3.D.3 The Proton-translocating Quinol:Cytochrome c Reductase (QCR) Superfamily
Proteins of the QCR family (also known as the cytochrome bc1 complex or the mitochondrial respiratory complex III) include three subunits in certain bacteria and eleven subunits in bovine heart mitochondria. Homologous complexes participate exclusively in respiration in eukaryotic mitochondria but participate in respiration, cyclic photosynthetic electron transfer, denitrification, and nitrogen fixation in phylogenetically diverse bacteria. In all cases, the complex transfers electrons from a quinol to cytochrome c and links electron transfer to proton translocation. There are two b cytochromes, one c1 cytochrome, one Reiske 2Fe-2S center and a bound ubiquinone per subunit. The structure of the bovine mitochondrial complex has been solved to 2.9 Å resolution. The complex is a dimer of ten subunits per monomer. There are 13 transmembrane helices per monomer. The cytochrome b protein had been predicted to possess 8 or 9 TMSs; the cytochrome c1 protein 1 TMS, and the Fe2S2 protein 1 or 2 TMSs. Most of the masses of core proteins I and II protrudes from the matrix side of the membrane whereas the cytochrome b protein is located primarily in the membrane, and most of the c1 and iron-sulfur proteins including their redox centers are located on the cytoplasmic side of the membrane. Electron flow from ubiquinol to cytochrome c is coupled to the electrogenic extrusion of protons, probably two per electron. The sequences of all eleven proteins of the bovine enzyme complex, of the three proteins of the Paracoccus denitrificans enzyme and of several other QCRs are known (Berry et al. 2013). Three of the eukaryotic subunits are homologous to the three Paracoccus subunits. A cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex (Moe et al. 2021).
The cytochrome b6f complex of the cyanobacterium, Synechocystis PCC6803, has four subunits, two of which are equivalent to the cytochrome b subunit of Paracoccus. The high-resolution (3 Å) 3-D structure of the b6f complex from the thermophilic cyanobacterium, Mastigocladus laminosus has been solved (Kurisu et al., 2003). This complex shuttles electrons between photosystems I and II reaction centers for oxygenic photosynthesis. It generated a pmf for ATP synthesis. The dimeric complex contains a large quinone exchange cavity where a heme is bound. The core of the b6f complex resembles the respiratory cytochrome bc1 complex, but the domain arrangement outside the core and the complement of prosthetic groups is strikingly different (Kurisu et al., 2003).
As much as two-thirds of the proton gradient used for transmembrane free energy storage in oxygenic photosynthesis is generated by the cytochrome b6f complex. The proton uptake pathway from the electrochemically negative (n) aqueous phase to the n-side quinone binding site of the complex, and a probable route for proton exit to the positive phase resulting from quinol oxidation, are defined in a 2.70-A crystal structure and in structures with quinone analog inhibitors at 3.07 A (tridecyl- stigmatellin) and 3.25-A (2-nonyl-4-hydroxyquinoline N-oxide) resolution (Hasan et al. 2013). The simplest n-side proton pathway extends from the aqueous phase via Asp20 and Arg207 (cytochrome b6 subunit) to quinone bound axially to heme c(n). On the positive side, the heme-proximal Glu78 (subunit IV), which accepts protons from plastosemiquinone, defines a route for H+ transfer to the aqueous phase. These pathways provide a structure-based description of the quinone-mediated proton transfer responsible for generation of the transmembrane electrochemical potential gradient in oxygenic photosynthesis.
The Q cycle, by which a proton motive force is believed to be generated, involves two distinct quinol/quinone binding sites. Quinol is first oxidized by the Rieske Fe2-S2 center at the Qo site to generate a reactive semiquinone which reduces a low potential cytochrome b heme (bL). bL quickly transfers an electron to the high potential cytochrome b (bH) located on the opposite side of the membrane. Reduced bH is then oxidized by a quinone or semiquinone at the Qi site. Proton release thus probably occurs as a result of both proton translocation and QH2 deprotonation at the Qo site coupled to protonation of the reduced Q at the Qi site. Both Qi and Qo are probably localized to the cytochrome b protein. The possible H+ translocation pathways and mechanisms have been reviewed by Schultz and Chan (2001). The Q cycle involving cytochrome bc1 operates reversibly on coupled electron and proton transfers of quinone at two binding sites on opposite membrane faces (Osyczka et al. 2005). Cytochrome b and the Q cycle may function in vectorial transmembane H+ transport as has been reported for acetogenic bacterial (Kremp et al. 2020; Kremp et al. 2022).
Electron transfer between respiratory complexes drives transmembrane proton translocation. Complex III uses the Q cycle, involving ubiquinol oxidation and ubiquinone reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. Di Trani et al. 2022 determined structures of Candida albicans CIII2 by cryo-EM, revealing endogenous ubiquinone and visualizing the continuum of Rieske head domain conformations. Analysis of these conformations does not indicate cooperativity in the Rieske head domain position or ligand binding in the two CIIIs of the CIII2 dimer. Cryo-EM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that Inz-5 inhibition alters the equilibrium of Rieske head domain positions (Di Trani et al. 2022).
The bifurcated electron transfer reaction, which is built into this mechanism, recycles one electron, thus allowing it to translocate two protons per one electron moving to the high-potential redox chain. Smirnov and Nori (2012) studied the Q-cycle mechanism in an artificial system that mimics the bf complex of plants and cyanobacteria in the regime of ferredoxin-dependent cyclic electron flow. They described a time sequence of electron and proton transfer reactions in the complex, finding energetic conditions when the bifurcation of the electron pathways at the positive side of the membrane occurs naturally, without additional gates. They showed that this system is able to translocate 1.8 protons, on average, per one electron, with a thermodynamic efficiency of ~32% or higher (Smirnov and Nori, 2012).
Key components of respiratory and photosynthetic energy-transduction systems, the cytochrome bc1 and b6f (Cytbc1/b6f) membranous multisubunit homodimeric complexes have been reviewed (Sarewicz et al. 2021). These molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient. Cytsbc1/b6f share many similarities but also have significant differences. Structural, mechanistic, and physiological aspects required for function of Cytbc1/b6f have been reviewed. The discussion covers (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc1/b6f (Sarewicz et al. 2021).
The different mitochondrial complexes, I, III and IV have been shown to interact physically, and a stable supercomplex of complex I with dimeric complex III has been isolated from plants (Dudkina et al., 2005). The cytochrome bc1 complexes have been reviewed from structure, function and mechanistic aspects (Xia et al. 2012). The cytochrome bc1-aa3 oxidase supercomplex is an emerging and potential drug target against tuberculosis (Sindhu and Debnath 2022). The enzymes of the mitochondrial electron transport chain associate into supercomplexes. Supercomplexes CIII2CIV1-2, CICIII2 and CICIII2CIV (respirasome) exist in mammals, but in contrast to CICIII2 and the respirasome, CIII2CIV requires a specific assembly factor (SCAF1) to be formed. Dr. Irene Vercellino solved the structures of mammalian (mouse and ovine) CIII2CIV and its assembly intermediates in different conformations. These allowed description of the assembly of CIII2CIV from the CIII2 precursor to the final CIII2CIV conformation, driven by the insertion of the N terminus of SCAF1 deep into CIII2, while its C terminus is integrated into CIV. The structures confirmed that SCAF1 is exclusively required for the assembly of CIII2CIV and has no role in the assembly of the respirasome. Further, CIII2 is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII2, explaining the presence of one copy of CIV in CIII2CIV in mammals. Biochemical analyses showed that CIII2 and CIV gain catalytic advantage when assembled into the supercomplex, suggesting a role for CIII2CIV in fine tuning the efficiency of electron transfer in the electron transport chain.
Tetrahymena thermophila, a ciliate model organism, has tubular mitochondrial cristae and highly divergent electron transport chain involving four transmembrane protein complexes (I-IV). Han et al. 2023 reported cryo-EM structures of its ~8 MDa megacomplex IV(2 )+ (I + III(2 )+ II)(2), as well as a ~ 10.6 MDa megacomplex (IV(2) + I + III(2 )+ II)(2) at lower resolution. In megacomplex IV(2 )+ (I + III(2 )+ II)(2), each CIV(2) protomer associates one copy of supercomplex I + III(2) and one copy of CII, forming a half ring-shaped architecture that adapts to the membrane curvature of mitochondrial cristae. Megacomplex (IV(2 )+ I + III(2 )+ II)(2) defines the relative position between neighbouring half rings and maintains the proximity between CIV(2) and CIII(2) cytochrome c binding sites. These findings expand the current understanding of divergence in eukaryotic electron transport chain organization and how it is related to mitochondrial morphology (Han et al. 2023).
The overall reaction catalyzed by the protein complexes of the QCR family are:
quinol (QH2) + 2 cytochrome c (ox) + 2H+ (in) → quinone (Q) + 2 cytochrome c (red) + 4H+ (out)
References:
Proton pumping cytochrome bc1 complex of 3 dissimilar subunits, PetABC. The pathway of transmembrane electron transfer has been determined and compared with that of the B6f complex from the same organism which is strikingly different (Bhaduri et al. 2017). The cryo-EM structure of the four-subunit Rhodobacter sphaeroides cytochrome bc1 complex in styrene maleic acid nanodiscs has been solved (Swainsbury et al. 2023). The additional subunit is called subunit IV which is one of up to eight supernumerary subunits that modify the function of the CytBC1 complex in mitochondria (Swainsbury et al. 2023).
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Cytochrome bc1 Complex of Rhodobacter capsulatus
Cytochrome b, PetB, of 437 aas and 10 TMSs in a 5 + 5 TMS arrangement (P0CY47)
Cytochrome c1, PetA, of 279 aas and 2 TMSs, N- and C-terminal (D5ANZ4)
Iron-sulfur subunit of 191 aas and 1 TMS (P0CY48)
Ubiquinol:cytochrome c oxidoreductase complex, including cytb, cytc1 and UCRI. The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly (Flores-Mireles et al. 2023).
Bacteria; eukaryotic mitochondria
Cytochrome bc1 complex of Bos taurus, Cytb, cytC1,
Proton-translocating cytochrome bc1/Rieske complex. Trophozoites of P. falciparum are inhibited by inhibitors such as atovaquone, buparvaquone and decoquinate (Meier et al. 2018).
Cytbc1 complex of Plasmodium falciparum
Cytb of 376 aas and 9 TMSs
cytc1 of 394 aas and 2 TMSsd
Rieske of 355 aas and 1 or 2 TMSs
Ubiquinol:cytochrome c oxidoreductase. The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as the center N (Qn site) and the center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome (Ding et al. 2006).
Bacteria; eukaryotic mitochondria
Cytochrome bc1 complex of Saccharomyces cerevisiae
Cytochrome b6f complex, PetB, PetC, PetD, PetM, Hcf164. The dimeric photosynthetic cytochrome b6f complex, a 16-mer of eight distinct subunits and 26 transmembrane helices, catalyzes transmembrane proton-coupled electron transfer for energy storage. Using a 2.5 Å crystal structure of the dimeric complex, Hasan and Cramer 2014 identified 23 distinct lipid-binding sites per monomer. Annular lipids provide a connection for super-complex formation with the photosystem-I reaction center and the LHCII kinase for transmembrane signaling. Internal lipids mediate crosslinking to stabilize the domain-swapped iron-sulfur protein subunit, dielectric heterogeneity within intermonomer and intramonomer electron transfer pathways, and dimer stabilization through lipid-mediated intermonomer interactions. In the cytochrome b6f complex with the quinol analog, stigmatellin, which partitions in the Qp portal of the bc1 complex, but not of b6f, the Qp portal is partially occluded in the b6f complex relative to bc1. Occlusion of the Qp portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Qp portal and is inferred to be a cause of enhanced superoxide production (Hasan et al. 2014). PetD subunit integration into the thylakoid membrane is a post-translational and an SRP-dependent process that requires the formation of a cpSRP-cpFtsY-ALB3-PetD complex (Króliczewski et al. 2017). The b6f complex plays a role in trans-membrane signal transduction from reductant. The effect of the p-side of the electron transport chain on the regulation of light energy to the two photosystems by trans-side phosphorylation of the light-harvesting chlorophyll protein has been discussed (Cramer 2018). The cryo-EM structure of the spinach cytochrome b6 f complex has been solved at 3.6 A resolution (Malone et al. 2019). The cytb6f complex links electron transfer between photosystems I and II and converting solar energy into a pmf for ATP synthesis. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses. Malone et al. 2019 presented a 3.6 Å resolution cryo-EM structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp), adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis (Malone et al. 2019). Cytochrome b6f (cytb6f) lies at the heart of the light-dependent reactions of oxygenic photosynthesis, where it serves as a link between photosystem II (PSII) and photosystem I (PSI) through the oxidation and reduction of the electron carriers plastoquinol (PQH2) and plastocyanin (Pc). A mechanism of electron bifurcation, known as the Q-cycle, couples electron transfer to the generation of a transmembrane proton gradient for ATP synthesis. Cytb6f catalyses the rate-limiting step in linear electron transfer, is pivotal for cyclic electron transfer and plays a key role as a redox-sensing hub involved in the regulation of light-harvesting, electron transfer and photosynthetic gene expression (Malone et al. 2021). Xiao et al. 2022 investigated the phytotoxicity of reduced graphene oxide (RGO), graphene oxide (GO) and amine-functionalized graphene (G-NH2) on Brassica napus L. RGO impaired photosynthesis mainly by decreasing the chlorophyll content and Rubisco activity. This effect of RGO could be due to its toxicity on sulfate transmembrane transporter and nitrogen metabolism, which ultimately led to nutrient imbalance. However, GO directly damaged the photosystem by disrupting the chloroplast structure, and a decrease in Rubisco activity indicated that GO also inhibits carbon fixation. Gene-level analysis demonstrated that GO has toxicity on the chloroplast membrane, photosystem, photosynthethic electron transport and F-type ATPase (Xiao et al. 2022). Regulation of the generation of reactive oxygen species during photosynthetic electron transport has been discussed (Krieger-Liszkay and Shimakawa 2022). Cyclic electron transfer and photoreduction of oxygen contribute to the size of the proton gradient. The yield of singlet oxygen production in photosystem II is regulated by changes in the midpoint potential of its primary quinone acceptor. In addition, numerous antioxidants inside the photosystems, the antenna and the thylakoid membrane quench or scavenge ROS (Krieger-Liszkay and Shimakawa 2022). Rieske FeS overexpression in tobacco provides increased abundance and activity of cytochrome b(6) f (Heyno et al. 2022).
Plants
Cytochrome b6f complex of Arabidopsis thaliana
Cytochrome b6/f complex, PetABCD of (cyt f; cyt b6, iron sulfur protein, and subunit 4, respectively, of 324, 215, 179, and 160 aas, respectively (Soo et al. 2017).
PetABCD of Synechococcus elongatus (strain PCC 7942) (Anacystis nidulans R2)
The three component QcrABC cytochrome bc1 (bcc) complex. The bc1 complex catalyzes the oxidation of
menaquinol and the reduction of a cytochrome c in the respiratory chain.
The bc1 complex operates through a Q-cycle mechanism that couples
electron transfer to generation of the proton gradient that drives ATP
synthesis. QcrA is an iron-sulfur (2Fe-2S) protein of 353 aas and 3 central TMSs; QcrB is a cytochrom b protein that contains two quinone binding sites, one for oxidations, and one for reduction of 545 aas and 9 TMSs, while QcrC is a membrane-bound diheme c-type cytochrome with 269 aas and 2 TMSs, one N-terminal, and one C-terminal. QcrABC forms a complex with CtaCDEF (TC# 3.D.4.4.5), a cytochrome aa3 oxidase complex (Falke et al. 2018). This supercomplex is required for spore-specific nitrate reductase 1 activity (Falke et al. 2019).
QcrABC of Streptomyces coelicolor
The Ubiquinol-cytochrome oxidase supercomplex with 8 subunits. In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation. Wiseman et al. 2018 isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. Menaquinone is bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection (Wiseman et al. 2018).
The III2/IV2 supercomplex of Mycobacterium smegmatis
Cytochrome b6f complex of 10 subunits, PetA, B, C1-3, D, E, G, J, L, M and N. It transports H+ and electrons across the membrane. Lipids contribute to the stability and activity of the enzyme complex (Bhaduri et al. 2019).
Cyt b6f of Nostoc sp. PCC7120
PetA, 333 aas
PetB, 215 aas
PetC3 = PetC4, 178 aas
PetD, 160 aas
PetE, 139 aas
PetG, 37 aas
PetJ, 111 aas
PetL, 31 aas
PetM, 34 aas
PetN, 29 aas
PetC1, 179 aas
PetC2, 178 aa
Cytochrome b6f complex including 11 subunits, A - K. CryoEM structures are known (Proctor et al. 2022).
Cytochrome b6f complex including 8 subunits, A - H.
7R0W_A, 222 aas and 5 TMSs
7R0W_B, 160 aas and 3 TMSs
7R0W_C, 328 aas and 2 TMSs, N- and C-terminal
7R0W_D, 192 aas and 1 N-terminal TMS
7R0W_E, 32 aas and 1 TMS
7R0W_F, 36 aas and 1 TMS
7R0W_G, 38 aas and 1 TMS
7R0W_H, 29 aas and 1 TM