The assembly of succinate dehydrogenase: a key enzyme in bioenergetics

Abstract

Succinate dehydrogenase (SDH) also known as complex II or succinate:quinone oxidoreductase is an enzyme involved in both oxidative phosphorylation and tricarboxylic acid cycle; the processes that generate energy. SDH is a multi-subunit enzyme which requires a series of proteins for its proper assembly at several steps. This enzyme has medical significance as there is a broad range of human diseases from cancers to neurodegeneration related to SDH malfunction. Some of these disorders have recently been linked to defective assembly factors, reinvigorating further research in this area. Apart from that this enzyme has agricultural importance as many fungicides have been/will be designed targeting specifically this enzyme in plant fungal pathogens. In addition, we speculate it might be possible to design novel fungicides specifically targeting fungal assembly factors. Considering the medical and agricultural implications of SDH, the aim of this review is an overview of the SDH assembly factors and critical analysis of controversial issues around them.

Introduction

Glucose metabolism, the main source of energy in eukaryotes provides carbon skeletons and ATP in three cellular processes: glycolysis, the tricarboxylic acid cycle (TCA cycle, Krebs cycle) and oxidative phosphorylation (OXPHOS) [1]. Enzymes catalyze the reactions in these pathways and succinate dehydrogenase (SDH) is one of the most important enzymes involved. SDH, also known as complex II or succinate:ubiquinone oxidoreductase (SQR) is a unique enzyme in four ways: first, it is involved in both the TCA and OXPHOS in mitochondria. Second, with a few exceptions all the genes for mitochondrial SDH are nuclear-encoded [2] (the exceptions include red algae such as Porphyra purpurea and heterotrophic zooflagellates such as Reclinomonas americana, in which the SDHB, SDHC, and SDHD genes are mitochondrially encoded [3]). Third, it is the only membrane-bound component of TCA cycle [4]. Fourth, it is the smallest and the only complex of mitochondrial electron transport chain (ETC) which does not directly extrude protons [5]. Although it contributes to the proton gradient by supplying reducing equivalents resulting from succinate metabolism. The reducing equivalents are then transported through the ubiquinone pool thereby enabling proton extrusion by complex III and IV.

The TCA cycle generates ATP by glucose oxidation through eight steps. It also engenders intermediate metabolites for numerous anabolic pathways [1]. SDH catalyzes one step of the TCA cycle, the oxidation of succinate to fumarate. Instead of reducing NAD⁺ like all the other oxidative steps of the TCA cycle, the electrons are transferred to ubiquinone. Presumably, this situation arose because the succinate/fumarate couple (Em7 ~ + 30 mv) is not strongly enough reducing, to reduce NAD⁺ to NADH (Em7 − 320 mv).

OXPHOS, on the other hand produces ATP through coupling the mitochondrial respiratory chain (MRC) to ATP synthesis [6–8]. The MRC comprises four complexes (complex I–IV [9]) along with mobile electron carriers, ubiquinone (coenzyme Q) and cytochrome c and is located in the inner mitochondrial membrane. The complexes catalyze the oxidation of reducing substrates, such as NADH, by O₂ as the terminal electron acceptor which yields H₂O at final step. At certain points in complexes I, III, and IV, electron transport is coupled to the translocation of protons from the mitochondrial matrix into the intermembrane space. This process drives phosphorylation of ADP by ATP synthase (complex V) leading to the formation of ATP [2, 10, 11]. The less energetic electrons from succinate oxidation reduce ubiquinone to ubiquinol, which is the substrate for complex III. They thus bypass complex I and are used to make ATP by only complexes III and IV. Since ubiquinone is a hydrophobic molecule existing in the mitochondrial inner membrane, complex II must transfer electrons from succinate in the matrix aqueous phase to ubiquinone in the membrane. The structure of complex II is ideally suited to do this.

Here, in this article, we review the SDH complex structure and assembly. Since the plant SDH assembly has recently been discussed extensively in Refs. [12, 13] we only briefly point out the key findings here.

The overall structure of SDH

Succinate dehydrogenase in eukaryotes is composed of four subunits SDHA-D (Sdh1–4 in yeast). They are localized to the inner mitochondrial membrane and cytoplasmic membrane in eukaryotes and prokaryotes, respectively. The catalytic domain (SDHA and B) is extrinsic on the matrix side, while the anchor subunits (SDHC and D) are intrinsic transmembrane proteins, allowing transfer of the electrons from succinate in the mitochondrial matrix to ubiquinone in the inner membrane.

The SDHA subunit is a flavoprotein containing a covalently bound FAD cofactor and the binding site for dicarboxylates (e.g., succinate). SDHB is an iron–sulfur cluster protein containing three Fe–S clusters. SDHA and SDHB make up the catalytic domain. They extend out into the matrix and constitute the hydrophilic head. SDHC and SDHD subunits are alpha-helical transmembrane proteins which ligate a single heme between them. SDHB is sandwiched between SDHA on the matrix side, and SDHC and SDHD in the membrane. These two transmembrane subunits thus form the hydrophobic anchor. A small patch of the anchor is exposed on the distal side, to the aqueous intermembrane space [7, 14]. Therefore, the structure of SDH can be divided into two main modules; SDHA and SDHB as the membrane extrinsic (soluble) domain, and SDHC and SDHD as the membrane domain [4, 15] (Fig. 1). In the structure, we can see a clear path for electrons from FAD in SDHA, via three Fe–S clusters in SDHB, to the quinone-binding site at the membrane interface.

Fig. 1.

The structure of complex II (succinate dehydrogenase). This enzyme links oxidative phosphorylation to TCA cycle. Succinate is oxidized to fumarate in the TCA cycle by SDHA-B and the electrons derived are transported to ubiquinone (coenzyme Q) and then to complex III. The electrons along the way reduce FAD of SDHA subunit and move through Fe–S clusters in SDHB subunit and then reduce ubiquinone before transfer to complex III

The interface between the catalytic head domain and the anchor subunits can be separated without the use of detergent, making the catalytic domain an “extrinsic” membrane protein. In fact, the earliest preparations of isolated succinate dehydrogenase consisted of the water-soluble two-subunit complex, stripped from the mitochondrial membrane in the absence of detergents by organic solvent [16–18] or chaotropic agents [19]. The soluble two-subunit catalytic domain of bovine complex II [19] or Escherichia coli fumarate reductase [20] can also be prepared by treating the isolated four-subunit complex with chaotropic agents. The two-subunit preparations were termed “succinate dehydrogenase” because they displayed succinate dehydrogenase activity with artificial electron acceptors: E.C. 1.3.99.1 (ubiquinone not discovered until 1957). Four-subunit preparations were termed complex II or succinate:ubiquinone oxidoreductase (SQR, E.C. 1.3.5.1), but now also succinate dehydrogenase (SDH) often is used for the four-subunit complex, as the two-subunit preparation is realized to be somewhat artefactual, and that is the way we will use it here.

While complex II specifically refers to mitochondrial succinate:ubiquinone oxidoreductase (SQR) [9] and homologous SQR found in some bacteria, it is a member of a superfamily of related membrane proteins coupling the succinate–fumarate and quinone–quinol redox pairs, collectively referred to as succinate:quinone oxidoreductases (SQORs) [21]. In some family members, the physiological direction of the reaction catalyzed is the reverse, i.e., quinol oxidation with reduction of fumarate to succinate. These enzymes are called fumarate reductase (FRD) or more specifically quinol:fumarate reductase (QFR).

The SQORs as a superfamily all have a flavoprotein and iron–sulfur protein with strong sequence (and where known, structural) homology. The great majority of well-characterized SQORs also have transmembrane anchors related to SDHC and SDHD, but with considerably more variation in sequence and structure [8, 21]. The membrane subunits vary in the number of membrane domain subunits (1–2), the number of transmembrane helices (5 or 6), and the number of b-type hemes (0–2) and the presence of one or two quinone-binding sites.

The canonical SQORs having transmembrane anchors have been classified by the number of hemes and the number of subunits making up the anchor. Class C, which includes complex II, has two anchor subunits and only one heme. Class D, which includes E. coli FRD, also has two anchor subunits but no heme. Class A and B both have two hemes but differ in that, while Class A has two membrane subunits like SDHC and SDHD, in Class B they are fused into a single subunit, with elimination of one transmembrane helix. Some of the Class A members function as succinate dehydrogenases, but in the rest of this text we will take SDH to mean complex II proper, i.e., Class C mitochondrial and bacterial SQR. Ascaris rhodoquinol:fumarate reductase also falls in Class C, and probably evolved from mitochondrial complex II [21]. Lemos and coworkers [22] have proposed inclusion of a number of less well-known SQORs, anchored by amphipathic helices that bind to the surface of the membrane, as Class E.

SdhA/FrdA has soluble homologs which catalyze α, β-dehydrogenase reactions. The fumarate reductase (flavocytochrome c) from Shewanella frigidimarina and Shewanella putrefaciens, and l-aspartate oxidase from E. coli are examples of soluble SdhA/FrdA homologs. The Shewanella enzyme consists of a single polypeptide chain harboring a domain similar to the flavoprotein (FrdA/SdhA) fused to a tetra-heme cytochrome. Consequently, they are termed as flavocytochrome c3 (Fcc3) fumarate reductases. E. colil-aspartate oxidase, on the other hand, does not contain any peripheral domain to accommodate redox cofactors. Interestingly, FAD is non-covalently bound in these soluble enzymes, while it is covalently bound in the SQORs of Classes A, B, C, or D. These enzymes are not SQORs since they do not oxidize or reduce quinones, but the high degree of homology with SDHA, especially around the dicarboxylate-binding site, together with the convenience of working with soluble, single-subunit proteins, makes them valuable in the effort to decipher mechanisms of dicarboxylate oxidoreduction in complex II (see Ref. [8] and references therein).

In principal under the same conditions an enzyme must catalyze the forward and reverse reactions equally, since it cannot change the equilibrium. However evolution has engineered SDH to favor the forward direction, by causing it to shut down under highly reducing conditions that would cause net reverse reaction. This is analogous to a ratchet: when the force is in one direction it slides, in the other direction it sticks. The electrical analogy of a diode has also been used to describe the phenomenon [23]. On the other hand, E. coli fumarate reductase, which is expressed under anaerobic conditions, is more active when reducing fumarate [8, 24]. The structural factors favoring each direction of the reaction has been detailed by Maklashina et al. [25].

Proteins required for the assembly of SDH

Having four subunits and several cofactors as well as being embedded in a specific position in inner mitochondrial membrane necessitate an intricate multi-step mechanism for the assembly and integration of SDH complex in its proper location. The number of assembly factors identified in this process has been increasing over recent years illustrating the complexity and the level of regulation effected. So far, several proteins have been known to be essential for the assembly of SDH. Here, we attempt to elaborate how these proteins may contribute to the SDH assembly at certain steps. A summary of these proteins with their roles is listed in Table 1.

Table 1.

The list and proposed functions of SDH complex assembly factors

Assembly factor	Location	Proposed function
Tcm62 (yeast), prohibitin? (mammalian cells)	Mitochondrial membrane	Chaperone for SDH and a group of mitochondrial proteins at physiological temperature and heat stress condition, respectively
Flx1 (yeast)	Mitochondria, cytoplasm?	FAD transporter? Regulator of Sdh1 level? Regulator of Sdh1 flavination? An aging determinant?
SDHAF1 or LYRM8 (humans)/Sdh6 (yeast)	Mitochondrial matrix	Maturation of SDHB (Sdh2), protection against oxidative damage together with SDHAF3, Fe–S cluster insertion into the SDH complex in humans but not in yeast
SDHAF3 (humans)/SDH7 (yeast and Drosophila)	Mitochondrial matrix	Similar to SDHAF1
SDHAF2 (humans)/Sdh5 (yeast)/SdhE (bacteria)	Mitochondrial matrix	Regulator/chaperone for SDHA flavination
SDHAF4 (humans and Drosophila)/Sdh8 (yeast)	Mitochondrial matrix	Chaperone for SDHA-FAD, promotes Sdh1–Sdh2 dimerization

Tcm62p

Tcm62 was the first protein which was identified to function as a chaperone required for the assembly of SDH. It was reported to interact with at least three subunits of SDH. It is a 64-kDa membrane-spanning protein, containing 572 amino acids and has 17.3% and 16% sequence identity with yeast chaperonin (hsp60) and E. coli GroEL, respectively, both acting as molecular chaperone. No close homologs have been reported so far, suggesting this protein is possibly yeast-specific [6, 15]. Tcm62 is initially synthesized as a precursor which is then transported to the mitochondria where the pre-sequence is cleaved to form the mature protein. It has been hypothesized that Tcm62 may be engaged in the formation/insertion of the [3Fe–4S] clusters into apo-Sdh2p or stabilization of the holo-Sdh2p subunit before it forms a stable complex with the Sdh1p subunit [15].

Tcm62 overexpression leads to the formation of insoluble large aggregates (inclusion bodies) which trap Sdh1 and Sdh2 but not unrelated proteins within the mitochondria. This phenomenon points to the specificity of its interaction with Sdh subunits. Tcm62 without Sdh forms a complex of about 450 kDa which might be a heptameric ring-like structure similar to the E. coli GroEL [15]. Further study has shown that Tcm62p also acts as a general chaperone, assuring the stability of mitochondrial proteins including the respiratory chain complexes at high temperatures. This function of Tcm62p resembles that of FMC1 which is required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions [26].

Subsequently, it was reported that similar to chaperonins, Tcm62p forms a soluble, high-molecular weight protein complex of 850 kDa in the mitochondrial matrix [27]. This observation was in contrast with an earlier finding claiming Tcm62p to be a 200-kDa transmembrane protein [15]. The reason for this discrepancy is not clear but it is possibly the consequence of application of two different assays (gel filtration and blue native gel electrophoresis). Despite its similarity with chaperonins, Tcm62p lacks the typical ATP-binding and -hydrolysis domain which is conserved in chaperonins and required for their function [27]. Therefore, it might be that Tcm62p exerts its function through a distinct mechanism.

The lack of Tcm62 leads to respiration deficiency, while its overexpression results in formation of intra-mitochondrial inclusion bodies that trap SDH subunits. Although Tcm62 is absolutely essential for the assembly of SDH, it is dispensable for the assembly of other respiratory chain components. It may be that other complexes fold spontaneously predominantly with less need to Tcm62, or there might be other chaperones governing the folding of these complexes. Intriguingly SDH2 is the only subunit in the whole respiratory chain which contains [3Fe–4S], and, therefore, it has been suggested that this type of cluster may pose a strict folding requirements which are possibly met by Tcm62 [15].

Tcm62 has several attributes in common with Abc1 which is essential for the assembly of complex III [15, 28–30]. First, Like Tcm62, Abc1 has minor similarity with the chaperonins. Second, both Abc1 and Tcm62 are specifically required for the assembly of a particular respiratory chain complex. Third, deletion of both ABC1 and TCM62 genes independently impair the function of respiratory chain complex [15, 29]. These common features, therefore, suggest that Tcm62 and Abc1 most likely have chaperone function in the assembly of complex II and complex III, respectively. The loss of Abc1 also affects the function of complex II (SDH) and complex IV which might be the consequence of its effect on complex III. This particularly might be the case in yeast as there is evidence that the respiratory chain complexes may exist in the form of a supermolecular complex in this microorganism [6, 31, 32]. Abc1 has also been found to be engaged in ubiquinone biosynthesis. Since Abc1 contains motifs usually existing in eukaryotic protein kinases, Abc1 may regulate ubiquinone biosynthesis through the phosphorylation of a biosynthetic enzyme [6, 33]. This suggests that ubiquinone may also have a role in the efficient biosynthesis of SDH. Correspondingly, the lack of COQ5 gene encoding a methyltransferase in the yeast ubiquinone biosynthetic pathway, also impairs SDH assembly [6, 34]. These results commend that Tcm62 and Abc1 may have similar functions as chaperones but also have distinct roles.

Another protein which has been found to be associated with Tcm62 is Bcl-X_L (a mitochondrial transmembrane protein involved in apoptosis inhibition). It has been shown that Bcl-X_L overexpression can partially rectify the defect in diauxic shift of tcm62Δ yeast cells. This is in line with the fact that Bcl-X_L facilitates diauxic shift in mammalian cells. Nevertheless, Bcl-X_L is unlikely to operate as a molecular chaperone, but rather it may compensate for the lack of Tcm62 through increasing the overall competence of mitochondria or endurance of mitochondria in stress conditions [35]. The same study has revealed that Tcm62 inhibits mammalian cell death following growth factor withdrawal. This function of Tcm62 is in common with prohibitin, the human analogue of Tcmp62 with a chaperone function. Thus, inhibition of mammalian cell death upon growth factor deprivation seems to occur by the chaperone function of Tcm62. Therefore, Tcm62 ostensibly facilitates cell survival in the absence of growth factors through promoting the efficiency of oxidative phosphorylation [35]. A more recent study has indicated that TCM62 deletion leads to a shorter replicative and chronological life span compared with wild-type yeast, and this is presumably due to several mechanisms such as low respiration rate and low-energy production rather than ROS-related toxicity or changes in mitochondrial membrane potential [36].

Overall, these findings suggest that Tcm62 most likely has a chaperone function for SDH and a group of mitochondrial proteins at physiological temperature and heat stress condition, respectively [15, 27], but leave several issues unresolved (see “Conclusion and future perspective”).

Flx1

Initial studies have shown that low levels of FAD in a mutant yeast strain dramatically reduce the enzymatic activity of Sdh1 subunit which employs FAD as a coenzyme [37]. Later, Tzagoloff et al.’s research has revealed that Flx1 can restore wild-type FAD/FMN ratio to the yeast mutant. The primary sequence of Flx1 suggests it is a carrier protein belonging to a family consisting of mitochondrial substrate and nucleotide carriers (IMM carriers). And the biochemical and genetics pieces of evidence show it has a role as a FAD carrier protein involved in transport of FAD from the cytosol into the mitochondria [38]. Further studies by Bafunno et al. and others have indicated that FLX1 deletion, in a different genetic background, leads to reduced levels of SDH, lipoamide dehydrogenase, and flavoproteins involved in ubiquinone biosynthesis, i.e., FAD-dependent enzymes [39–41]. However, in contrast with Tzagoloff et al., Bafunno et al. did not detect any changes in flavin cofactor levels or flavination (incorporation of the flavin adenine dinucleotide cofactor) of SDH inside mitochondria. Besides, they showed FAD was synthesized both in the yeast cytoplasm and mitochondria, although the latter had a minor contribution to the overall FAD synthesis [39]. Similarly the mammalian orthologue of the FAD1 gene (which in yeast is responsible for encoding FAD synthetase which performs the second step in synthesis of FAD from riboflavin), i.e., FLAD1, was later shown to produce two transcripts encoding two isoforms of FAD synthetase, one of which is cytosolic and the other is mitochondrial [42, 43]. This implied that mammalian cells might also synthesize FAD inside the mitochondrial matrix thereby rendering any IMM carrier dispensable [43]. These observations were contrary to an earlier finding claiming FAD synthetase activity being limited to the cytosolic fraction [37]. Based on their results, Bafunno et al. proposed that Flx1 might have a regulatory role in synthesis or degradation of SDH rather than FAD carrier role [39]. Further investigation by the same group suggested this regulatory function is implemented at the post-transcription level and involves regulatory sequences located upstream of the SDH1-coding sequence. Moreover, they illustrated that the SDH1-coding sequence and the regulatory sequences located downstream of the SDH1-coding region as well as protein import and cofactor attachment do not play a notable role in regulating SDH1 expression level [44].

In other studies, yet three more controversial observations with regard to the role of Flx1 were made. First, it was reported yeast strains lacking FLX1 displayed a significant defect in Sdh1 flavination [45–47]. However, Sdh5 (another SDH assembly factor) overexpression enhanced Sdh1 flavination to 50% of WT levels but could not restore the growth of flx1Δ yeast cells in non-fermentable carbon sources. This, therefore, implied that Flx1 might have a role in Sdh1 flavination but also an extra role in other as yet unknown cellular process. In addition, it hinted that in the absence of Flx1, FAD is still available in the matrix possibly as a result of FAD release from the FAD-containing enzymes [47]. Second, only a minor reduction in SDH1 expression was detected upon FLX1 deletion. This again entails that the regulatory role of Flx1, if any at all, for SDH1 expression is dispensable [45]. The third point of dispute was that while the Winge group [46] suggested the defect in Sdh1 flavination in a flx1Δ yeast strain led to an unstable apo-Sdh1, another study showed apo-Sdh1 was fairly stable in the absence of Flx1 [45]. The disparities between the two studies might originate, at least partially, from the difference in the yeast strains which were used.

Recently, Flx1 has been shown to be involved in general processes such as the efficiency of ATP production, ROS homeostasis, and chronological lifespan of S. cerevisiae [48]. This is the reminiscent of the role of Tcm62 and Sdh5 in aging [36, 45], and perhaps points to a role of mitochondrial flavoproteome [48] or ATP synthesis as a major regulator of aging process rather than certain specific respiratory chain elements, i.e., Tcm62, Flx1 or Sdh5. The role of Flx1 in aging is apparently exerted through changing the cellular levels of succinate and it seems that even the level of FLX1 expression is influenced by metabolic variations as a dozen of hypothetical regulator motifs have been revealed upstream of the FLX1 gene by bioinformatics approaches [48].

Altogether these data suggest different roles for Flx1p as a FAD transporter, regulator of Sdh1 level, or regulator of Sdh1 flavination. The source of these controversies is not yet clear, but it might be that the yeast strains used in these studies have alternative FAD transporters or various levels of FAD in the matrix [47]. Furthermore, Flx1 may have several roles in mitochondria and cytoplasm, with each role being more prominent in certain yeast strains or under specific conditions. The role of Flx1 orthologue in mammalian SDH1 expression is not entirely clear. Therefore, further investigation is warranted to understand the roles of Flx1 in SDH biology.

SDHAF1 or LYRM8 (in humans)/Sdh6 (in yeast)

SDHAF1 is a soluble, mitochondrial matrix protein with 115 residue [49]. This protein was initially found as a specific human SDH assembly factor which was defective in two families with specific infantile leukoencephalopathy syndrome. Impaired function of this protein in humans and its homolog in yeast led to severe reduced SDH activity and assembly. Accordingly, a yeast strain lacking SDHAF1 homolog (Sdh6) was found to be OXPHOS incompetent. Transformation of this strain with YDR379C-A variants corresponding to the human mutant alleles did not recover OXPHOS growth, indicating that these mutations cause the disease. However, the absence of SDHAF1 had no effect on other mitochondrial respiratory chain complexes, demonstrating its specificity to the SDH assembly [50]. Biochemical studies in humans and yeast showed a significant reduction in the SDH enzyme activity for the mutant alleles, while the K_m value of SDH for succinate in the SDH6 null mutant was almost similar to the one in wild-type SDH6, indicating the mutant allele causes a deficiency in the number of SDH units rather than qualitative remodeling of the enzyme [50]. Further clinical studies reported more cases of infantile leukoencephalopathy syndrome with similar or novel mutations in SDHAF1 gene particularly Gly57Arg. However, the severity of clinical symptoms of the patients were different, depending upon the mutation involved. Similar to the previous cases, these patients showed accumulation of succinate detectable by in vivo proton MR spectroscopy of the brain [51, 52].

Several lines of evidence hint that Sdh6 interacts with a Sdh1/Sdh2 subcomplex and the interface for the interaction locates within Sdh2: (1) the lack of Sdh6 leads to reduced steady-state levels of Sdh2, while steady state and flavination of Sdh1 remains unperturbed. (2) The cells lacking Sdh3 or Sdh4 have increased levels of Sdh1–Sdh2 subcomplex and Sdh6. (3) Sdh6 co-precipitates with Sdh1 and Sdh2 in sdh4Δ mutant cells, while Sdh6 does not co-precipitate with Sdh1 and Sdh2 in WT or sdh2Δ mutant cells in immunoprecipitation experiment. (4) Sdh6 overexpression leads to accumulation of Sdh2 in sdh1Δ mutant, in which Sdh2 is extremely unstable.

SDHAF1 contains a LYR motif; LX(L/A)YRXX(L/I)(R/K) which seems to be the signature for proteins involved in Fe–S metabolism. Therefore, NDUFA6, NDUFB9 and possibly SDHAF1 which harbor LYR motif may have a role in insertion or retention of the Fe–S centers within the complex I and SDH structure, respectively. The loss of SDHAF1 incorporation into the apo-enzyme structure may, therefore, destabilize the holo-enzyme or inhibit its formation [50]. A more recent study illustrated that SDHAF1 through its LYR motif, interacts with the co-chaperone HSC20 which itself is part of the complex ISCU-HSC20-HSPA9. This complex was then hypothesized to be responsible for incorporation of three Fe–S clusters within the final structure of SDH [53]. Later studies by the same group confirmed this hypothesis with a minor modification and showed that [Fe–S] is first assembled on ISCU scaffold protein. SDHAF1 then recruits the Fe–S transfer complex (which includes HSC20-HSPA9-holo-ISCU) to the C-terminus of SDHB through direct binding of its N-terminal LYR motif to the co-chaperone HSC20 (Fig. 2). The region L53-R65 of SDHAF1 interacts with SDHB at three binding sites. Binding of SDHAF1 to SDHB and recruitment of the HSC20-HSPA9-holo-ISCU complex by its first LYR motif facilitates Fe–S cluster incorporation into SDHB. Defect in the function of SDHAF1 significantly debilitates the biogenesis of SDHB as a result of sequential rapid degradation by the mitochondrial protease, LONP1 [54]. Furthermore defect in ISC machinery was shown to cause oxidative stress through an iron-dependent mechanism which eventually leads to dysfunction of respiratory complexes [55].

Fig. 2.

The ISCU complex involved in inserting Fe–S clusters into SDHB. First, [Fe–S] clusters are assembled on ISCU scaffold protein. Then SDHAF1 recruits the Fe–S transfer complex, comprised of HSC20-HSPA9-holo-ISCU, to SDHB. The N-terminal LYR motif of SDHAF1 directly binds the co-chaperone HSC20

Another set of experiments in yeast, fly and mammalian cells showed that SDHAF1 in concert with SDHAF3 (details of the latter protein described in the next section) is involved in maturation of SDH2 during oxidative metabolism and particularly protection against ROS damage. These experiments showed that overexpression of yeast Yap1 (a transcription factor which induces the expression of a set of antioxidant genes) or supplementation of the growth media with antioxidants restores the growth defect of the sdh6Δ yeast cells in media containing the non-fermentable carbon source acetate [56]. The details of how SDHAF1 and SDHAF3 might protect SDH2 against oxidative damage and potential sources of ROS have not been completely examined; however, some speculations have been propounded (details in Van Vranken et al. [43]). Besides, contrary to the results from human SDHAF1 [53, 54], Na et al. did not identify any direct role for the yeast SDH6 in Fe–S cluster insertion into the SDH complex [48]. The reason for this discordance might be that the SDHB assembly in human cells studied by Maio et al. likely differs from the yeast counterpart studied by Na et al. 2014. The observations that human SDHAF1 has minor similarity to the yeast orthologue Sdh6 and that the human SDHAF1 cannot reestablish respiration in a Sdh6 null yeast strain [50] support this hypothesis. Therefore, there might be some differences in the function of SDH6/SDHAF1 amongst species.

SDHAF3 (in humans)/SDH7 (in yeast and Drosophila)

SDHAF3 together with SDHAF1 was asserted as factors required for maturation of Sdh2/SDHB [56]. Yeast and Drosophila lacking SDHAF3 exhibit defective SDH activity and reduced levels of Sdh2. The lack of Sdh7 in Drosophila renders it hypersensitive to oxidative stress and leads to muscular and neuronal dysfunction. Sdh6 and Sdh7 in yeast act in conjunction to facilitate Sdh2 maturation by binding to an Sdh1/Sdh2 intermediate (interface of the reaction resides within Sdh2), shielding it from the damage by oxidants. Yap1 overexpression and antioxidant supplementation reinstate SDH activity in sdh7Δ yeast cells similarly to sdh6Δ cells. Yet, Sdh2 overexpression is unable to restore SDH activity in sdh7Δ cells. On the other hand, supplementation of the growth media with paraquat (an oxidant) enfeebles the SDH activity in sdh7Δ yeast cells. The respiratory defect resulting from the lack of Sdh7 can be partially rescued by overexpression of Sdh6, while the same defect arising from the loss of Sdh6 cannot be amended by overexpression of Sdh7. And when Sdh7 is absent, the steady state of Sdh2 amongst all other SDH subunits is perturbed the most. Together, these results from yeast and Drosophila suggest that SDHAF3 mutations may be the cause of some of idiopathic SDH-associated disease [43, 48]. This hypothesis was recently supported by isolation of an impaired (hypomorphic) variant of SDHAF3, c.157 T>C (p.Phe53Leu) in pheochromocytoma and paraganglioma patients. The sdhaf3 mutant was not able to restore SDH function in sdh7 null yeast cells. Furthermore, while WT SDHAF3 was able to interact directly with SdhB in vitro, the defective variant was not fully capable of such interaction. The residues 46 and 242 on SdhB were found to be required for this interaction [57].

SDHAF2 (in humans)/Sdh5 (in yeast)/SdhE (in bacteria)

As stated earlier, SDH has a catalytic flavoprotein subunit, termed SDHA. The latter incorporates a covalently bound redox cofactor flavin adenine dinucleotide (FAD) that is mandatory for enzyme activity [7, 45, 58]. Under aerobic conditions, the FAD moiety catalyzes succinate oxidation, upon which FAD is itself reduced to FADH₂. Changes in redox potential prompt electrons to move from the reduced FADH₂ through Fe−S clusters in SdhB to eventually reduce ubiquinone at the ubiquinone-binding site formed by SdhC and SdhD [7, 59, 60]. How FAD is integrated into SdhA? Many flavoproteins bind FAD through an autocatalytic process without any assembly factors or auxiliary enzymes [59, 61–66]. But it turned out in yeast, a soluble, mitochondrial matrix protein named Sdh5 was required for the activation and flavination of Sdh1 (SDH flavoprotein subunit homolog) as well as for SDH-dependent respiration [45]. The yeast cells lacking SDH5 gene can grow in fermentative mode (i.e., in glucose), but fail to grow in respiratory mode (e.g., in glycerol) which is an indication of a defective oxidative phosphorylation. This phenotype can be rescued by expression of SDH5. Other phenotypes of sdh5Δ yeast cells include: substantially decreased levels of all four SDH subunits, impaired oxygen consumption (similar to the respiratory-deficient sdh1Δ cells), respiration-related phenotypes of H₂O₂ hypersensitivity and reduced chronological life-span. Another phenotype is acetate hyper-excretion which is shared by four other TCA cycle mutants [45].

Sdh5 has also been shown to interact with Sdh1 (SDHA homolog) and this interaction is necessary for Sdh5 stability as Sdh5 similar to Sdh2 is fully degraded in sdh1Δ cells [45, 67]. Conversely, the steady-state levels of Sdh1 are diminished only about 50% in the absence of Sdh5. The lack of Sdh5–Sdh1 interaction also compromises the SDH activity, similar to the phenotype observed in sdh1Δ. This investigation also proposed that in the absence of Sdh5, although SDH complex is formed, it is unfunctional and Sdh1 is not strongly incorporated into the SDH complex [45]. Sdh5 is not a stable part of SDH complex, as it migrates distinctly from SDH complex during Blue Native-PAGE; a method for isolation of native protein complexes [68]. However, Sdh1–Sdh5 complex is formed in early steps of SDH complex assembly, because the complex exists independent of other SDH subunits and accumulates when SDH assembly is inhibited [43, 45, 46].

The effect of Sdh5 on SDH stability is specific, as the stability and activity of other ETC complexes remained intact [45]. In the absence of Sdh5, Sdh1 flavination was lost without affecting the flavination of other mitochondrial flavoproteins. Furthermore, overexpression of Sdh5 reconstitutes flavination of the yeast strain lacking Flx1, which otherwise shows almost no flavination. These data imply that Sdh5 is necessary and sufficient for Sdh1 flavination in yeast. The observation that only SDHAF2 but no other SDH subunits or even Tcm62p can complement the yeast homolog in sdh5Δ strain, to grow in glycerol media, is in line with this proposal. The human homolog of Sdh5 (SDHAF2) has been shown to have a similar role in SdhA flavination [69]. Yet the data from this study could not clarify whether Sdh5 catalyzes FAD incorporation into Sdh1 or it maintains Sdh1 in a conformation competent for autocatalytic FAD binding [45].

In an attempt to gain more insights into the role of Sdh5 in SDH1 flavination, chemical shift perturbation measurements indicated that Sdh5 does not bind FAD in vitro, implying Sdh5 most likely does not act simply as a FAD transporter for delivering FAD to Sdh1 [70]. Consistent with this finding, another inquiry using E. coli indicated that the interaction between SdhE (Sdh5 homolog) and SdhA facilitates flavination by orienting amino acid residues near the dicarboxylate and FAD-binding site on SdhA, entailing SdhE most likely does not interact directly with FAD to promote SdhA flavination [71]. Another study revealed that SDHE gene or its homologs, in addition to many eukaryotes, could be found in α-, β-, and γ-proteobacteria, and evolved prior to the evolution of mitochondria [58]. Serratia one example of γ-proteobacteria, is an opportunistic pathogens of animals, plants and insects and a member of enterobacteriaceae family. Using Serratia as a model, McNeil et al. (2012) showed SdhE (Sdh5 homolog), similar to its homologs in other organisms, is essential for SdhA flavination. However, they showed SdhE, in contrast to Eletsky et al. and Maklashina et al. could bind to FAD directly, albeit SdhE was largely found free from FAD. The authors suggested SdhE binds FAD either covalently or non-covalently and integrates FAD into SdhA. And since this binding is lost upon dialysis, the interaction is likely weak or of low affinity. They further suggested while SdhE might be often in a FAD-free state, it binds FAD when required. FAD-bound SdhE then interacts with SdhA which has a high affinity for FAD, resulting in integration of FAD into SdhA. Alternatively, SdhE might remodel the structure of SdhA to promote FAD incorporation. According to the authors, these two scenarios are not mutually exclusive and might both occur. Furthermore, they did not rule out the possibility that SdhE might only bind FAD when in complex with SdhA [58]. Intriguingly, while SDH was destabilized and disassembled in sdh5Δ yeast cells [45], SDH assembly was not compromised in sdhEΔ bacterial cells. Furthermore, while SdhE function in Serratia and other Gram-negative bacteria found to be important for pathogenicity (Sdh5 did not have such an established role in S. cerevisiae which is typically a non-pathogen. Nevertheless, this possibility should be further investigated particularly in cases where S. cerevisiae turns into pathogen). These disparities may reflect the difference in the role of SdhE in flavination of its targets in bacterial versus yeast cells [58].

McNeil and Fineran subsequently proceeded their investigation to identify residues on bacterial SdhE important for SdhA flavination, and they discovered that RGxxE motif is essential for SdhA flavination and activity, but not for the interaction between SdhE and SdhA. Furthermore, they realized that SdhE can interact with and flavinate a folded form of SdhA without the need for other SDH subunits. Remarkably, they found out SdhA can also covalently bind FAD through an inefficient autocatalytic mechanism in the absence of SdhE [59]. Additional research from the same group revealed that SdhE is also required for the complete flavination and activation of another bacterial SQOR, fumarate reductase (see section “The overall structure of SDH”), under anaerobic conditions in E. coli and Serratia bacterial species. They detected lower levels of FAD incorporation in the absence of SdhE. This background flavination could possibly account for partial FRD activity and growth under anaerobic conditions. They concluded that FrdA binds FAD covalently by virtue of an inefficient autocatalytic mechanism when SdhE was absent. This was in line with their earlier findings showing SdhA covalently attaches FAD without SdhE being present, through an inefficient autocatalytic mechanism [59, 72]. Consequently, they propounded that SdhE increases the rate of an otherwise autocatalytic covalent FAD incorporation into complex II enzymes (i.e., SDH and FRD) required for α-, β-, and γ-proteobacterial growth in diverse environmental conditions.

In Arabidopsis thaliana, there is a human SDHAF2 homolog, though with partial sequence homology. The lack of SDHAF2 leads to a reduced activity of SDH, however, this is not as dramatic as it is in other species, presumably due to the presence of significant residual levels of SDHAF2 protein in sdhaf2Δ. Nevertheless, it is expected that complete loss of SDHAF2 to be lethal for the plant.

In vitro studies on recombinant human SdhA also showed the necessity for the presence of SDHAF2 for flavination, however unlike Hao et al. [45] suggested human SdhA binds FAD first and then SDHAF2 (Sdh5 homolog). FAD stabilized SdhA in the absence of SDHAF2, perhaps by stabilizing the fold of protein or by providing a core around which folding occurs. This suggested that SADHF2 might have either a chaperone or catalytic role rather than flavin delivery [69].

So far, it seems that all studies have agreed upon the necessity of Sdh5/SdhE/SDHAF2 for SdhA flavination and the interaction between them. Notwithstanding this is not quite the case, as there are three examples which suggest otherwise, they have illustrated that SDHAF2/SdhE is dispensable for SdhA flavination:

(1) This was first shown in two hyperthermophilic bacteria/archaea lacking SdhE; Thermus thermophilus and Sulfolobus tokodaii, in which SdhA apparently requires only heat and dicarboxylic acid [73]. Furthermore, bacteria other than α-, β-, and γ-proteobacteria although possessing SDH, do not encode a known SdhE homolog. While an alternative, as yet unidentified FAD assembly factors may exist in these species [58, 59, 74], one cannot exclude the possibility that the rate of SdhA autocatalytic flavination might be sufficient for SDH activity in these SdhE-negative species [59].

(2) The second study was carried out in complex II homolog quinol:fumarate reductase (QFR or Frd). The results from this research pointed to redox environment of bacteria as the determinant of necessity for SdhA flavination by SdhE [75]. According to this investigation, flavination of QFR and its homolog SDH in sdhEΔ is retained during anaerobic condition, although at a lower magnitude compared to when SdhE is present. On the contrary when bacteria are grown under aerobic condition, flavination is significantly reduced in the absence of SdhE. It thus seems that SdhE is mainly required for SdhA flavination of E. coli under aerobic condition. Overall, these results led the authors to propose that bacterial flavination occurs mainly autocatalytically and that SdhE is merely a stimulator of flavination [75]. A function which they believe, is similar to the function of known activators of SdhA flavination such as dicarboxylates, including citric acid cycle intermediates [76, 77], and the iron–sulfur protein [46, 77].

Nevertheless different activators possibly re-arrange SdhA active site residues to an orientation competent for flavination through multiple complementary mechanisms [75]. This hypothesis was recently confirmed in a study on the structure of an assembly intermediate of fumarate reductase from E. coli. The most critical findings of this study is that the two stimulators of flavination, namely Sdh5/SdhE/SDHAF2, and Fe–S subunit (FrdB/SdhB) facilitate distinct substrate-selectivity and enzymatic mechanism. In this scenario, Sdh5/SdhE/SDHAF2 acts in two ways, first chaperoning the hydrophobic surface of FrdA subunit, and second but perhaps more importantly, rearranging the conformational equilibrium of the malleable flavoprotein subunit toward an architecture that favors covalent flavination. And this architecture can be further promoted by bound dicarboxylate but not by Fe–S subunit (FrdB/SdhB). The latter most likely enhances flavination by a distinct mechanism. These observations explain why the lack of SdhE does not completely impair flavination and predict an inherent malleability for the FrdA/SdhA subunit to irregularly sample the conformation that suits to covalent flavination spontaneously [78]. In spite of this illumination on the flavination of FrdA from E. coli, the debate on the mechanism of flavination is far from complete as there might be some differences between bacterial FrdA, and Sdh1 flavination as well as amongst bacterial, yeast and mammalian Sdh1/SdhA flavination.

(3) The third controversial observation with regard to the necessity of Sdh5/SdhE/SDHAF2 for SdhA flavination was made in humans and that added even more complexity to SdhA flavination; Bezawork-Geleta et al. found that SdhA flavination in breast cancer cells also occurs in the absence of SDHAF2. The authors proposed SdhA flavination in breast cancer cells and possibly some other mammalian cells may take place through alternative, as yet unknown routes [79].

A separate matter of debate is the difference between flavination under anaerobic versus aerobic condition. To explain this, Starbird et al. resorted to an evolution-based hypothesis. They suggested the evolution of SdhE during transition and adaptation of cells to an aerobic environment might be the cause of variations in flavination. A proposition which they assumed is in accord with the idea that SDH might have evolved from a QFR through transition from anaerobic to aerobic life. And that the covalent bond meets the need for higher redox potential to oxidize succinate rather than reduce fumarate [75]. This idea discords with a recent hypothesis, laid out based on the phylogenetic studies of whole family of FRD/SDH in different organisms that these enzymes most likely emerged from a common ancestor [21, 80]. These evolutionary hypotheses are not fully conclusive and further inquiries are required.

All in all, while studies in bacteria might represent species-specific evolution of flavination in certain kinds of microorganisms inhabiting hot environment or habitats with varied levels of oxygen availability, studies in humans point to the possibility of functional redundancy for SdhA flavination in certain mammalian cells [79] or in specific cellular milieu. In addition, the data emerging from these studies hint to a regulatory/chaperoning role for SDHAF2 rather than an FAD-transporting function. Apart from SDHAF2, a mitochondrial hsp60 has been found to assist in covalent flavination process through its direct interaction with SDHA subunit, although this chaperone is not absolutely essential for flavination [4, 81]. Hsp60 is distinct from Tcm62p mentioned earlier.

We, here mainly focus on the flavination of SDHA. The detailed steps of SDHA maturation (which occurs prior to flavination), including import of SDHA into the mitochondrion, folding, and processing as well as FAD biosynthesis have been described by Sharma et al. [82].

The significance of FAD covalent bond versus non-covalent bond

The covalent nature of the FAD link provides two advantages for the SDH enzyme; first is the enhancement of the redox potential of the enzyme. A compendium of redox potentials of flavoproteins comprising either a non-covalent or a covalent bond indicates that the covalent mode increases the redox potential dramatically, thereby increasing the oxidative capacity of the enzyme. In line with this, flavoproteins incorporating two covalent linkages (a N₁-histidyl-C6-cystinyl-8α-FAD linkage) bear stronger redox potential. Similarly, synthetic model compounds with modified forms of riboflavin possess higher midpoint potentials compared to the free forms. As expected, the impairment of covalent bond as a result of mutation gives rise to a weaker redox potential. Correspondingly, the disruption of the covalent bond in Sdh1 by replacing the linking His residue to a Ser residue renders the SDH enzyme incompetent to oxidize succinate, demonstrating the significance of covalent bond in catalytic activity of the enzyme [47, 63, 83–85]. The same observation has been made in fumarate reductase (FRD) which has the same N₃-histidyl-8α-FAD linkage in its FrdA subunit (Sdh1 equivalent); the mutation of the linking His residue to a Ser leads to the loss of succinate oxidation. Nevertheless, the mutation does not perturb fumarate reductase activity, targeting to mitochondria and translocation across the mitochondrial membrane where the mutant Sdh1 binds FAD non-covalently and is assembled with other SDH subunits. These observations suggest (1) the covalent bond likely increases the FAD redox potential to enable succinate oxidation [47, 86]. This is further evidenced by the observation that the redox state of FAD drives substrate binding and product release in E. coli SDH [87]. (2) The covalent binding of FAD is negligible for both fumarate reduction and for the import and assembly of the flavoprotein subunit [77]. This latter observation was later confirmed by Hao et al. [45].

The second advantage of the FAD covalent link is conferring stability to the SDH enzyme complex. Although mutation of the covalently linking His90 to a Ser residue in yeast Sdh1 prevents the FAD covalent linkage, Sdh1 can still fold and bind FAD non-covalently. This has been shown by radiolabeled FAD that co-migrates with the holo-SDH complex on a blue native gel electrophoresis (BN-PAGE) [88, 89]. Similarly while FAD covalent bond is disrupted by deletion of SDH5, Sdh1 can still assemble into a mature complex presumably with a non-covalently-bound FAD [46, 47, 84]. These results imply that the covalent bond is not required for promoting proper folding of Sdh1 and ensuing assembly of the SDH complex. Nevertheless, the absence of the covalent bond in Sdh1 destabilizes the mature SDH complex. This has been demonstrated by disruption of the covalent bond through either mutation of the His90 residue or deletion of SDH5, resulting in reduced steady-state levels of the mature SDH complex. The instability of the enzyme is possibly due to enhanced sensitivity to proteases or inherent defect in formation of a thoroughly stable complex [47, 84, 90].

Residues on SDH1 and Sdh5/SDHAF2 required for their interaction

Before structures of the SdhA:SdhE complex became available, there were several studies that provided some insights into how this interaction might occur. The structures of SdhE from Neisseria [91] and E. coli [92] were available before the function of protein was known. That of yeast Sdh5 was determined by NMR in 2012 [70]. In this structure, the authors identified a conserved surface region which might represent a hypothetical Sdh5:Sdh1 interaction interface. The conserved region encompasses a mostly positive surface (Arg 68, 74, Lys 75), a negatively charged periphery (Glu 60, 80, Asp 82, 105, 112, 114), and a hydrophobic patch (Ile78, Leu 79, 83) at the C-terminus. Point mutations in this region led to the loss of covalent flavination of Sdh1. This effect was not due to SDH1 destabilization, although SDH5 mutations had opposite effects on the mutant stability. The low stability of Sdh5 in one of the mutants was rescued by SDH1 overexpression which implies that the loss of Sdh5–Sdh1 interaction may destabilize some SDH5 mutants [70]. The disease-causing mutation G78R (G77 in yeast SDH5) lies within the central positive area of the proposed interface.

This predicted interface was nicely confirmed by X-ray structures of the E. coli SdhE in complex with flavinated FrdA [78] or SdhA [93]. Residues 11–22 (corresponding to 72–83 of Sdh5 and including the site of the disease-causing G78R mutation—G16 in E. coli SdhE) are at the elbow between helix 1 and helix 2, with the critical G16 in the turn. This region makes intimate contact with the flavoprotein in both structures. Residues 49–51 (corresponding to yeast 110–112 in the acidic region identified, and forming helix 3) also make contact, as do several residues near the C-terminus.

As for the interaction site on SdhA, site-directed mutagenesis experiments identified two Arg residues near the C-terminus to be required for SDH1 flavination and to affect the attachment and stability of Sdh5 [46, 94]. These were residues 582 and 638 in the yeast SDH1 sequence, and they are conserved through bacterial, fungal, vertebrate, and plant SdhA. An R589 W mutation in the C-terminal tail of human SDH1 (SDHA), like the G78R mutation in human SDHAF2, gives rise to paraganglioma [94, 95]. While it was felt likely that these residues and the C-terminal stretch in which they reside would be involved in the contact with Sdh5, this was not supported by the structures of the complex when they became available. In both the SdhE/FrdA [78] and SdhE/SdhA [93] complexes from E. coli, the interface revealed by the structures is on the opposite side of the protein from the C-terminus and these arginines. Consistent with this, these Arg residues are also conserved in non-proteobacteria such as Thermus thermophilus, Sulfolobus tokodaii, and Wolinella succinogenes, which are believed not to have an SdhE Homolog.

The fact that these mutations also prevent assembly of the complex might suggest that flavination of SDH1 is needed for stabilization of proper SDH1 conformation which in turn is required for its attachment to other SDH subunits, but other results show covalent flavination is not required for assembly of SDH in yeast [84] or bacteria [58] or for assembly of E. coli FRD [86]. Perhaps instead these Arg residues are important for stabilizing a conformation of the flavoprotein that is required for binding Sdh5 and becoming flavinated, and also required for assembly into the complex.

Interestingly, in both structures of the SdhE:flavoprotein complex the stretch containing Arg 638 (Arg 586 and 578 in E. coli SdhA and FrdA, respectively) was disordered, and so omitted from the models. This is surprising because in structures of mature SDH [7, 14, 96] these regions are well ordered. In SDH, Arg 586 ion pairs with a conserved Asp 287 in the CAP domain, adjacent to the catalytic base Arg 286. Two residues after this Arg is a conserved Tyr 588, which is the C-terminal residue in SdhA. The terminal carboxylate binds to backbone amides in the CAP domain residue 280, fixing the tail in place. The stretch 280–287 is at the end of the CAP domain that moves when the CAP opens, with the Ca atoms of 280 and 287 moving by 16 and 6°, respectively. Aligning the high-resolution E. coli SDH structure 2wdq [97] with the SdhA:SdhE complex, based on the FAD domain exclusive of the cap, it can be seen that the CAP domain moves away from Arg 586 and the terminal carboxyl in the open position making these interactions impossible. This suggests that the interactions with the C-terminal stretch serve like a “magnetic latch” to catch the CAP in its closed position and hold it there. If flavination requires the closed position, failure in the absence of the Arg may be because without the latch, the complex does not spend enough time in the closed conformation. Arg 586 may also be important for precisely locating the catalytic base Arg to carry out its function during catalysis. Arg 568 does not interact with the cap domain but may be important to hold the C-terminus in position and prevent unraveling when the CAP dissociates.

In addition to defining the interfaces between SdhE and SdhA or FrdA, the above-mentioned structures suggest a mechanism by which the assembly factor promotes catalysis of covalent bond formation in flavination. SdhE binds to SdhA or FrdA in approximately the same area that the Fe–S protein does in the mature enzyme (the interface for SdhE binding to SdhA or FrdA overlaps largely with that for binding the Fe–S protein). The elbow between the first and second helices of SdhE, including the critical stretch 11–22, is located where the loop bearing the Fe2S2 cluster contacts the flavoprotein. This brings the conserved Gly16 of SdhE close to the flavin isoalloxazine-binding site. The carbonyl O of Gly16 makes an H-bond with N^d1 atom of the histidine that is covalently bound to FAD. It must be remembered that both of these structures [78, 93] have flavin already covalently bound, so they represent the product complex after bond formation. But assuming the same H-bond is formed in the reaction complex, with non-covalent flavin bound in the same position, this H-bond between G16 O of SdhE and the His residue could promote the bond formation in two ways [78, 93]. First, it would stabilize the N^δ1-protonated π tautomer of histidine, making the N^ε2 nitrogen available as a strong nucleophile to attack the C8 alpha atom of FAD. Second, it would hold the His sidechain in the correct rotamer and direct its N^e2 atom’s lone pair toward that atom for the attack. This could account for the great acceleration of the reaction by the assembly factor, while still allowing that the reaction could proceed in its absence at a slower rate under certain conditions. Note that this interaction would not directly explain the requirement for G16, as mutation would not alter the O atom. However, it is likely that a large, charged side chain like Arg would interfere with binding and prevent the backbone from approaching the His closely enough to form the H-bond. It could also interfere with folding, as glycines are often found in sharp turns, presumably due to their relaxed backbone dihedral (Ramachandran) restraints. However, in the SdhE structures of the protein alone [92] or the SdhA:SdhE complex [93], G16 has dihedral angle well inside the allowed region for general amino acids. Mutation of this His in FrdA disrupts interaction with SdhE as measured by crosslinking [75].

In both of the SdhE:flavoprotein structures, the CAP domain is rotated significantly from its position in structures of the mature complex with OAA bound. The extent of the rotation is different in the two structures, and may result from crystal packing artifacts in some cases. Reports that dicarboxylates stimulate flavination would seem to indicate that the cap is closed during catalysis, since the dicarboxylate-binding site is made up of residues in the CAP and FAD domain and, therefore, only exists in the closed state. Maintaining an open state after bond formation was suggested to be a way of preventing succinate oxidation with resulting ROS production before the flavoprotein is joined to the Fe–S protein. Experiments with purified overexpressed flavinated SdhA that has been stripped of SdhE showed limited catalysis with PMS as acceptor and very low but not zero ROS production. Even with the covalent bond, the midpoint potential of FAD in SDH is much lower than that of the succinate/fumarate couple, so in the absence of Fe–S clusters to immediately accept the electron from FAD, the equilibrium level of reduction of FAD would be low, resulting in little ROS production [98].

The cap domains of both complexes in the SdhA:SdhE structure are involved in crystal contacts. However, the contacts are different for the two protomers, so the fact that the orientation of the cap domain is essentially the same suggests that this is in fact the stable conformation resulting from binding to SdhE.

Although the CAP domain is in the open seems to be stable position in the crystal, this does not preclude it taking a closed position while SdhE is bound. It can be modeled in the closed position based on the position in 2wdq with no serious clashes that could not be resolved by side chain reorientation.

The role of SDH2/SDHB in flavination

Additional to the role of Sdh5 and Flx1 in Sdh1 flavination, Sdh2 appears to play a role in the same. Sdh1 covalent flavination in sdh2Δ is reduced to half of the same in WT cells, while the total level of Sdh1 remains fairly stable. This effect is specific to SDH2 as deletion of SDH3 or SDH4 hardly reduces flavination. How exactly SDH2 plays a role in flavination yet to be understood [46, 47, 77].

The fact that SdhE and flavinated SdhA form a stable complex suggests that SdhE remains bound to the flavoprotein after flavination [93], as observed also for the yeast Sdh5/Sdh1 [45]. Since binding surfaces of SdhE and SDHB on the flavoprotein overlap, it is likely that binding of SDHB competitively displaces SdhE [93]. In the case of limiting SdhE, displacement by SdhB could make it available for flavination of another molecule of SDHA, increasing the level of flavination reached. Otherwise the rate of catalysis could decrease greatly after one turnover of the available SdhE. If instead of SDHB, another chaperone (SDHAF4 homolog) displaces SdhE from SDHA, the same explanation would still apply provided that the other chaperone is in limited supply, and it remains bound to SDHA until displaced by SDHB.

SDHAF4 (in humans and Drosophila)/Sdh8 (in yeast)

Sdh8, a mitochondrial matrix protein is an evolutionary conserved SDH assembly factor which binds directly to Sdh1-FAD, and in doing so, acts as a chaperone to prevent the potentially damaging effects of Sdh1-FAD. This damage is likely the result of ROS generation caused by solvent-accessible FAD covalently bound to Sdh1. FAD cofactor can oxidize succinate to fumarate regardless of SDH complex. This reaction leads to FAD reduction followed by auto-oxidization by molecular oxygen to yield superoxide.

Sdh8, therefore, acts a chaperone to prevent FAD-mediated oxidative stress. This hypothesis was further corroborated by two observations; first, Sdh8 interacts with covalently flavinated Sdh1 but not with apo-Sdh1 (in sdh5Δ, or in a strain with flavination-deficient sdh1 mutant; H90S and H90A). And second, the stability of Sdh8 relies on its interaction with Sdh1, and that the Sdh1–Sdh8 dimer remarkably accumulates in sdh2Δ and sdh4Δ strains, which is likely the result of enhanced pool of unbound but flavinated Sdh1. The function of Sdh8 signifies the importance of devoted chaperones for ETC complex assembly to shield redox-active cofactors existing in many ETC complex subunits.

A second function for Sdh8 is inferred from the observation that SDH8 deletion leads to destabilization of Sdh2 protein, while Sdh1 protein levels are not perturbed which is in accord with the idea that chaperoning function of Sdh8 for Sdh1 promotes Sdh1–Sdh2 dimer formation. Therefore, it seems that Sdh8 have two functions. First, it prevents oxidative damage by FAD, and second it facilitates Sdh1–Sdh2 dimer formation and thereby maintains SDH complex stability. And while oxidative stress caused by deletion of SDH8 can be rescued by overexpression of Yap1 (a transcription factor involved in mediating oxidative stress), SDH deficiency cannot be rescued by the same treatment [99].

The steady-state levels of the SDH complex and SDH enzyme activity are diminished upon mutating SDH8 in yeast/fly and siRNA-mediated knockdown of SDHAF4 displays the same defects in mammalian cells. But the presence of Sdh8/SDHAF4 is not an absolute requirement for SDH activity, as some levels of activity are maintained in the absence of Sdh8/SDHAF4 in yeast/fly/mammalian cell models. Nevertheless, the loss of SDHAF4 has more drastic effects on Drosophila than in yeast and mammalian cells. In line with this while sdhaf4Δ Drosophila preserves only about 10% of SDH activity with almost undetectable SDH complex, the sdh8Δ yeast strain keeps about 40% of SDH activity and complex. In addition, deletion of SDHAF4 in Drosophila unlike yeast destabilizes Sdh1 dramatically and leads to both muscular and behavioral dysfunction. The reason for this discrepancy is not yet clear but it might be the result of functional redundancy in yeast and mammalian cells or the buffering capacity of yeast growth media against oxidative stress [99].

Similar to other SDH subunit mutations studied in model organisms, the physiological abnormalities of sdhaf4Δ Drosophila can possibly be linked to the corresponding human diseases, as defects in various SDH subunits have already been associated with human diseases. In line with this there are reports of SDH-defective diseases such as Leigh’s syndrome and wild-type gastrointestinal stromal tumors (WT GIST) without any mutations in all known genes encoding SDH subunits [99]. How defects in the function of SDH complex might be linked to neurodegeneration is not fully understood. Neurodegeneration phenotype might be the result of a defect in mitochondrial ATP production; however, ROS production may play a more dominant role as this was evidenced by the study on Sdh8/SDHAF4, where the latter acts as an assembly factor for SDH and consequently prevents ROS production, while the lack of SDHAF4 does not lead to ATP depletion [99]. Other possible mechanisms may also contribute to the pathogenesis.

Other assembly factors

The production of iron–sulfur clusters is an essential function of yeast mitochondria involving several enzymes [6, 100, 101]. Apart from mutations in the genes encoding these proteins which lead to the loss of SDH activity (probably as a result of structural instability of the Sdh2 subunit and of the catalytic dimer) (see Lemire and Oyedotun [6] and references therein), mutations in the chaperone proteins Ssq1 (an Hsp70-type chaperone) and Jac1 (a 22-kDa co-chaperone that may regulate Ssq1) also give rise to the loss of SDH activity. Thus, these chaperones may have a role in the assembly or repair of iron–sulfur clusters [6, 102]. The human homologs of Ssq1 and Jac1 are mortalin and Hsc20, respectively [103]. Both cancer and neurodegenerative diseases have been found to be linked with the deregulation of expression and/or activity of these two proteins [104–109]. Detailed accounts of the synthesis of iron–sulfur clusters and the role of chaperones at different steps of synthesis have been provided by several reviews [100, 103, 110–112] and, therefore, are not addressed here.

The structure and assembly of SDH in plants

The structure and assembly of SD have been recently reviewed [13] and, therefore, we here only sketch out some key aspects of it. pSDH, in addition to four typical subunits observed in eukaryotes contains four other subunits; SDH5-8 (these are not related to assembly factors with the same nomenclature in yeast and should not be mistaken with them). Consequently, the size of pSDH is larger than SDH from other species, raising the possibility of having yet unknown extra roles. pSDH is involved in stromal opening, the plant defense response, and reactive oxygen species-mediated stress responses. Furthermore, salicylic acid and nitric oxide, two signaling molecules interact with the ubiquinone-binding site, thereby modulating signal transduction [13].

Although SDH1-4 subunits of animals and fungi are nuclear-encoded, SDH3 and SDH4, in some plant species, are nuclear-encoded and in other species mitochondrial-encoded. Regarding the assembly factors, orthologs of SDHAF1, SDHAF2, and SDHAF4 have been identified in Arabidopsis [13, 113, 114]. The plant SDHAF2 and SDHAF4 have similar functions with their human orthologs. However, the function of SDHAF1 has not been determined, nor any orthologs has been found for SDHAF3 in plants as yet (discussed in detail in Ref. [13]). Remarkably, reduced levels of SDH (as a result of SDHAF2 deletion) in Arabidopsis leads to the inhibition of primary root elongation with early lateral root emergence [114].

The current model of SDH assembly

Recently, with the discovery of novel SDH assembly factors, our understanding of SDH assembly has been remarkably advanced. This though has posed a challenge of how these cofactors act coordinately in the process of SDH assembly and how this process is regulated. The current model suggests the assembly occurs in distinct, subunit-specific stages, including cofactor insertion, stabilization of sub-complex assembly intermediates to prevent detrimental solvent interactions and subsequently maturation of individual subunits which promote the complete assembly of SDH [43]. The following is the outline of the main steps of SDH assembly that has perceived so far (Fig. 3).

Fig. 3.

The current model of complex II (succinate dehydrogenase) assembly. Sdh5 bound to Sdh1 facilitates flavination of Sdh1. Sdh5 is then released, while Sdh8 chaperone binds the flavinated Sdh1 to facilitate the dimerization of Sdh1 and Sdh2. Sdh6 and Sdh7 assist in either insertion or retention of [Fe–S] clusters within Sdh2. Sdh2 then forms a dimer with Sdh1, while Sdh6/Sdh7 and Sdh8 are released from Sdh2 and Sdh1, respectively. The dimer is subsequently integrated into the membrane where Sdh3-Sdh4 dimer containing a heme b is formed. There is not much information regarding the formation of Sdh3-Sdh4 dimer

SDH subunits are first translated in the cytosol and then translocated to the mitochondria by TOM (the general import pore for mitochondrial proteins in the mitochondrial outer membrane) and TIM (the translocase of the inner membrane) complexes. Maturation of Sdh1 and Sdh2 occurs simultaneously. In the case of Sdh1, Sdh5 binds apo-Sdh1 inside mitochondria and forms a dimer which is competent to covalently attach to the FAD cofactor. Flavination leads to the break-down of the Sdh1–Sdh5 dimer and release of flavinated-Sdh1. Sdh1 preserves the redox-active FAD cofactor from potentially detrimental solvent interactions. The flavinated-Sdh1 then forms a complex with Sdh8 chaperone. The formation of the complex with Sdh8 facilitates the subsequent constitution of Sdh1–Sdh2 soluble dimer, while prevents the generation of superoxide by flavinated Sdh1. Maturation of Sdh2 requires apo-Sdh2 to mature into a complex-competent subunit. First, three Fe–S clusters generated by ISCU and ISCA complexes are inserted into Sdh2. Consequently, Sdh2 interacts with Sdh6 and Sdh7, which protect exposed Fe–S clusters. Sdh2 then adjoins a mature Sdh1 subunit forming a heterotetrameric assembly intermediate. The Sdh1–Sdh2 hydrophilic head then docks to the inner mitochondrial membrane (IMM) via interactions with the Sdh3–Sdh4 subcomplex (hydrophobic anchor), which has or has not pre-assembled at the IMM [43, 99]. An unassembled SdhA (Sdh1 homolog) and its homolog fumarate reductase in E. coli (FrdA) as well as human SdhA were found to have minor catalytic activity and to generate little ROS. This corroborates the idea that aligning active site residues from both SdhA and SdhB is essential to warrant a robust catalytic activity [98].

Regarding the assembly of Sdh3–Sdh4 subcomplex (hydrophobic anchor) information is much scarce compared with the assembly of Sdh1–Sdh2 subcomplex (hydrophilic domain). It is largely believed that after translation, Sdh3 and Sdh4 are translocated into mitochondria through the TOM complex. Subsequently, they are transported into Tim23 complex and then released to mitochondrial inner membrane [43, 115]. SDHC and D have similar fold, both with N-terminus on the proximal (matrix or cytoplasm) side and three transmembrane helices. The first two transmembrane helices of each come together in a symmetrical fashion to form a 4-helix bundle crosslinked by heme via histidines in the second helix of each [21, 116]. It is thought that this heterodimeric membrane anchor may have arisen from a primordial homodimeric structure by gene duplication and further specialization. In the case of the E. coli SQR, SDHD overexpression forms a b cytochrome in the membrane, however, it is not competent for reconstituting SQR activity from SDHA+B [117].

Dimerization of Sdh3–Sdh4 requires the presence of both hydrophilic domain subunits, i.e., Sdh1 and Sdh2. Therefore, the biogenesis of the hydrophobic anchor is somewhat tied to the biogenesis of the hydrophilic domain. Furthermore, the integrity of the hydrophobic anchor in mammalian cells relies on the existence of heme b. The exact role of heme b in the stability of the Sdh3–Sdh4 subcomplex or electron transport is not yet known, neither how heme b is assembled into the Sdh3–Sdh4 subcomplex. However, since several assembly factors such as Coa1, Coa2 and Shy1 are required for heme insertion into Cox1, a subunit of complex IV, there might be such an assembly factor for heme intercalation into Sdh3–Sdh4 subcomplex [43, 118–121]. In E.coli, a variant of SDH lacking heme b has been constructed which surprisingly assembles and functions properly. Furthermore, the variant does not affect ROS production. The mutants, however, is sensitive to typical detergents used for isolation of SDH from the mitochondrial membrane. Thus, it seems that the presence of heme b is not absolutely essential for the assembly and function of SDH, but the presence of heme b may enhance the efficacy of SDH folding in this microorganism [122].

The assembly of SDH, i.e., the formation of a four-subunit SDH through the steps described, was regarded a universal phenomenon in humans until recently, when a functional role has been identified for alternative assembly of SDH [123]. The latter entails formation of a smaller complex (CII_low) comprising SDHA and two assembly factors SDHAF2 and SDHAF4. Presumably bioenergetic stress promotes formation of CII_low, which in turn inhibits the anabolic activity of TCA, DNA synthesis and cell cycle progression. Therefore, it seems that this alternative SDH assembly links energy stress to metabolic checkpoints. This function of CII_low seems to support the growth of SDHB-deficient tumoral cells under suboptimal condition. The function of the two assembly factors in CII_low is still under investigation [123].

SDH inhibitors as fungicides

SDH inhibitors (SDHIs) have been designed to act as fungicide in the agricultural industry for decades. The development of these fungicides historically is divided into two time-frames. The first generation came to existence between 1966 and 1997 with limited target spectrum namely basidiomycete pathogens such as rusts or Rhizoctonia sp. Carboxins were the most prominent SDHIs in this period and the second generation which was developed in 2003 and beyond with broad-spectrum target specificity and boscalid as the representative [124]. The first generation initially termed as carboxamides, however, both groups are now commonly known as SDHIs [125].

SDHIs have been considered a favorable agricultural fungicides and that is because of several advantages they have over other fungicides; these include broad-spectrum efficacy (including those fungi resistant to other fungicides), distinctive crop selectivity, bearing only mild environmental side effects, low application rate, and minor toxicity [125–127].

SDH has two binding sites, one for succinate (linked with TCA cycle) and another for ubiquinone (in association with oxidative phosphorylation). While there are SDHIs which bind succinate-binding site (e.g., malonate; a natural inhibitor), all SDHI fungicides target ubiquinone-binding pocket [124, 128]. The SDHIs acting as fungicides are effective against a series of ascomycete and basidiomycete pathogens which infect crops such as cereals, ryegrass seed, apples, pears, grapes, stone fruit, cucurbits, potato, vegetable crops and kiwifruit [129, 130].

Conclusion and future perspective

Succinate dehydrogenase enzyme is a unique enzyme which connects TCA cycle to oxidative phosphorylation. It is composed of hydrophilic and hydrophobic subdomains each containing two subunits. Years have been passed since the revelation of SDH structure and during this period much has been learned about the function of SDH. Recent discoveries of assembly factors and their roles in cancer development reenvigorated research on SDH and its assembly. While many puzzles had remined regarding the function of known assembly factors such as Tcm62 and Flx1, new discoveries posed new questions adding to the complexity of SDH assembly. Regarding Tcm62, although it is believed to have a chaperone function for SDH complex (at physiological temperature) and other mitochondrial proteins (under heat stress) several issues have remaind unanswered: for instance, it is not clear whether Tcm62 at physiological temperature has only one substrate, SDH. And at what steps of SDH assembly it may play a role. Given that under heat stress condition, Tcm62 has possibly many substrates, another question that may arise is that what is the nature of its specificity under physiological condition versus its somewhat promiscuity under heat stress? Furthermore, the discrepancy on molecular weight and exact location of Tcm62 should be addressed. As for Flx1, controversial data suggest different roles for it including FAD transporter, regulator of Sdh1 level, and regulator of Sdh1 flavination and it is not certain whether Flx1 plays different roles under diverse circumstances or not. Furthermore, it is not clear what other organisms, apart from yeast express it and if they do not why?

The roles of SDHAF1 and SDHAF3 in humans do not exactly match the roles of their homologs in yeast, i.e., Sdh6 and Sdh7, although in both organisms the two proteins were found to be essential for SDH2/SDHB maturation during oxidative metabolism. The details of such differences thus need to be addressed. The controversy around the role of SDHAF2 in SDHA flavination is even more intensive as different species show distinct dependencies on SDHAF2 for flavination (from absolutely unessential to completely necessary) and SDH complex stability. Furthermore, the direct interaction between FAD and SDHAF2 homolog in various species is still debated. All or at least some of these discrepancies could merely reflect evolutionary and environmental differences.

Additional to the role of Sdh5 and Flx1 in Sdh1 flavination, Sdh2 appears to play a role in the same, yet this role has not been fully investigated. Another assembly factor, Sdh8/SDHAF4 has two functions. First, it prohibits oxidative damage by FAD, and second it promotes Sdh1–Sdh2 dimer formation and in doing so stabilizes SDH complex. Similar to other assembly factors, there are some disparities amongst species regarding the role of this protein which ought to be settled.

So far, cases of human diseases have been or predicted to be linked with the assembly factors. This has been particularly evident amongst the cases where no typical dysfunction has been detected in SDH subunits. This necessitates further investigation of the role of the assembly factors. Furthermore, several fungicides have been developed/are being developed to specifically inhibit the function of SDH complex. As described, some functional differences have been identified between yeast, plant and human SDH structure and assembly factors. This may, therefore, open a new window to design novel fungicides against the fungal-specific SDH complex (or their assembly factors) which may prevent a broad toxic effect amongst non-target population. This is particularly relevant as recently cases of health risk for humans and worms reported upon application of a few marketed SDHIs [131].

Abbreviations

ETC

Electron transport chain

FAD

Flavin adenine dinucleotide

FADH₂

Flavin adenine dinucleotide hydrogen

FCC3

Flavocytochrome c3

FMN

Flavin mononucleotide

FRD

Fumarate reductase

IMM

Inner mitochondrial membrane

OXPHOS

Oxidative phosphorylation

QFR

Quinol:fumarate reductase

SDH

Succinate dehydrogenase

SQR

Succinate:quinone oxidoreductase

SQORs

Succinate:quinone oxidoreductases

TCA

Tricarboxylic acid cycle

TIM

Translocase of the inner membrane

TOM

Translocase of the outer membrane