Google Scholar]

[R35] [35].Wang TY, Tsou CL, Wang YL. Scientia Sinica. 1956;5:73–90. [Google Scholar]

[R36] [36].Singer TP, Kearney EB, Bernath P. Studies on succinic dehydrogenase. II. Isolation and properties of the dehydrogenase from beef heart. J Biol Chem. 1956;223:599–613. [PubMed] [Google Scholar]

[R37] [37].Basford RE, Tisdale HD, Green DE. Studies on the terminal electron transport system. VIII. Conversion of succinic dehydrogenase complex to soluble succinic dehydrogenase. Biochim Biophys Acta. 1957;24:290–4. [DOI] [PubMed] [Google Scholar]

[R38] [38].Davis KA, Hatefi Y. Succinate dehydrogenase. I. Purification, molecular properties, and substructure. Biochemistry. 1971;10:2509–16. [DOI] [PubMed] [Google Scholar]

[R39] [39].Ackrell BA, Kearney EB, Coles CJ. Isolation of reconstitutively active succinate dehydrogenase in highly purified state. J Biol Chem. 1977;252:6963–5. [PubMed] [Google Scholar]

[R40] [40].Yu CA, Yu L. Resolution and reconstitution of succinate-cytochrome c reductase: preparations and properties of high purity succinate dehydrogenase and ubiquinol-cytochrome c reductase. Biochim Biophys Acta. 1980;591:409–20. [DOI] [PubMed] [Google Scholar]

[R41] [41].King TE. Reconstitution of Respiratory Chain Enzyme Systems. Xii. Some Observations on the Reconstitution of the Succinate Oxidase System from Heart Muscle. J Biol Chem. 1963;238:4037–51. [PubMed] [Google Scholar]

[R42] [42].Ohnishi T, Lim J, Winter DB, King TE. Thermodynamic and EPR characteristics of a HiPIP-type iron-sulfur center in the succinate dehydrogenase of the respiratory chain. J Biol Chem. 1976;251:2105–9. [PubMed] [Google Scholar]

[R43] [43].Keilin D, King TE. Reconstitution of the succinic oxidase system from soluble succinic dehydrogenase and a particulate cytochrome system preparation. Nature. 1958;181:1520–2. [DOI] [PubMed] [Google Scholar]

[R44] [44].King TE, Takemori S. Reconstitution of Respiratory Chain Enzyme Systems. Xiv. Reconstitution of Succinate-Cytochrome C Reductase from Soluble Succinate Dehydrogenase and the Cytochrome B-C1 Particle. J Biol Chem. 1964;239:3559–69. [PubMed] [Google Scholar]

[R45] [45].Yu CA, Yu L, King TE. Soluble cytochrome b-c1 complex and the reconstitution of succinate- cytochrome c reductase. J Biol Chem. 1974;249:4905–10. [PubMed] [Google Scholar]

[R46] [46].Yu CA, Yu L, King TE. Reconstitution of succinate-Q reductase. Biochem Biophys Res Commun. 1977;79:939–46. [DOI] [PubMed] [Google Scholar]

[R47] [47].Ackrell BA, Ball MB, Kearney EB. Peptides from complex II active in reconstitution of succinate-ubiquinone reductase. J Biol Chem. 1980;255:2761–9. [PubMed] [Google Scholar]

[R48] [48].Hatefi Y, Galante YM. Isolation of cytochrome b560 from complex II (succinateubiquinone oxidoreductase) and its reconstitution with succinate dehydrogenase. J Biol Chem. 1980;255:5530–7. [PubMed] [Google Scholar]

[R49] [49].Yu CA, Yu L. Isolation and properties of a mitochondrial protein that converts succinate dehydrogenase into succinate-ubiquinone oxidoreductase. Biochemistry. 1980;19:3579–85. [DOI] [PubMed] [Google Scholar]

[R50] [50].Vinogradov AD, Gavrikov VG, Gavrikova EV. Studies on the succinate dehydrogenating system. II. Reconstitution of succinate-ubiquinone reductase from the soluble components. Biochim Biophys Acta. 1980;592:13–27. [DOI] [PubMed] [Google Scholar]

[R51] [51].Davis KA, Hatefi Y. Resolution and reconstitution of complex II (succinate-ubiquinone reductase) by salts. Arch Biochem Biophys. 1972;149:505–12. [DOI] [PubMed] [Google Scholar]

[R52] [52].Lancaster CR. Wolinella succinogenes quinol:fumarate reductase and its comparison to E. coli succinate:quinone reductase. FEBS Lett. 2003;555:21–8. [DOI] [PubMed] [Google Scholar]

[R53] [53].Hards K, Rodriguez SM, Cairns C, Cook GM. Alternate quinone coupling in a new class of succinate dehydrogenase may potentiate mycobacterial respiratory control. FEBS Lett. 2019;593:475–86. [DOI] [PubMed] [Google Scholar]

[R54] [54].Schirawski J, Unden G. Menaquinone-dependent succinate dehydrogenase of bacteria catalyzes reversed electron transport driven by the proton potential. Eur J Biochem. 1998;257:210–5. [DOI] [PubMed] [Google Scholar]

[R55] [55].Matsson M, Tolstoy D, Aasa R, Hederstedt L. The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone. Biochemistry. 2000;39:8617–24. [DOI] [PubMed] [Google Scholar]

[R56] [56].Ohnishi T, Moser CC, Page CC, Dutton PL, Yano T. Simple redox-linked proton-transfer design: new insights from structures of quinol-fumarate reductase. Structure Fold Des. 2000;8:R23–32. [DOI] [PubMed] [Google Scholar]

[R57] [57].Lancaster CR, Herzog E, Juhnke HD, Madej MG, Muller FG, Paul R, et al. Electroneutral and electrogenic catalysis by dihaem-containing succinate:quinone oxidoreductases. Biochem Soc Trans. 2008;36:996–1000. [DOI] [PubMed] [Google Scholar]

[R58] [58].Lancaster CR. The di-heme family of respiratory complex II enzymes. Biochim Biophys Acta. 2013;1827:679–87. [DOI] [PubMed] [Google Scholar]

[R59] [59].Hinkle PC, Kumar MA, Resetar A, Harris DL. Mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Biochemistry. 1991;30:3576–82. [DOI] [PubMed] [Google Scholar]

[R60] [60].Ernster L P/O ratios--the first fifty years. Faseb J. 1993;7:1520–4. [DOI] [PubMed] [Google Scholar]

[R61] [61].Mowery PC, Steenkamp DJ, Ackrell AC, Singer TP, White GA. Inhibition of mammalian succinate dehydrogenase by carboxins. Arch Biochem Biophys. 1977;178:495–506. [DOI] [PubMed] [Google Scholar]

[R62] [62].Lisse D, Huang L-s, Gasteiger T, Berry EA, Lümmen P. Kinetic and structural analysis of succinate:ubiquinone oxidoreductase (complex II) inhibition by thiapronil. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2010;1797, Supplement:18–9. [Google Scholar]

[R63] [63].Osanai A, Harada S, Sakamoto K, Shimizu H, Inaoka DK, Kita K. Crystallization of mitochondrial rhodoquinol-fumarate reductase from the parasitic nematode Ascaris suum with the specific inhibitor flutolanil. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009;65:941–4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R64] [64].Scalliet G, Bowler J, Luksch T, Kirchhofer-Allan L, Steinhauer D, Ward K, et al. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola. PLoS One. 2012;7:e35429. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R65] [65].Fraaije BA, Bayon C, Atkins S, Cools HJ, Lucas JA, Fraaije MW. Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat. Mol Plant Pathol. 2012;13:263–75. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R66] [66].Zhu XL, Xiong L, Li H, Song XY, Liu JJ, Yang GF. Computational and experimental insight into the molecular mechanism of carboxamide inhibitors of succinate-ubquinone oxidoreductase. ChemMedChem. 2014;9:1512–21. [DOI] [PubMed] [Google Scholar]

[R67] [67].Xiong L, Zhu XL, Shen YQ, Wishwajith WK, Li K, Yang GF. Discovery of N-benzoxazol-5-yl-pyrazole-4-carboxamides as nanomolar SQR inhibitors. Eur J Med Chem. 2015;95:424–34. [DOI] [PubMed] [Google Scholar]

[R68] [68].Xiong L, Zhu XL, Gao HW, Fu Y, Hu SQ, Jiang LN, et al. Discovery of Potent Succinate-Ubiquinone Oxidoreductase Inhibitors via Pharmacophore-linked Fragment Virtual Screening Approach. J Agric Food Chem. 2016;64:4830–7. [DOI] [PubMed] [Google Scholar]

[R69] [69].Xiong L, Li H, Jiang LN, Ge JM, Yang WC, Zhu XL, et al. Structure-Based Discovery of Potential Fungicides as Succinate Ubiquinone Oxidoreductase Inhibitors. J Agric Food Chem. 2017;65:1021–9. [DOI] [PubMed] [Google Scholar]

[R70] [70].Grivennikova VG, Vinogradov AD. [Interaction of mitochondrial succinate:ubiquinone reductase with thenoyltrifluoroacetone and carboxin]. Biokhimiia. 1985;50:375–83. [PubMed] [Google Scholar]

[R71] [71].Tushurashvili PR, Gavrikova EV, Ledenev AN, Vinogradov AD. Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. Biochim Biophys Acta. 1985;809:145–59. [DOI] [PubMed] [Google Scholar]

[R72] [72].Xu JX, King TE. Two-site property of thenoyltrifluoroacetone inhibiting succinate-ubiquinone reductase. Sci China B. 1992;35:162–8. [PubMed] [Google Scholar]

[R73] [73].Yankovskaya V, Sablin SO, Ramsay RR, Singer TP, Ackrell BA, Cecchini G, et al. Inhibitor probes of the quinone binding sites of mammalian complex II and Escherichia coli fumarate reductase. J Biol Chem. 1996;271:21020–4. [DOI] [PubMed] [Google Scholar]

[R74] [74].Matsson M, Ackrell BA, Cochran B, Hederstedt L. Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase. Arch Microbiol. 1998;170:27–37. [DOI] [PubMed] [Google Scholar]

[R75] [75].Maklashina E, Cecchini G. Comparison of catalytic activity and inhibitors of quinone reactions of succinate dehydrogenase (Succinate-ubiquinone oxidoreductase) and fumarate reductase (Menaquinol-fumarate oxidoreductase) from Escherichia coli. Arch Biochem Biophys. 1999;369:223–32. [DOI] [PubMed] [Google Scholar]

[R76] [76].Oyedotun KS, Lemire BD. The Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. Identification of Sdh3p amino acid residues involved in ubiquinone binding. J Biol Chem. 1999;274:23956–62. [DOI] [PubMed] [Google Scholar]

[R77] [77].Grigolava IV, Konstantinov AA, Ksenzenko M, Ruuge EK, Tikhonov AN. [Interaction of ubisemiquinone with succinate dehydrogenase and the cytochrome chain of mitochondria]. Biokhimiia. 1982;47:1970–82. [PubMed] [Google Scholar]

[R78] [78].Westenberg DJ, Gunsalus RP, Ackrell BA, Sices H, Cecchini G. Escherichia coli fumarate reductase frdC and frdD mutants. Identification of amino acid residues involved in catalytic activity with quinones. J Biol Chem. 1993;268:815–22. [PubMed] [Google Scholar]

[R79] [79].Yang X, Yu L, He D, Yu CA. The quinone-binding site in succinate-ubiquinone reductase from Escherichia coli. Quinone-binding domain and amino acid residues involved in quinone binding. J Biol Chem. 1998;273:31916–23. [DOI] [PubMed] [Google Scholar]

[R80] [80].Oyedotun KS, Lemire BD. The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase. J Biol Chem. 2001;276:16936–43. [DOI] [PubMed] [Google Scholar]

[R81] [81].Broomfield PL, Hargreaves JA. A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Curr Genet. 1992;22:117–21. [DOI] [PubMed] [Google Scholar]

[R82] [82].Skinner W, Bailey A, Renwick A, Keon J, Gurr S, Hargreaves J. A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Curr Genet. 1998;34:393–8. [DOI] [PubMed] [Google Scholar]

[R83] [83].Matsson M, Hederstedt L. The carboxin-binding site on Paracoccus denitrificans succinate:quinone reductase identified by mutations. J Bioenerg Biomembr. 2001;33:99–105. [DOI] [PubMed] [Google Scholar]

[R84] [84].Lee GY, He DY, Yu L, Yu CA. Identification of the ubiquinone-binding domain in QPs1 of succinate-ubiquinone reductase. J Biol Chem. 1995;270:6193–8. [DOI] [PubMed] [Google Scholar]

[R85] [85].Shenoy SK, Yu L, Yu C. Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase. J Biol Chem. 1999;274:8717–22. [DOI] [PubMed] [Google Scholar]

[R86] [86].Cecchini G, Maklashina E, Yankovskaya V, Iverson TM, Iwata S. Variation in proton donor/acceptor pathways in succinate:quinone oxidoreductases. FEBS Lett. 2003;545:31–8. [DOI] [PubMed] [Google Scholar]

[R87] [87].Maklashina E, Cecchini G. The quinone-binding and catalytic site of complex II. Biochim Biophys Acta. 2010;1797:1877–82. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R88] [88].Nasiri HR, Madej MG, Panisch R, Lafontaine M, Bats JW, Lancaster CR, et al. Design, synthesis, and biological testing of novel naphthoquinones as substrate-based inhibitors of the quinol/fumarate reductase from Wolinella succinogenes. J Med Chem. 2013;56:9530–41. [DOI] [PubMed] [Google Scholar]

[R89] [89].Hederstedt L Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis. Biochim Biophys Acta. 2002;1553:74–83. [DOI] [PubMed] [Google Scholar]

[R90] [90].Tappel AL. Inhibition of electron transport by antimycin A, alkyl hydroxy naphthoquinones and metal coordination compounds. Biochem Pharmacol. 1960;3:289–96. [DOI] [PubMed] [Google Scholar]

[R91] [91].Maklashina E, Cecchini G. The quinone-binding and catalytic site of complex II. Biochim Biophys Acta. 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R92] [92].Inaoka DK, Shiba T, Sato D, Balogun EO, Sasaki T, Nagahama M, et al. Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria. Int J Mol Sci. 2015;16:15287–308. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R93] [93].Miyadera H, Hiraishi A, Miyoshi H, Sakamoto K, Mineki R, Murayama K, et al. Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity. Eur J Biochem. 2003;270:1863–74. [DOI] [PubMed] [Google Scholar]

[R94] [94].Amino H, Wang H, Hirawake H, Saruta F, Mizuchi D, Mineki R, et al. Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit. Mol Biochem Parasitol. 2000;106:63–76. [DOI] [PubMed] [Google Scholar]

[R95] [95].Iwata F, Shinjyo N, Amino H, Sakamoto K, Islam MK, Tsuji N, et al. Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host. Parasitol Int. 2008;57:54–61. [DOI] [PubMed] [Google Scholar]

[R96] [96].Grivennikova VG, Gavrikova EV, Timoshin AA, Vinogradov AD. Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction. Biochim Biophys Acta. 1993;1140:282–92. [DOI] [PubMed] [Google Scholar]

[R97] [97].Cecchini G, Schroder I, Gunsalus RP, Maklashina E. Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta. 2002;1553:140–57. [DOI] [PubMed] [Google Scholar]

[R98] [98].Cheng VW, Tran QM, Boroumand N, Rothery RA, Maklashina E, Cecchini G, et al. A conserved lysine residue controls iron-sulfur cluster redox chemistry in Escherichia coli fumarate reductase. Biochim Biophys Acta. 2013;1827:1141–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R99] [99].Maklashina E, Berthold DA, Cecchini G. Anaerobic expression of Escherichia coli succinate dehydrogenase: functional replacement of fumarate reductase in the respiratory chain during anaerobic growth. J Bacteriol. 1998;180:5989–96. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R100] [100].Ball EG, Anfinsen CB, Cooper O. The inhibitory action of naphthoquinones on respiratory processes. J Biol Chem. 1947;168:257–70. [PubMed] [Google Scholar]

[R101] [101].Takemori S, King TE. Reconstitution of Respiratory Chain Enzyme Systems. 13. Sequential Fragmentation of Succinate Oxidase: Preparation and Properties of Succinate-Cytochrome C Reductase and the Cytochrome B-C1 Particle. J Biol Chem. 1964;239:3546–58. [PubMed] [Google Scholar]

[R102] [102].Fry M, Pudney M. Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem Pharmacol. 1992;43:1545–53. [DOI] [PubMed] [Google Scholar]

[R103] [103].Kessl JJ, Moskalev NV, Gribble GW, Nasr M, Meshnick SR, Trumpower BL. Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex. Biochim Biophys Acta. 2007;1767:319–26. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R104] [104].White GA. Substituted Benzanilides: Structural Variation and Inhibition of Complex II Activity in Mitochondria from a Wild-Type Strain and a Carboxin-Selected Mutant Strain of Ustilago maydis. Pestic Biochem Physiol. 1987;27:249–60. [Google Scholar]

[R105] [105].Horsefield R, Yankovskaya V, Sexton G, Whittingham W, Shiomi K, Omura S, et al. Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction. J Biol Chem. 2006;281:7309–16. [DOI] [PubMed] [Google Scholar]

[R106] [106].Berman HM, Kleywegt GJ, Nakamura H, Markley JL. The future of the protein data bank. Biopolymers. 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R107] [107].Zhou Q, Zhai Y, Lou J, Liu M, Pang X, Sun F. Thiabendazole inhibits ubiquinone reduction activity of mitochondrial respiratory complex II via a water molecule mediated binding feature. Protein Cell. 2011;2:531–42. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R108] [108].Huang L-S, Borders TM, Shen JT, Wang C-J, Berry EA. Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å. Acta Cryst D. 2005;D61. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R109] [109].Tomasiak TM, Archuleta TL, Andrell J, Luna-Chavez C, Davis TA, Sarwar M, et al. Geometric restraint drives on- and off-pathway catalysis by the Escherichia coli menaquinol:fumarate reductase. J Biol Chem. 2011;286:3047–56. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R110] [110].White GA, Thorn GD. Structure-Activity Relationships of Carboxamide Fungicides and the Succinic Dehydrogenase Complex of Cryptococcus laurentii and Ustilago maydis’. Pestic Biochem Physiol. 1975;5:380–95. [Google Scholar]

[R111] [111].Mowery PC, Ackrell BA, Singer TP, Thorn GD, White GA. Carboxins: powerful selective inhibitors of succinate oxidation in animal tissues. Biochem Biophys Res Commun. 1976;71:354–61. [DOI] [PubMed] [Google Scholar]

[R112] [112].Coles CJ, Singer TP, White GA, Thorn GD. Studies on the binding of carboxin analogs to succinate dehydrogenase. J Biol Chem. 1978;253:5573–8. [PubMed] [Google Scholar]

[R113] [113].Ramsay RR, Ackrell BA, Coles CJ, Singer TP, White GA, Thorn GD. Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II. Proc Natl Acad Sci U S A. 1981;78:825–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R114] [114].White GA. Pyrazole Carboxanilide Fungicides. I. Correlation of Mitochondrial Electron Transport Inhibition and Anti-fungal Activity. Pestic Biochem Physiol. 1986;25:163–8. [Google Scholar]

[R115] [115].White GA. Substituted 2-methylbenzaniIides and Structurally Related Carboxamides: Inhibition of Complex II Activity in Mitochondria from a Wild-Type Strain and a Carboxin-Resistant Mutant Strain of Ustilago maydis. Pestic Biochem Physiol. 1989;34:255–76. [Google Scholar]

[R116] [116].Miyadera H, Shiomi K, Ui H, Yamaguchi Y, Masuma R, Tomoda H, et al. Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase). Proc Natl Acad Sci U S A. 2003;100:473–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R117] [117].Silkin Y, Oyedotun KS, Lemire BD. The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase. Biochim Biophys Acta. 2007;1767:143–50. [DOI] [PubMed] [Google Scholar]

[R118] [118].Tran QM, Rothery RA, Maklashina E, Cecchini G, Weiner JH. The quinone binding site in Escherichia coli succinate dehydrogenase is required for electron transfer to the heme b. J Biol Chem. 2006;281:32310–7. [DOI] [PubMed] [Google Scholar]

[R119] [119].Milunsky JM, Maher TA, Michels VV, Milunsky A. Novel mutations and the emergence of a common mutation in the SDHD gene causing familial paraganglioma. Am J Med Genet. 2001;100:311–4. [DOI] [PubMed] [Google Scholar]

[R120] [120].De Miccolis Angelini RM, Habib W, Rotolo C, Pollastro S, Faretra F. Selection, characterization and genetic analysis of laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to the fungicide boscalid. European Journal of Plant Pathology. 2010;128:185–99. [Google Scholar]

[R121] [121].Leroux P, Gredt M, Leroch M, Walker AS. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Appl Environ Microbiol. 2010;76:6615–30. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R122] [122].Yin YN, Kim YK, Xiao CL. Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple. Phytopathology. 2011;101:986–95. [DOI] [PubMed] [Google Scholar]

[R123] [123].Veloukas T, Leroch M, Hahn M, Karaoglanidis GS. Detection and Molecular Characterization of Boscalid-Resistant Botrytis cinerea Isolates from Strawberry. Plant Dis. 2011;95:1302–7. [DOI] [PubMed] [Google Scholar]

[R124] [124].Manodori A, Cecchini G, Schroder I, Gunsalus RP, Werth MT, Johnson MK. [3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis. Biochemistry. 1992;31:2703–12. [DOI] [PubMed] [Google Scholar]

[R125] [125].Hägerhäll C, Sled V, Hederstedt L, Ohnishi T. The trinuclear iron-sulfur cluster S3 in Bacillus subtilis succinate:menaquinone reductase; effects of a mutation in the putative cluster ligation motif on enzyme activity and EPR properties. Biochim Biophys Acta. 1995;1229:356–62. [DOI] [PubMed] [Google Scholar]

[R126] [126].Ishii N, Fujii M, Hartman PS, Tsuda M, Yasuda K, Senoo-Matsuda N, et al. A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes. Nature. 1998;394:694–7. [DOI] [PubMed] [Google Scholar]

[R127] [127].Amiri A, Zuniga AI, Peres NA. Mutations in the Membrane-Anchored SdhC Subunit Affect Fitness and Sensitivity to Succinate Dehydrogenase Inhibitors in Botrytis cinerea Populations from Multiple Hosts. Phytopathology. 2020;110:327–35. [DOI] [PubMed] [Google Scholar]

[R128] [128].Lovell SC, Word JM, Richardson JS, Richardson DC. The penultimate rotamer library. Proteins. 2000;40:389–408. [PubMed] [Google Scholar]

[R129] [129].Oyedotun KS, Lemire BD. The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562. FEBS Lett. 1999;442:203–7. [DOI] [PubMed] [Google Scholar]

[R130] [130].Tron T, Crimi M, Colson AM, Degli Esposti M. Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor-resistant mutants. Eur J Biochem. 1991;199:753–60. [DOI] [PubMed] [Google Scholar]

[R131] [131].Yun CH, Wang Z, Crofts AR, Gennis RB. Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides. J Biol Chem. 1992;267:5901–9. [PubMed] [Google Scholar]

[R132] [132].Crofts A, Hacker B, Barquera B, Yun CH, Gennis R. Structure and function of the bc-complex of Rhodobacter sphaeroides. Biochim Biophys Acta. 1992;1101:162–5. [PubMed] [Google Scholar]

[R133] [133].Berry EA, Walker FA. Bis-histidine-coordinated hemes in four-helix bundles: how the geometry of the bundle controls the axial imidazole plane orientations in transmembrane cytochromes of mitochondrial complexes II and III and related proteins. J Biol Inorg Chem. 2008;13:481–98. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R134] [134].Scheidt WR, Kirner JF, Hoard JL, Reed CA. Unusual orientation of axial ligands in metalloporphyrins. Molecular structure of low-spin bis(2-methylimidazole)(meso-tetraphenylporphinato)iron(III) perchlorate. Journal of the American Chemical Society. 1987;109:1963–8. [Google Scholar]

[R135] [135].Walker FA, Huynh BH, Scheidt WR, Osvath SR. Models of the Cytochromes b. The Effect of Axial Ligand Plane Orientation on the EPR and Mössbauer Spectra of Low-Spin Ferrihemes. J Am Chem Soc. 1986;108:5288–97. [Google Scholar]

[R136] [136].Yu L, Xu JX, Haley PE, Yu CA. Properties of bovine heart mitochondrial cytochrome b560. J Biol Chem. 1987;262:1137–43. [PubMed] [Google Scholar]

[R137] [137].Peterson J, Vibat C, Gennis RB. Identification of the axial heme ligands of cytochrome b556 in succinate: ubiquinone oxidoreductase from Escherichia coli. FEBS Lett. 1994;355:155–6. [DOI] [PubMed] [Google Scholar]

[R138] [138].Crouse BR, Yu CA, Yu L, Johnson MK. Spectroscopic identification of the axial ligands of cytochrome b560 in bovine heart succinate-ubiquinone reductase. FEBS Lett. 1995;367:1–4. [DOI] [PubMed] [Google Scholar]

[R139] [139].Waldeck AR, Stowell MH, Lee HK, Hung SC, Matsson M, Hederstedt L, et al. Electron paramagnetic resonance studies of succinate:ubiquinone oxidoreductase from Paracoccus denitrificans. Evidence for a magnetic interaction between the 3Fe-4S cluster and cytochrome b. J Biol Chem. 1997;272:19373–82. [DOI] [PubMed] [Google Scholar]

[R140] [140].Moss GP. Nomenclature of tetrapyrroles. Recommendations 1986 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). Eur J Biochem. 1988;178:277–328. [DOI] [PubMed] [Google Scholar]

[R141] [141].Miki T, Yu L, Yu CA. Characterization of ubisemiquinone radicals in succinate-ubiquinone reductase. Arch Biochem Biophys. 1992;293:61–6. [DOI] [PubMed] [Google Scholar]

[R142] [142].Shima Y, Ito Y, Hatabayashi H, Koma A, Yabe K. Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides. Biosci Biotechnol Biochem. 2011;75:181–4. [DOI] [PubMed] [Google Scholar]

[R143] [143].Ruprecht J, Iwata S, Rothery RA, Weiner JH, Maklashina E, Cecchini G. Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster. J Biol Chem. 2011;286:12756–65. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R144] [144].Shima Y, Ito Y, Kaneko S, Hatabayashi H, Watanabe Y, Adachi Y, et al. Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae. Fungal Genet Biol. 2009;46:67–76. [DOI] [PubMed] [Google Scholar]

[R145] [145].Hauska G Plasto- and ubiquinone as translocators of electrons and protons through membranes: a facilitating role of the isoprenoid side chain. FEBS Lett. 1977;79:345–7. [DOI] [PubMed] [Google Scholar]

[R146] [146].Teixeira MH, Arantes GM. Effects of lipid composition on membrane distribution and permeability of natural quinones. RSC Adv. 2019;2019:16892–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R147] [147].Cheng VW, Johnson A, Rothery RA, Weiner JH. Alternative sites for proton entry from the cytoplasm to the quinone binding site in Escherichia coli succinate dehydrogenase. Biochemistry. 2008;47:9107–16. [DOI] [PubMed] [Google Scholar]

[R148] [148].Iverson TM, Luna-Chavez C, Schroder I, Cecchini G, Rees DC. Analyzing your complexes: structure of the quinol-fumarate reductase respiratory complex. Curr Opin Struct Biol. 2000;10:448–55. [DOI] [PubMed] [Google Scholar]

[R149] [149].Kita K, Vibat CR, Meinhardt S, Guest JR, Gennis RB. One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556. J Biol Chem. 1989;264:2672–7. [PubMed] [Google Scholar]

[R150] [150].Yang X, Yu L, Yu CA. Resolution and reconstitution of succinate-ubiquinone reductase from Escherichia coli. J Biol Chem. 1997;272:9683–9. [DOI] [PubMed] [Google Scholar]

[R151] [151].Stroh A, Anderka O, Pfeiffer K, Yagi T, Finel M, Ludwig B, et al. Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans. J Biol Chem. 2004;279:5000–7. [DOI] [PubMed] [Google Scholar]

[R152] [152].Otwinowski Z, Minor W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology. 1997;276:307–26. [DOI] [PubMed] [Google Scholar]

[R153] [153].Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68:352–67. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R154] [154].Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R155] [155].Jones TA, Zhou J-Y, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors on these models. Acta Crystallogr. 1991; A47,:110–9. [DOI] [PubMed] [Google Scholar]

[R156] [156].Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R157] [157].Schüttelkopf AW, van Aalten DM. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2004;60:1355–63. [DOI] [PubMed] [Google Scholar]

[R158] [158].Kraulis PJ. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr. 1991; 24:946–50. [Google Scholar]

[R159] [159].Merritt EA, Murphy ME. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994;50:869–73. [DOI] [PubMed] [Google Scholar]

[R160] [160].Combet C, Blanchet C, Geourjon C, Deleage G. NPS@: network protein sequence analysis. Trends Biochem Sci. 2000;25:147–50. [DOI] [PubMed] [Google Scholar]

[R161] [161].Gong H, Gao Y, Zhou X, Xiao Y, Wang W, Tang Y, et al. Cryo-EM structure of trimeric Mycobacterium smegmatis succinate dehydrogenase with a membrane-anchor SdhF. Nat Commun. 2020;11:4245. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Crystallographic Investigation of the Ubiquinone binding site of Respiratory Complex II and its Inhibitors

Li-shar Huang

Peter Lümmen

Edward A Berry

Abstract

1. Introduction:

1.1. Complex II

Figure 1. Overall structure of Mitochondrial Complex II (Stereo projection).

1.2. Q-site inhibitors.

Figure 2.

Figure 3. Structures of Ubiquinone, HQNO, and Complex II Q-site inhibitors.

1.3. X-ray studies.

2. Results and Discussion

2.1. Structure in the region of the Q-site

Location of the Q site and structural elements involved in its formation.

Table.

Figure 4. Location of the Q site in the overall structure showing two modes of binding.

2.2. Overview of binding interactions: the role of common features of the inhibitors.

Figure 5. Superposition of bound inhibitors in the Q site to identify common features (Stereo figure).

Figure 6. Cross-sectioned electrostatic surfaces of the Q-site binding pocket.

2.2.1. Universal interactions with inhibitor keto or hydroxyl group.

2.2.2. Possible H-bonds to SdhC from the other (inward-facing) edge of the Inhibitor

2.2.3. Lining of the central pocket; interface with the Fe3S4 cluster.

2.2.4. Head pocket

2.2.5. Heme propionates.

2.2.6. Cavity for Ortho-substituent of head ring

2.2.7. Tail pocket:

2.2.8. How does the substrate ubiquinone bind?

2.2.9. H-bond with B:His216?

2.2.10. Substrate and proton access to the Q-site.

Figure 7. Transmembrane level of ubiquinone binding positions or entry points in the respiratory chain.

2.3. Differences between the E. coli and mitochondrial Q-sites, resulting from different binding of the extrinsic subunits to the membrane anchor domain.

Figure 8. Comparison of the Q site architecture in Complex II from mitochondria (colored as in Figure 4) and E. coli (gray).

2.4. Details of the individual inhibitors.

Figure 9. Individual inhibitors in context of the structure, with electron density.

2.5. Conclusion

3. Methods

Supplementary Material

4. Acknowledgements

Footnotes

5. References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

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2.2.3. Lining of the central pocket; interface with the Fe₃S₄ cluster.