3.D.4 The Proton-translocating Cytochrome Oxidase (COX) Superfamily
Multi-subunit enzyme complexes of the COX superfamily are found in bacteria, archaea and eukarya. These enzyme complexes reduce O2 to water and concomitantly pump four protons across the membrane. Specific proposals, based on the three-dimensional structure of the enzyme complex, have been put forth to explain proton pumping (Branden et al., 2006; Brzezinski, 2004; Namslauer et al., 2007). They possess a unique bimetallic active site consisting of a heme and a closely associated copper atom where O2 is reduced. The COX superfamily is therefore also called the heme-copper superfamily. Mitochondrial COX is also referred to as Mitochondrial Respiratory Complex IV. There are two main enzyme types in the COX superfamily which have distinct substrate specificities: cytochrome c oxidases and quinol oxidases. The reduced substrates of the latter enzymes are ubiquinol or menaquinol rather than reduced cytochrome c. Both types of oxidases pump protons. COX pumps one H+ per electron transferred to O2 (Brzezinski and Johansson, 2010). X-ray structures of catalytic intermediates of cytochrome c oxidase have provided insight into its O2-activation and unidirectional proton-pump mechanisms (Shimada et al. 2020). Allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane pmf (Capitanio et al. 2020). Letts et al. have presented structures of a supercomplex, which suggest how the interaction choreography may control overall functionality (Letts and Sazanov 2017; Letts et al. 2019). A mechanism for proton pumping, involving a specific role for the active site tyrosine, conserved in all CcOs, has been suggested (Blomberg 2020). The structure of cytochrome c oxidase Subunit 1 from Aedes aegypti has been proposed (Josyula and Mutheneni 2022). Relatively weak protein-protein and protein-lipid interactions exert effects on the enzymatic activities of transmembrane electron carriers such as CcO (Kato et al. 2022). The catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria has been identified (Covian et al. 2023).
Eukaryotic COX complexes contain 3 mitochondrially encoded subunits (I, II and III) and up to ten nuclearly-encoded subunits. All prokaryotic members of the COX superfamily contain homologues of subunit I and most also contain homologues of subunits II and III. The nuclearly-encoded subunits of the eukaryotic complexes are not found in bacteria. The cytochrome oxidases of Paracoccus denitrificans and Rhodobacter sphaeroides contain only 3 subunits, but they function as efficiently as the 13-subunit bovine enzyme complex. Some evidence suggests that the monomeric complex of the Rhodobacter enzyme is fully functional for electron flow and proton pumping in artificial membrane (Cvetkov and Prochaska, 2007). For Rhodobacter sphaeroides CytcO (cytochrome aa3), the E286 side chain of subunit I appears to be a branching point from which protons are shuttled either to the catalytic site for O2 reduction or to the acceptor site for pumped protons (Busenlehner et al., 2008). The 3-dimensional structures of the bovine and Paracoccus enzymes have been elucidated by x-ray crystallography. Liu et al. (2011) published crystallographic and online spectral evidence for the roles of conformational changes and conserved water in the Rhodobacter spheroides cytochrome oxidase proton pump. The mechanism of membrane potential generation by heme-copper respiratory oxidases has been reviewed (Siletsky 2023).
The E. coli homologue, a ubiquinol oxidase, cytochrome bo, has homologues of subunits I, II and III. Subunits I of various COXs have at least twelve (but not more than 20) transmembrane spanners. Subunits I bind crucial prosthetic groups and probably provide the proton channel. The probable pathways of H+ translocation and the mechanism involved has been reviewed by Schultz and Chan (2001). Most phylogenetic analyses have been conducted with the sequences of subunits I and II. The cytochrome bd complex includes one of two homologous small subunit, either AppX or CydX. Members of the CydX/AppX family of small proteins (30 - 50 aas with 1 TMS) interact with and activate the Cytochrome bd oxidase complex (TC# 3.D.4.3.2) (VanOrsdel et al. 2013).
In the chemolithotrophic, thermophilic, acidophilic crenarchaeota, Acidianus ambivalens, the essential residues comprising the H+ channel of the quinol oxidase (TC #3.D.4.9.1) are lacking, and the enzyme is believed to pump protons purely by chemical charge separation. In this six subunit enzyme complex, only the heme-bearing subunit I (DoxB) is demonstrably homologous to subunits in other quinol oxidases. The cytochrome aa3 oxygen reductase utilizes the tunnel observed in the crystal structures to deliver O2 for catalysis (Mahinthichaichan et al. 2018).
The bacterial respiratory nitric oxide reductase (NOR (3.D.4.10.1 and 2)) is a member of the super-family of O2-reducing, proton-pumping, heme-copper oxidases. Even though NO reduction is a highly exergonic reaction, NOR is not a proton pump and rather than taking up protons from the cytoplasmic (membrane potential negative) side of the membrane, like the heme-copper oxidases, NOR derives its substrate protons from the periplasmic (membrane potential positive) side of the membrane. In these complexes, Glu-122 in NorB contributes to defining the aperture of a non-electrogenic 'E-pathway' that serves to deliver protons from the periplasm to the buried active site in NOR (Flock et al., 2007).
Cytochrome c oxidase catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O2 to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O2 reduction are transferred though a specific intraprotein (D) pathway. Replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O2 reduction. Introduction of the acidic residue results in an increase of the apparent pK(a) of E286, an internal proton donor to the catalytic site, from 9.4 to ~11. Lepp et al (2008) investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation resulted in uncoupling of proton pumping from O2 reduction with a decrease in the apparent pK(a) of E286 from 9.4 to 7.6.
Human diseases associated with COX deficiency including encephalomyopathies, Leigh syndrome, hypertrophic cardiomyopathies, and fatal lactic acidosis are caused by mutations in COX subunits or assembly factors (Diaz, 2010). A mutation in COA3 causes a phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature (Ostergaard et al. 2015). Structures of respiratory supercomplex I+III2 revealed functional and conformational crosstalk (Letts et al. 2019). ATP interactions with Cox13 in S. cerevisiae (CoxVIa in mammals) is inhibitory of catalytic activity (Zhou et al. 2021).
Heme-copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Structural and functional investigations, focussing on proton transfer in the B, C and NOR families are discussed by Lee et al. (2012).
Most proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of these enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. Xu et al. 2020 reported the 3.6-Å-resolution X-ray structure of the cytochrome aa 3-600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which had been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to aas shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.
The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea3(2+) and CuB(1+), suggesting that a peroxide-bound state (Fea33+-O--O--CuB2+) rather than an O2-bound state (Fea32+-O2) is the initial catalytic intermediate. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism was proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces (Shimada et al., 2020; 33516318). An essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe-O distance of the ferryl center could best be described as Fea34+-O2-, not as Fea34+-OH-. Shimada et al, 2020 found an interstitial water molecule that could trigger rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+-O--O--CuB2+ state, preventing its detection. The H-pathway structures of both intermediates indicate that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.
Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Steimle et al. 2021 reported the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra TMSs attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria (Steimle et al. 2021).
Cytochrome c oxidase (CcO) (mitochondrial electron-transport-chain complex IV) is a multi-subunit transmembrane protein responsible for catalyzing the last step of the electron transport chain, dioxygen reduction to water, which is essential to the establishment and maintenance of the membrane proton gradient that drives ATP synthesis (Panda et al. 2023). Novel findings regarding (a) proton and electron transfer (PT/ET) processes, together with their contributions to exogenous phenol induced O-O cleavage, (b) the stereo-electronic tunability of the secondary coordination sphere (especially hydrogen-bonding) on the geometric and spin state alteration of the heme-peroxo‑copper unit, and (c) a plausible mechanism for the Tyr-His cofactor biogenesis, are discussed in detail. Additionally, since the ferric-superoxide and the ferryl-oxo (Compound II) species are critically involved in the CcO catalytic cycle, this review also highlights a few fundamental aspects of these heme-only (i.e., without copper) species, including the structural and reactivity influences of electron-donating trans-axial ligands and Lewis acid-promoted H-bonding (Panda et al. 2023).
The generalized transport reaction catalyzed by cytochrome c (Cyt c) oxidases is:
2Cyt c (red) + 1/2 O2 + 6H+ (in) → 2Cyt c (ox) + H2O + 4H+ (out).
The generalized transport reaction catalyzed by quinol oxidases is:
quinol + 1/2 O2 + 4H+ (in) → quinone + H2O + 4H+ (out)
References:
The cytochrome ba complex consisting of the Sox/CbsA/cytb protein of 553 aas and 12 TMSs, and the CbsB or cytochrome b573 protein of 311 aas and 9 TMSs (Bandeiras et al. 2009). May function with SoxL (Q3LCJ1; 329 aas and 2 TMSs) and CbsB (Q3LCJ3; 311 aas and 2 TMSs).
Cytba of Acidianus ambivalens (Desulfurolobus ambivalens)
Nitric oxide reductase (EC #1.7.99.7) (NorBC) (component of the anerobic, respiratory chain that converts NO3- to N2; denitrification) [reaction catalyzed by Nor: 2 nitric oxide (NO) + 2e- + 2H+ → nitrous oxide (N20) + H2O]. This enzyme does not pump protons across the bacterial membrane (Reimann et al. 2007), but the protons needed for the reaction are taken from the periplasmic side of the membrane (from which side the electrons are donated). P. denitrificans NOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance (ter Beek et al. 2016). norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and is similar to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ or norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity (de Boer et al. 1996).
Bacteria
NorBC of Paracoccus denitrificans
NorB (Q51603; 462 aas; 12 TMSs)
NorC (Q51662; 150 aas; 1 N-terminal TMS)
NorD (Q51665;638 aas; 0 TMSs)
NorE (Q51666; 167 aas; 5 TMSs)
NorF (Q51667; 77 aas and 2 TMSs)
Bacterial respiratory, anaerobic, nitric oxide reductase (NorBC) (not a proton pump; Flock et al., 2008 )
Gram-negative bacteria
NorBC of Pseudomonas stutzeri
NorB (cytochrome b subunit; 474 aas) (P98008)
NorC (cytochrom c subunit, 146 aas) (Q52527)
Nitric oxide reductase, NorBC. 3-d structure known (PDB# 3o0R) (Lee et al., 2012)
Bacteria
NorBC of Pseudomonas aeruginosa
NorB (Chain B) (Q59647)
NorC (Chain C) (Q59646)
Nitric oxide reductase of 787 aas and 14 TMSs, NorZ. This copper-A-dependent NOR uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity (Al-Attar and de Vries 2015). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, suggesting that bacilli using Cu(A)Nor to exploit NO reduction to increase cellular ATP production (Al-Attar and de Vries 2015).
NOR of Bacillus azotoformans
Nitric oxide reductase large subunit, NorB, of 753 aas and 14 TMSs (Al-Attar and de Vries 2015).
NorB of Bacillus azotoformans
Cytochrome oxidase (Cox or CcO). Reversible hydration-level changes of the cavity can be a key factor that regulates the branching of proton transfer events and therefore contributes to the vectorial efficiency of proton transport (Son et al. 2017). Cox16 is required for the assembly of the mitochondrial cytochrome c oxidase (respiratory chain complex IV (CIV)), possibly by promoting the insertion of copper into the active site of cytochrome c oxidase subunit II (MT-CO2/COX2) (Cerqua et al. 2018; Aich et al. 2018). Lipid composition affects the efficiency of the functional reconstitution of the cytochrome c oxidase (Hugentobler et al. 2020). The DeepCys program has been used to predict the functions of cysteine residues in Cox2 (Nallapareddy et al. 2021).
Animals
Cox of Homo sapiens (CoxI-VIII3)
CoxI (Cox1) (P00395)
CoxII (Cox2) (P00403)
CoxIII (Cox3) (P00414)
CoxIV-1 (isoform 1) (Cox41) (P13073)
CoxIV-2 (isoform 2) (Cox42) (Q96KJ9)
CoxVa (Cox5a) (P20674)
CoxVb (Cox5b) (P10601)
CoxVIa (Cox6A2) (Q02221)
CoxVIb (Cox6B2) (Q6YFQ2)
CoxVIIa-H (Cox7A1) (P24310)
CoxVIIa-L (Cox7A2) (P14406)
CoxVIIb2 (Cox7B2) (Q8TF08)
CoxVIIc (Cox7c) (P15954)
CoxVIII-1 (Cox 81) (P48772) (Mouse; human not available)
CoxVIII-2 (Cox82) (P10176)
CoxVIII-3 (Cox83) (Q7Z4L0)
Cox 16, auxillary subunit (Q9P0S2)
Three subunit cytochrome c oxidase including subunits I (524 aas and 13 TMSs in a 1 + (2)6 TMS arrangement); II (232 aas and 3 - 5 TMSs in a 2 TMSs (N-terminal) + 1 - 3 TMSs (C-terminal) arrangement), and III (256 aas and 7 TMSs about equally spaced). It is a biological temperature-sensing electrical regulator of the devil worm, Halicephalobus mephisto (Guerin et al. 2024). This extremophile metazoan was isolated 1.3 km underground in a South African goldmine, where it adapted to heat and potentially to hypoxia, making its mitochondrial sequence a likely target of adaptational change. The complete mitochondrial genome sequence of this organism was obtained, and it showed through dN/dS analysis evidence of positive selection in H. mephisto cytochrome c oxidase subunits. Seventeen of the positively selected amino acid substitutions were located in proximity to the H- and K-pathway proton channels of the complex. The H. mephisto cytochrome c oxidase completely shuts down at low temperatures (20 °C), leading to a 4.8-fold reduction in the transmembrane proton gradient (ΔΨm) compared to the optimal temperature (37 °C). Direct measurement of oxygen consumption found a corresponding 4.6-fold drop at 20 °C compared to 37 °C. Correspondingly, the lifecycle of H. mephisto takes four times longer at low temperature than at higher. This elegant evolutionary adaptation creates a finely-tuned mitochondrial temperature sensor, allowing this ectothermic organism to maximize its reproductive success across varying environmental temperatures (Guerin et al. 2024).
Cytochrome oxidase of Halicephalobus mephisto (a subterrestrial
Cytochrome ubiquinol oxidase subunits I and II of 878 aas and 8 or 9 N-terminal TMSs (I), and 342 aas and 9 TMSs. While subunit I is very distant to many other subunits I from other bacteria, subunit II is much more similar.
Cyt Ox of Desulfococcus multivorans
Cytochrome ba3 oxidase, CbaABC or CcO. The 3-d structure is known (PDB# 1EHK) (Lee et al., 2012). Proton transfer has been reviewed (von Ballmoos et al., 2012). A mutation in subunit A, D372I, a probable pump H+ binding site, uncouples H+ transport from electron flow (von Ballmoos et al. 2015). In this cytochrome ba3, O2 molecules that arrive at the reduction site diffuse through the X-ray-observed tunnel, supporting its role as the main O2 delivery pathway in this cytochrome ba3 as well as the cytokchrome aa3 of Rhodobacter spheroides (Mahinthichaichan et al. 2018). The proton loading site cluster in the ba3 cytochrome c oxidase that loads and traps protons has been identified (Cai et al. 2020).
Thermus/Deinococcus
CbaABC of Thermus thermophilus
CbaA (Q56408)
CbaB (P98052)
CbaC (P82543)
Nitrous oxide reductase, NosZ, of 652 aas with one N-terminal TMS. The NosZ enzyme is the terminal reductase of anaerobic N2O
respiration. Electrons derived from a donor substrate are
transferred to NosZ by means of an electron transport chain (ETC) that
conserves energy through proton motive force generation (Hein and Simon 2019). In both clade I and clade II NosZs, proton motive quinol oxidation by N2O is thought to be catalyzed by the Q cycle mechanism of a membrane-bound Rieske/cytochrome bc complex (Hein and Simon 2019). Nitrous-oxide reductase is part of a bacterial
respiratory system which is activated under anaerobic conditions in the
presence of nitrate or nitrous oxide. NosZ is similar in sequence to cytochrome c oxidase component 2 only in the C-terminal 100 aa residues of both proteins which contains a pair of cysteyl residues.
NosZ of Paracoccus denitrificans
Cytochrome bd quinol oxidoreductase, CydA/CydB. Borisov et al. (2011) have presented evidence concerning a proton channel connecting the site of oxygen reduction to the bacteria cytoplasm and the molecular mechanism by which a membrane potential is generated. The CydX protein of 37 aas and 1 TMS, is encoded in the cydAB operon and functions as a subunit of the Cytochrome bd oxidase complex, activating its activity (VanOrsdel et al. 2013). The AppX protein of 30 aas and 1 TMS, is a paralogue of CydX and can substitute for it in activating the Cytochrome bd oxidase complex (VanOrsdel et al. 2013). Cytochrome bd-type quinol oxidases are structurally unrelated to mitochondrial cytochrome c oxidases. Safarian et al. 2019 determined the structure of the E. coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. The structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase revealed structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site (Safarian et al. 2019). Subunit I of the cytochrome bd quinol oxidase from E. coli has nine transmembrane helices with the O2 reactive site near the periplasmic surface (Zhang et al. 2004). Two small proteins, YtkA (CtaK; 145 aas; P40768) and YczB (CtaM; 70 aas; O31467; TatAd) facilitate the biogenesis of cytochrome c oxidase in Bacillus subtilis (von Wachenfeldt et al. 2021). The latter protein may also function as a constituent of the Tat system (TC# 2.A.64.3.1).
Bacteria
CydA/CydB/CydX/AppX of E. coli
CydA (P0ABJ9)
CydB (P0ABK2)
CydX (P56100)
AppX (P24244)
CydH (YnhF) (A5A618)
Cbb3 cytochrome c oxidase (COX; Cbb3; CcoNOP). The 3-d structure is known to 3.2 Å resolution (PDB# 3MK7; 5DJQ) (Buschmann et al. 2010; Lee et al., 2012). The structure explains a proton-pumping mechanism and the high activity of family-C heme-copper oxidases compared to that of families A and B (Buschmann et al., 2010; Lee et al., 2012). A small subunit of 36 aas and 1 TMS, CcoM, was identified in the structure and plays a role in assembly and stability (Kohlstaedt et al. 2016; Carvalheda and Pisliakov 2017). CcoQ, another small protein of 62 aas (acc # F8H837) is an assembly factor for Cbb3-1 and Cbb3-2 (Kohlstaedt et al. 2017). The A-, B- and C-type oxygen reductases each have an active-site tyrosine that forms a unique cross-linked histidine-tyrosine cofactor. In the C-type oxygen reductases (also called cbb3 oxidases), this post-translationally generated co-factor occurs in a different TMS than for the A- and B-type reductases (Hemp et al. 2006).
Bacteria
CcoNOP of Pseudomonas stutzeri
CcoN (Chain A) (H7F0T0)
CcoO (Chain B) (F8H841)
CcoP (Chain C) (D9IA45)
CcoM (Chain D) (H7ESS5)
CcoQ (assembly factor) (Q8KS20)
Cytochrome oxidase subunit I (CydA) of 481 aas and 9 or 10 TMSs, and subunit II (CydB) of 337 aas and 9 TMSs (Soo et al. 2017).
CydAB of Thermosynechococcus elongatus
Cytochrome bd, AppB (CbdB, CyxB) of 378 aas and 9 TMSs. It is a terminal oxidase that catalyzes quinol-dependent, Na+-independent oxygen uptake. It prefers menadiol over other quinols although ubiquinol was not tested (Sturr et al. 1996). It generates a proton motive force using protons and electrons from opposite sides of the membrane to generate H2O, transferring 1 proton/electron. The mechanism of generation of a transmembrane electric potential difference (Deltapsi) during the catalytic cycle of a bd-type triheme terminal quinol oxidase hasbeen reviewed (Borisov 2023).
Cyt bd of E. coli
Cytochrome oxidase including heme A synthase (HAS) and 4 subunits of the cytochrome oxidase. The 3-d structure of heme A synthase (of 306 aas and 8 TMSs) at 2.2 Å resolution has been solved revealing that the N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant (Niwa et al. 2018). The Sco1 protein, YpmQ, is an accessory protein involved in the assembly of cytochrome c oxidase (Andrews et al. 2004).
Bacteria
CtaACDEF of Bacillus subtilis
The proton pumping Caa3-type cytochrome oxidase chains A-F. The crystal structure (PDB: 2YEV) is known (2.36Å resolution; Lyons et al., 2012). It has a covalently teathered cytochrome c domain. In the cytochrome aa3, O2 molecules that arrive at the reduction site diffuse through the X-ray-observed tunnel, supporting its role as the main O2 delivery pathway in this cytochrome ba3 as well as the cytochrome aa3 of Rhodobacter spheroides (Mahinthichaichan et al. 2018).
Bacteria
Caa(3)-type cytochrome oxidase of Thermus thermophilus
Subunit I + III, Chain A 791aas; 19 TMSs. (P98005)
Subunit II; Chain B 337aas; 2 TMSs. (Q5SLI2)
Subunit IV; Chain C 66aas; 2 TMSs. (Q5SH67)
Cytochrome c oxidase, subunits CtaC (337 aas) CtaD (552 aas) and CtaE (201 aas) (also called CoxABC; Soo et al. 2017).
CtaCDE of Thermosynechococcus elongatus
Cytochrome oxidase complex, CtaCDEF with CtaC being subunit 2 or Cox2 (319 aas and 3 TMSs), CtaD being subunit 1 or Cox1 (578 aas and 12 TMSs), CtaE being subunit 3 or Cox3 (206 aas and 5 TMSs), and CtaF being subunit 4 or Cox4 (132 aas with 4 TMSs in a 2 + 2 TMS arrangement). This complex forms a supercomplex with QcrABC (TC# 3.D.3.5.4) (Falke et al. 2018). The supercomplex is required for nitrate reductase 1 activity and is encoded within a single gene cluster (Falke et al. 2019).
CtaCDEF of Streptomyces coelicolor
Quinol oxidase (CyoABCD). This cytochrome bo3 ubiquinol oxidase is a transmembrane protein of 663 aas and 14 or 15 TMSs, which oxidizes ubiquinone and reduces oxygen, while pumping protons (Marušič et al. 2020). The cytochrome bo3 complex is the component of the aerobic respiratory chain of E. coli that predominates when cells are grown under high aeration conditions. It has proton pumping activity, pumping 2 protons/electron. Protons are probably pumped via D- and K-channels found in the cyoB subunit (Abramson et al. 2000). Two independent structures of the proton-pumping, respiratory cytochrome bo 3 ubiquinol oxidase (cytbo3) have been determined by cryo-EM in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively (Li et al. 2021). The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by TMS0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75(I) and R71(I). In both structures, residue H98(I) occupies two conformations. In conformation 1, H98(I) forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98(I) has flipped to form a hydrogen bond with E14(I) at the N-terminal end of TMS0. The authors proposed that H98(I) dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TMS0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98(I) (Li et al. 2021). Jose et al. 2021 investigated the key PM intermediate, which forms after O–O bond cleavage and precedes proton pumping, using magnetic circular dichroism spectroscopy. The authors observed features demonstrating that PM is a three-spin system, consistent with a consensus model including an iron(IV)-oxo species, a copper(II) ion, and a tyrosyl radical. These results provide validation of the O–O cleavage mechanism and open the door to understanding the proton pumping step (Jose et al. 2021).
Bacteria
CyoABCD of E. coli
CyoA, 315 aas and 3 N-terminal TMSs
CyoB, 663 aas and 14 TMSs
CyoC, 204 aas and 5 TMSs
CyoD, 109 aas and 3 TMSs
Cytochrome aa3 quinol oxidase subunits I- IV (E - H). The 3-D structure has been determined (PDB# 6KOE). The quinol oxidases have an additional transmembrane helix (TMS0) in subunit I that is not present in the related cytochrome c oxidases. Xu et al. 2020 reported the 3.6 Å-resolution X-ray structure of the cytochrome aa3-600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which had been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO revealed a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate (Xu et al. 2020).
Cytochrome aa3 quinol oxidase subunits I- IV (E - H) of Bacillus subtilis
Subunit I (1 or E) of 649 aas and 14 - 16 TMSs, WP_063334853.1
Subunit II (2 or F) of 296 aas and 3 TMSs, WP_003227405
Subunit III (3 or G) of 204 aas and 5 TMSs, WP_088327222
Subunit IV (4 or H) of 123 aas and 3 TMSs, WP_069837848.1
Cytochrome c aa3 oxidase (COX). The 3-d structure is known (PDB# 1M56) (Lee et al., 2012). There are three hydrophobic channels connecting the hydrophobic membrane through the protein to the heme A3/CuB binuclear center (BNC), two of which are probably preferred for O2 diffusion (Oliveira et al. 2014). The D channel is the proton transporting channel, and mutations in residues along this channel, especially N139 in subunit 1, uncouple H+ transport from electron flow (Han et al. 2005). Liang et al. 2017 provided insight into the decoupling mechanisms of CcO mutants, and explained how kinetic gating in the D-channel is imperative to achieving high proton-pumping efficiency in the WT CcO. The O2 molecules that arrived in the reduction site diffuse through the X-ray-observed tunnel, despite its apparent constriction, supporting its role as the main O2 delivery pathway in cytochrome aa3 (Mahinthichaichan et al. 2018).
Bacteria
COX chains A-D of Rhodobacter spheroides
Chain A (P33517)
Chain B (Q03736)
Chain C (Q3J5F6)
Chain D (Q3IZW6)
Cytochrome oxidase (Cox1-3). A radical in the peroxide-produced F-type ferryl form of bovine cytochrome c oxidase has been identified (Sztachova et al. 2022).
Eukaryotes
Coxl-3 of Bos taurus
Cytochrome oxidase (Cox). The factors limiting the allotopic production of the Cox2 subunit of yeast cytochrome c oxidase have been discovered (Nieto-Panqueva et al. 2024). The biogenesis pathway followed by the allotopically produced Cox2 subunit based on the participation of the 2 different structural/functional forms of the TIM23 translocon, TIM23MOTOR and TIM23SORT must follow a concerted and sequential mode of action to insert Cox2W56R into the inner mitochondrial membrane in the correct Nout-Cout topology (Nieto-Panqueva et al. 2024). The Plasmodium falciparum putative SURF1 protein can functionally replace SHY1, a COX assembly factor of complex IV of the mitochondrial electron transfer complex of Saccharomyces cerevisiae (Chellappan et al. 2021).
Yeast
Cytochrome oxidase (Cox) of Saccharomyces cerevisiae
Cox1p; Cox subunit I [Q0045] (NP_009305)
Cox2p; Cox subunit II [Q0250] (NP_009326)
Cox3p; Cox subunit III [Q0275] (NP_009328)
Cox4p; Cox subunit IV [YGL187c] (NP_011328)
Cox5Ap; Cox subunit Va [YNL052w] (aerobically induced) (NP_014346)
Cox5Bp; Cox subunit Vb [YIL111w] (anaerobically induced) (NP_012155)
Cox6p; Cox subunit VI [YHR051w] (NP_011918)
Cox7p; Cox subunit VII [YMR256c] (NP_013983)
Cox8p; Cox subunit VIII [YLR395c] (NP_013499)
Cox9p; Cox subunit VIIa [YDL067c] (NP_010216)
Cox11p; Cox assembly protein [YPL132w] (NP_015193)
Cox12p; Cox subunit VIb [YLR038c] (NP_013139)
Cox13p; Cox subunit VIa [YGL191w] (NP_011324)
Shylp; Cox chaperone [YGR112w] (NP_011627)
Quinol oxidase (proton gradient generated only by chemical charge separation) (Purschke et al., 1997) [DoxA + DoxD comprise a novel membrane-bound thiosulfate: quinone oxidoreductase, Dox (Müller et al., 2004)]
Archaea
DoxABCDEF of Acidianus ambivalens
DoxA (173 aas) (CAA69987)
DoxB (587 aas) (CAA69980)
DoxC (344 aas) (CAA69981)
DoxD (174 aas) (CAA69986)
DoxE (64 aas) (CAA69982)
DoxF (67 aas) (CAA69983)