Recent Advances in Cytochrome bc₁: Inter monomer Electronic Communication?

Abstract

The ubihydroquinone: cytochrome c oxidoreductase, or cytochrome bc₁, is a central component of photosynthetic and respiratory energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy production (ATP). The three-dimensional structures of cytochrome bc₁ indicate that its two monomers are intertwined to form a symmetrical homodimer. This unusual architecture raises the issue of whether the monomers operate independently, or function cooperatively during the catalytic cycle of the enzyme. In this review, recent progress achieved in our understanding of the mechanism of function of dimeric cytochrome bc₁ is presented. New genetic approaches producing heterodimeric enzymes, and emerging insights related to the inter monomer electron transfer between the heme b cofactors of cytochrome bc₁ are described.

Introduction

The ubihydroquinone: cytochrome c oxidoreductase (cytochrome bc₁ or complex III) is a multi-subunit membrane bound enzyme central to photosynthetic and respiratory electron transport chains of many organisms, including bacteria, archaea, eukaryotic mitochondria and chloroplasts [1–3]. This enzyme catalyzes oxidation of hydroquinone (QH₂) to quinone (Q) and transfers the resulting electrons, usually via c-type cytochrome, to the reaction centers in photosynthetic and cytochrome c oxidases in aerobic respiratory growth conditions. It couples the free energy released by these reactions to the translocation of electrons and protons across membrane to contribute to the formation of proton motive force used for ATP synthesis [4].

The static three dimensional (3D) structures of cytochrome bc₁ indicate that it is a symmetrical homodimer [5–9] with at least three catalytic subunits: the Rieske Fe/S protein with a high potential [2Fe2S] cluster, the cytochrome b with two b-type (one low potential (b_L) and one high potential (b_H)) hemes, and the cytochrome c₁ with a c-type heme [10] (Fig. 1). Additional subunits in variable numbers are also present in some species [6–8,11]. The Q-cycle model is generally used to describe the mechanism of function of cytochrome bc₁ [4,12,13]. Accordingly, two QH₂ molecules are oxidized at a QH₂ (Q_o) site on the positive side, and one Q molecule is reduced at a Q reduction (Q_i) site on the negative side, of the membrane. Each QH₂ oxidation is initiated by the oxidized Fe/S protein, which accepts one electron and transfers it to cytochrome c₁ via a large-scale movement of its cluster bearing extrinsic domain [14,15]. This electron is then transferred from cytochrome c₁ to other downstream electron carriers and terminal acceptors. The second electron from QH₂ oxidation is conveyed to the low potential hemes b_L and b_H, and then to a Q forming a stable semiquinone (SQ) at the (Q_i) site (Fig. 1). Following a second turnover of a Q_o site the SQ at the Q_i site is converted to QH₂ and released from the enzyme, completing the catalytic cycle [12]. This model accounts well for the electron transfer pathways between the cofactors of a monomeric cytochrome bc₁. However, it does not describe whether the two consecutive QH₂ oxidations, which are required to complete a full catalytic cycle, take place at the Q_o site of a given monomer or at the two distinct Q_o sites of the dimeric enzyme. The 3D structures of cytochrome bc₁ revealed that its cofactors, except the mobile [2Fe2S] clusters, are distributed symmetrically within the dimeric enzyme. Moreover, the distances separating the hemes b_L–b_H in a given monomer, and the hemes b_L–b_L between the two monomers are quasi-identical (Fig. 1). These findings raised a number of intriguing questions (e.g., see [16,17]), including the independent or co-ordinated functions of cytochrome bc₁ monomers. The architecture of cytochrome bc₁ might play a functional role in addition to the structural stability of the enzyme if the two consecutive QH₂ oxidations alternate between the two Q_o sites of the dimeric enzyme, or if inter monomer electronic communication occurs between the heme cofactors. A number of hypothetical models, including “activated Q cycle” [18], “alternating Q cycle” [19] or “heterodimeric Q cycle” [20], invoking different structural and functional interactions between the Q_o and Q_i sites within one monomer, or between both monomers, of cytochrome bc₁ have been described. Initially, peculiar transient kinetics data were interpreted as indication of a dimeric Q cycle mechanism for QH₂ oxidation [18] despite other possible explanations. In recent years, more convincing experimental approaches [21–23] and theoretical calculations [24] were reported. In this review, we survey recent studies on inter monomer electronic communication within cytochrome bc₁. We describe the different genetic systems used to produce asymmetric cytochrome bc₁ heterodimers, and critically assess the outcomes of these efforts in understanding the mechanism of function of dimeric cytochrome bc₁.

Fig. 1. Structure and function of heterodimeric cytochromes bc₁.

A. A hypothetical structural model for a non-native heterodimeric cytochrome b-bc₁. The 3D structure of cytochrome bc₁ (PDB entry 1ZRT) was modified to include the Q_i site H212N mutation (blue) on one monomer (M1) and the Q_o site G158W mutation (red) on the other (M2) to depict a heterodimeric enzyme produced by the one-plasmid system [29]. The carboxyl end of one cytochrome b (blue) is linked to the amino end of the other (yellow) with a peptide (green) indicated by an arrow to create cytochrome b-b that is not naturally present in cytochrome bc₁. S refers to the Strep–tag on the carboxyl terminal end of cytochrome b-b. The Fe/S and cytochrome c₁ subunits are shown in light blue and light brown, with the cofactors ([2Fe2S] and hemes b_H, b_L and c₁) in monomer M1 in blue and those in M2 in red. The black arrows indicate the electron transfer pathways across the monomers, Q and QH₂ correspond to quinone and hydroquinone, respectively. The black ellipsoid indicates the absence of heme b_H on monomer M1. B. A hypothetical structural model for a native-like heterodimeric cytochrome bc₁. The 3D structure of cytochrome bc₁ (PDB entry 1ZRT) was modified to include the Q_i site H212N mutation (blue) on one monomer (M1) and the Q_o site Y147A mutation (red) on the other (M2) to depict a heterodimeric enzyme produced by the two-plasmids system [32]. The labels are as in A, and note the absence (indicated by an arrow) of the peptide linking together the cytochromes b, and the presence of Flag- (F) and Strep (S)-tags on the carboxyl terminals of two cytochrome b on monomers M1 and M2, respectively.

Recent approaches to probe inter monomer electronic communication in cytochrome bc₁

Half of the sites reactivity

Early on, Trumpower and colleagues described that one molecule of stigmatellin (a Q_o site inhibitor) per dimer was sufficient to completely inhibit cytochrome bc₁ [25], and that when yeast cytochrome bc₁ is inhibited by antimycin A (a Q_i site inhibitor) the amount of cytochrome c₁ reduced corresponded to half of that present in cytochrome bc₁ [21]. These studies assumed that equilibrium between the two hemes b_H is attained via electron transfer between the hemes b_L to support the “half of the sites reactivity” model for yeast [26] and bacterial [27] cytochrome bc₁. More recently, a Paracoccus denitrificans strain that produces a quasi-wild type and a Q_o site defective mutant variants of cytochrome bc₁, tagged with His- and Strep-epitopes, respectively, (see below “two-plasmids system” for details) was described [28]. The quasi-wild type form of cytochrome bc₁ used in this study lacked the amino terminal acidic part of cytochrome c₁ and produced a dimeric enzyme, which otherwise forms a tetramer in its native state. From this strain, homodimeric (with two wild type Q_o, or two mutant Q_o sites) and heterodimeric (with a wild type Q_o site in one monomer, and a mutant Q_o site in the other monomer) cytochrome bc₁ were purified by affinity chromatography with appropriate epitope-tags. Purified homodimeric wild type and heterodimeric mutant cytochromes bc₁ exhibited comparable extents of cytochrome c₁ and cytochrome b_H reductions and enzyme activities, but antimycin-mediated stimulation was observed only with the homodimeric wild type enzyme. These findings suggested that only one of the two Q_o sites of the wild type homodimer, or the mutant heterodimer is active, and that inter monomer electron transfer occurs in cytochrome bc₁ [28]. These interesting findings are consistent with the alternating Q cycle model [19], although they do not prove directly the occurrence of either an inactive Q_o site in wild type, or inter monomer electron transfer between the hemes b_L in the heterodimeric mutant cytochrome bc₁. In addition, this P. denitrificans mutant provides no information about the physiological ability of a heterodimeric cytochrome bc₁ to support cellular growth.

One-plasmid system

More recently, a different genetic system to probe inter monomer electron transfer in dimeric cytochrome bc₁ was reported by Osyczka and collaborators [29]. They adapted a previously described Rhodobacter capsulatus genetic system [30] to carry an ingenuously modified petABC (structural genes of cytochrome bc₁) operon. Two copies of petB (encoding cytochrome b) were connected to each other via a designed linker sequence to form a fused petB-B gene on a single plasmid, to produce a ‘cytochrome b-b’ subunit, which is tagged with Strep-epitope at its carboxyl end [29] (Fig. 2A). This “one-plasmid” system was considered to produce a non-native but fully active dimeric “cytochrome b-bc₁” in which the two monomers are covalently linked to each other via a linker between two cytochromes b (Fig. 1A). The system was used to generate single and double mutations on either or both cytochrome b copies to yield homodimeric and heterodimeric cytochrome b-bc₁ variants with active or inactive Q_o and Q_i sites. The cytochrome b G158W substitution that inhibits QH₂ oxidation at the Q_o site without affecting the other cofactors [30,31], and the H212N substitution that eliminates heme b_H [16,22] were used to inactivate one or both monomers to produce heterodimers (Figs. 1A and 2A). Using appropriate cytochrome b-bc₁ mutants and measuring their activities, the authors concluded that electrons move freely between the hemes b_L and distribute without regulation among the hemes b_H using an H-shaped electron transfer path (a “bus bar”) within a dimeric cytochrome b-bc₁ [29].

Fig. 2. Genetic systems producing heterodimeric cytochromes bc₁.

A. One-plasmid system is genetically less stable possibly due to high frequency intra-molecular rearrangements between petB genes (encoding cytochrome b) duplications carried by the plasmid, but produces only heterodimeric cytochromes bc₁ [29]. petB-B encodes cytochrome b-b, formed by linking two cytochrome b copies by a dodecameric peptide. petA and petC refer to the structural genes of Fe/S protein and cytochrome c₁, respectively. S indicates the Strep-tag epitope added to the carboxyl end of the second copy of cytochrome b. B. Two-plasmids system is genetically more stable, as only one copy of petABC (structural genes encoding cytochrome bc₁) is carried by each plasmid. Various subunits of cytochrome bc₁ are produced and re-assorted independently, leading the cells harboring this system to produce a combination of homodimeric (M1/M1 and M2/M2) and heterodimeric (M1/M2) cytochrome bc₁ that are differentially tagged by the Flag (F) and Strep (S) epitopes, [32], as shown. Apparently no high frequency inter-molecular rearrangements are observed in this system, but the heterodimeric cytochromes bc₁ thus produced need to be separated from homodimeric variants by affinity chromatography via appropriate epitope tags.

Our ongoing experiments indicate that the R. capsulatus one-plasmid system with cytochrome b-b duplications, constructed as reported in [29], exhibit extreme genetic instability (~ >10⁻²) and does not confer normal photosynthetic growth to R. capsulatus cytochrome bc₁-minus mutant. Even under respiratory conditions, where growth can be maintained via a cytochrome bc₁ independent branch (i.e., via an alternate hydroquinone oxidase), selective pressure against intra-molecular direct cytochrome b repeats appears to be strong. Plasmids seem to rearrange immediately to discard one of the cytochrome b copies to yield wild type-like homodimeric cytochrome bc₁. We also observe that high frequency intra-molecular rearrangements similar to those described above (petAB-BC) (Fig. 2A) occur when the entire petABC operon is duplicated as either direct (petABC-ABC) or inverted (petABC-CBA) repeats on a single plasmid. These strains also exhibit poor photosynthetic growth and high frequency of intra-molecular rearrangements, to quickly generate uncontrollable mixtures of homo and heterodimeric cytochromes bc₁ (manuscript in preparation). Until we understand and control the occurrence of frequent intra-molecular rearrangements, these undesirable features restrict severely the use of the one-plasmid system for studies of heterodimeric cytochromes bc₁.

Two plasmids system

A genetic system, similar to that used in [28], for studying inter monomer electron transfer in dimeric cytochromes bc₁ was initiated about a decade ago by our group, and reported recently [32]. This system also derives from earlier described R. capsulatus genetic system [30] and harbors two different plasmids (marked with different antibiotic resistance, Kan^R and Tet^R). Each of the plasmids has a single copy of petABC operon where each petB is carboxyl-terminally tagged with a different (Strep and Flag) epitope [32] (Fig. 2B). The cytochrome bc₁ subunits being produced and assorted independently in cells, the “two-plasmids” system yields two sets of homodimeric cytochrome bc₁ with the same epitope at their monomers (Strep-Strep or Flag-Flag) together with a set of heterodimeric cytochrome bc₁ with a different tag at each monomer (Flag-Strep) (Fig. 1B). Unlike the one-plasmid system, the two-plasmids system carrying wild type petABC operons confers quasi-wild type like photosynthetic growth to R. capsulatus cytochrome bc₁-minus mutants. Inter-molecular genetic rearrangements among the two plasmids appear to be less frequent (~ < 10⁻⁴) than intra-molecular events seen with the one-plasmid system and intra-molecular duplications. With two-plasmids system, use of different replicons of different plasmid copy numbers changes steady-state amounts of homodimeric and heterodimeric cytochrome bc₁ variants in cells. In addition, the two-plasmid system is not restricted to heteromerization of cytochrome b only as it can yield heterodimers with different combinations of all cytochrome bc₁ subunits. However, extensive purification is required to isolate the desired heterodimeric cytochrome bc₁ variant among its multiple combinations produced.

This two-plasmids genetic system was used to probe inter monomer electronic communication between the hemes b_L of cytochrome bc₁, by selective inactivation of one Q_o site of one monomer and one Q_i site of the other monomer in a heterodimeric construct (Fig. 1B and 2B). Based on trials and errors, the previously characterized cytochrome b Y147A and H212N substitutions for inactivating the Q_o and Q_i sites, respectively were chosen. The former mutation affects drastically the rate of electron transfer from QH₂ oxidation to heme b_L at the Q_o site [33], whereas the latter causes loss of heme b_H [16,22], eliminating SQ formation at the Q_i site. Strains that produced only homodimeric mutant cytochromes bc₁ (i. e, carrying cytochrome b Y147A or H212N mutations on both plasmids) were unable to grow in photosynthesis and produced inactive enzymes. In contrast, strains producing heterodimeric cytochrome bc₁ (i.e., carrying cytochrome b Y147A and H212N mutations on two different plasmids) exhibited slow photosynthetic growth compared with a wild type strain and had cytochrome bc₁ activity [32]. Thus, electron transfer between the hemes b_L can interconnect across the enzyme the active Q_o site on one monomer to the active Q_i site on the other monomer of the heterodimeric cytochrome bc₁ to generate enough enzyme activity to support photosynthetic growth. Detailed, molecular genetic, biochemical and biophysical studies of cells with two-plasmids revealed semi-quantitatively that inter molecular heme b_L–b_L–b_H electron transfer occurs more slowly than intra-monomer heme b_L–b_H electron transfer, but is sufficient to sustain slow photosynthetic growth. Precise determination of inter molecular hemes b_L–b_L electron transfer rates is complicated due to electronic coupling between the photosynthetic reaction centers and cytochromes bc₁ in membranes. Vigorous photosynthetic growth was observed when higher amounts of heterodimeric cytochromes bc₁ were produced [32] (manuscript in preparation), documenting the physiological relevance of the two-plasmids system with respect to the efficiency and amount of a heterodimeric cytochrome bc₁.

Summary and future studies

Genetic approaches that were initiated in recent years are now probing functional roles of the dimeric architecture of cytochrome bc₁ [28,29,32]. Experiments are revealing, with varying degrees of rigor and reliability, the occurrence of slow but appreciable inter monomer electron transfer between the hemes b_L, at least in mutant heterodimeric cytochrome bc₁ enzymes that are forced to support photosynthetic growth [32]. Further studies of electronic communication between the hemes b_L, especially in a wild type cytochrome bc₁, is of paramount importance to our understanding of the mechanism of function of a dimeric enzyme, but this requires additional work. First, currently available heterodimeric cytochrome bc₁ production systems need improvements. Choosing appropriate mutations and respiration-deficient backgrounds might assess the physiological capability of P. denitrificans heterodimeric mutant cytochrome bc₁ enzyme reported in [28]. Inactivating RecA enzyme, which is known to reduce the frequency of homologous recombination in many organisms [34], might reduce molecular rearrangements to increase genetic stability of the one-plasmid system. Using different kinds of epitope tag or different Q_o and Q_i site mutations might improve the stability of purified active heterodimeric cytochromes bc₁ produced by the two-plasmids R. capsulatus [32] (manuscript in preparation).

Second, inter monomer electronic communication per se deserves further studies. Direct determinations of inter monomer hemes b electron transfer rates might be achieved using photo-activatable ruthenium cytochrome c derivatives [35]. The role of aromatic residues, like F195 and Y199 of cytochrome b, located between the hemes b_L at the interface of the monomers in the dimeric cytochrome bc₁ with respect to inter monomer electron transfer [36,37] need reexamination. A two-plasmids system producing appropriate heterodimeric enzymes with an inactive Q_o site in one monomer and an inactive Q_i site in the other monomer, together with selected cytochrome b F195 or Y199 substitutions, might probe better the role of these residues on inter monomer electronic communication in dimeric cytochrome bc₁. Moreover, availability of a reliable heterodimeric cytochrome bc₁ with one fully active and one fully inactive monomer generated via the two-plasmids system might probe if intra-monomer hemes b_L–b_H electron transfer alone can support photosynthetic growth without any hemes b_L-b_L electronic coupling. Comparing the amounts of non-productive bypass reactions (i.e., ROS production) generated by an appropriate heterodimeric cytochrome bc₁ having only one active monomer with those produced by a wild type homodimeric enzyme with two active monomers, might be eye opening in respect to physiological regulation of cytochrome bc₁. Could steady state inter monomer electronic communications within a dimeric cytochrome bc₁ minimize unproductive by-pass reactions? Could it play a protective role to minimize ROS mediated oxidative damages under specific physiological conditions, like high membrane potential or limited oxygen availability, rendering hemes b_L partially reduced [24]?

Third, time is ripe to start using heterodimeric cytochromes bc₁ to probe intra and inter monomer Q_o–Q_i sites interactions in particular for initiation and regulation of the first versus the second QH₂ oxidation during cytochrome bc₁ catalytic cycle. Recent structural rationalization [38] of the heterodimeric Q cycle model [20] begs experimental testing. Inspection of cytochrome b structure indicates that its “rotating” E helix (providing all necessary ligand interactions at the Q_i site) and its ef loop extension on the Q_o site might be tethered to the more “static” D helix upon SQ formation at the Q_i site. Faster electron transfer rates between hemes b_L–b_H as compared to those between hemes b_L–b_L–b_H [32] might favor intra, instead of inter, monomer SQ formation at the cognate Q_i sites of cytochrome bc₁ monomers. Consequently, following the first QH₂ oxidation conformation of the ef loop could change upon SQ production on the other side of the membrane, and inhibit access of oxidized Fe-S protein to the cognate Q_o site on the same monomer [20,38]. After the second QH₂ oxidation, only dismutation of the SQ molecules via inter monomer electronic communication would then complete the catalytic cycle to produce Q and QH₂, and relax the SQ-tethered Q_i sites, to render the cognate Q_o sites active for the next turnover of the enzyme. Production of appropriate heterodimeric cytochrome bc₁ variants should be invaluable to assess the validity, if any, of various aspects of speculative models [20].

In summary, the establishment of a dimeric architecture for cytochrome bc₁ raised in recent years interesting questions aimed at complementing the Q-cycle scheme generally used to describe the mechanism of function of a monomeric enzyme. In particular, do the monomers operate independently or function cooperatively? Do the two consecutive QH₂ oxidations required to complete the catalytic cycle occur in the same or different monomers? Does electronic communication occur between the hemes b cofactors of cytochrome bc₁? If so, is there any selective or beneficial advantage? These and other related outstanding questions awaiting answers enticed the development of new genetic approaches to produce novel heterodimeric cytochromes bc₁. Ongoing studies of these variant enzymes are progressing on our understanding of the structure-function relationship of dimeric cytochrome bc₁. Hopefully, future studies will better define whether or not the mechanism of function of a dimeric cytochrome bc₁ invokes intra and inter monomer cooperation and electronic communications.

Abbreviations

Q

quinone

SQ

semiquinone

QH₂

hydroquinone