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 Q_o site to generate a reactive semiquinone which reduces a low potential cytochrome b heme (b_L). b_L quickly transfers an electron to the high potential cytochrome b (b_H) located on the opposite side of the membrane. Reduced b_H is then oxidized by a quinone or semiquinone at the Q_i site. Proton release thus probably occurs as a result of both proton translocation and QH₂ deprotonation at the Q_o site coupled to protonation of the reduced Q at the Q_i site. Both Q_i and Q_o 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 CIII₂CIV_1-2, 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 (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 CIII₂CIV_1-2, 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)