cytochrome c oxidase cytochrome c oxidase the enzyme cytochrome c oxidase or complex iv, (was ec 1.9.3.1, now reclassified as a translocase ec 7.1.1.9) is a large transmembrane protein complex found in bacteria, archaea, and mitochondria of eukaryotes. cytochrome c oxidasethe crystal structure of bovine cytochrome c oxidase in a phospholipid bilayer. the intermembrane space lies to top of the image. adapted from pdb: 1occ​ (it is a homodimer in this structure)identifiersec no.1.9.3.1cas no.9001-16-5 databasesintenzintenz viewbrendabrenda entryexpasynicezyme viewkeggkegg entrymetacycmetabolic pathwaypriamprofilepdb structuresrcsb pdb pdbe pdbsumgene ontologyamigo / quickgosearchpmcarticlespubmedarticlesncbiproteins cytochrome c oxidasesubunit i and ii of complex iv excluding all other subunits, pdb: 2eik​identifierssymbolcytochrome c oxidaseopm superfamily4opm protein2dyrmembranome257 it is the last enzyme in the respiratory electron transport chain of cells located in the membrane. it receives an electron from each of four cytochrome c molecules and transfers them to one oxygen molecule and four protons, producing two molecules of water. in addition to binding the four protons from the inner aqueous phase, it transports another four protons across the membrane, increasing the transmembrane difference of proton electrochemical potential, which the atp synthase then uses to synthesize atp. structure the complex the complex is a large integral membrane protein composed of several metal prosthetic sites and 14 protein subunits in mammals. in mammals, eleven subunits are nuclear in origin, and three are synthesized in the mitochondria. the complex contains two hemes, a cytochrome a and cytochrome a3, and two copper centers, the cua and cub centers. in fact, the cytochrome a3 and cub form a binuclear center that is the site of oxygen reduction. cytochrome c, which is reduced by the preceding component of the respiratory chain (cytochrome bc1 complex, complex iii), docks near the cua binuclear center and passes an electron to it, being oxidized back to cytochrome c containing fe3+. the reduced cua binuclear center now passes an electron on to cytochrome a, which in turn passes an electron on to the cytochrome a3>-cub binuclear center. the two metal ions in this binuclear center are 4.5 å apart and coordinate a hydroxide ion in the fully oxidized state. crystallographic studies of cytochrome c oxidase show an unusual post-translational modification, linking c6 of tyr(244) and the ε-n of his(240) (bovine enzyme numbering). it plays a vital role in enabling the cytochrome a3- cub binuclear center to accept four electrons in reducing molecular oxygen and four protons to water. the mechanism of reduction was formerly thought to involve a peroxide intermediate, which was believed to lead to superoxide production. however, the currently accepted mechanism involves a rapid four-electron reduction involving immediate oxygen–oxygen bond cleavage, avoiding any intermediate likely to form superoxide.: 865–866 the conserved subunits table of conserved subunits of cytochrome c oxidase complex no. subunit name human protein protein description from uniprot pfam family with human protein 1cox1cox1_humancytochrome c oxidase subunit 1pfam pf00115 2cox2cox2_humancytochrome c oxidase subunit 2pfam pf02790, pfam pf00116 3cox3cox3_humancytochrome c oxidase subunit 3pfam pf00510 4cox4i1cox41_humancytochrome c oxidase subunit 4 isoform 1, mitochondrialpfam pf02936 5cox4a2cox42_humancytochrome c oxidase subunit 4 isoform 2, mitochondrialpfam pf02936 6cox5a cox5a_human cytochrome c oxidase subunit 5a, mitochondrialpfam pf02284 7cox5b cox5b_human cytochrome c oxidase subunit 5b, mitochondrialpfam pf01215 8cox6a1cx6a1_humancytochrome c oxidase subunit 6a1, mitochondrialpfam pf02046 9cox6a2cx6a2_humancytochrome c oxidase subunit 6a2, mitochondrialpfam pf02046 10cox6b1cx6b1_humancytochrome c oxidase subunit 6b1pfam pf02297 11cox6b2cx6b2_humancytochrome c oxidase subunit 6b2pfam pf02297 12cox6c cox6c_humancytochrome c oxidase subunit 6cpfam pf02937 13cox7a1cx7a1_humancytochrome c oxidase subunit 7a1, mitochondrialpfam pf02238 14cox7a2cx7a2_humancytochrome c oxidase subunit 7a2, mitochondrialpfam pf02238 15cox7a3cox7s_humanputative cytochrome c oxidase subunit 7a3, mitochondrialpfam pf02238 16cox7b cox7b_humancytochrome c oxidase subunit 7b, mitochondrialpfam pf05392 17cox7c cox7c_humancytochrome c oxidase subunit 7c, mitochondrialpfam pf02935 18cox7rcox7r_humancytochrome c oxidase subunit 7a-related protein, mitochondrialpfam pf02238 19cox8a cox8a_humancytochrome c oxidase subunit 8a, mitochondrial ppfam pf02285 20cox8c cox8c_humancytochrome c oxidase subunit 8c, mitochondrialpfam pf02285 assembly subunits 1coa1 coa1_human cytochrome c oxidase assembly factor 1 homologpfam pf08695 2coa3 coa3_human cytochrome c oxidase assembly factor 3 homolog, mitochondrialpfam pf09813 3coa4 coa4_human cytochrome c oxidase assembly factor 4 homolog, mitochondrialpfam pf06747 4coa5 coa5_human cytochrome c oxidase assembly factor 5pfam pf10203 5coa6 coa6_human cytochrome c oxidase assembly factor 6 homologpfam pf02297 6coa7 coa7_human cytochrome c oxidase assembly factor 7,pfam pf08238 7cox11 cox11_human cytochrome c oxidase assembly protein cox11 mitochondrialpfam pf04442 8cox14 cox14_human cytochrome c oxidase assembly proteinpfam pf14880 9cox15 cox15_human cytochrome c oxidase assembly protein cox15 homologpfam pf02628 10cox16 cox16_human cytochrome c oxidase assembly protein cox16 homolog mitochondrialpfam pf14138 11cox17 cox17_human cytochrome c oxidase copper chaperonepfam pf05051 12cox18 cox18_human mitochondrial inner membrane protein (cytochrome c oxidase assembly protein 18)pfam pf02096 13cox19 cox19_human cytochrome c oxidase assembly proteinpfam pf06747 14cox20 cox20_human cytochrome c oxidase protein 20 homologpfam pf12597 assembly cox assembly in yeast is a complex process that is not entirely understood due to the rapid and irreversible aggregation of hydrophobic subunits that form the holoenzyme complex, as well as aggregation of mutant subunits with exposed hydrophobic patches. cox subunits are encoded in both the nuclear and mitochondrial genomes. the three subunits that form the cox catalytic core are encoded in the mitochondrial genome. hemes and cofactors are inserted into subunits i & ii. the two heme molecules reside in subunit i, helping with transport to subunit ii where two copper molecules aid with the continued transfer of electrons. subunits i and iv initiate assembly. different subunits may associate to form sub-complex intermediates that later bind to other subunits to form the cox complex. in post-assembly modifications, cox will form a homodimer. this is required for activity. dimers are connected by a cardiolipin molecule, which has been found to play a key role in stabilization of the holoenzyme complex. the dissociation of subunits viia and iii in conjunction with the removal of cardiolipin results in total loss of enzyme activity. subunits encoded in the nuclear genome are known to play a role in enzyme dimerization and stability. mutations to these subunits eliminate cox function. assembly is known to occur in at least three distinct rate-determining steps. the products of these steps have been found, though specific subunit compositions have not been determined. synthesis and assembly of cox subunits i, ii, and iii are facilitated by translational activators, which interact with the 5’ untranslated regions of mitochondrial mrna transcripts. translational activators are encoded in the nucleus. they can operate through either direct or indirect interaction with other components of translation machinery, but exact molecular mechanisms are unclear due to difficulties associated with synthesizing translation machinery in-vitro. though the interactions between subunits i, ii, and iii encoded within the mitochondrial genome make a lesser contribution to enzyme stability than interactions between bigenomic subunits, these subunits are more conserved, indicating potential unexplored roles for enzyme activity. biochemistry the overall reaction is 4 fe2+ – cytochrome c + 4 h+ + o2 → 4 fe3+ – cytochrome c + 2 h2o δfgo' = - 218 kj/mol two electrons are passed from two cytochrome c's, through the cua and cytochrome a sites to the cytochrome a3–cub binuclear center, reducing the metals to the fe2+ form and cu+. the hydroxide ligand is protonated and lost as water, creating a void between the metals that is filled by o2. the oxygen is rapidly reduced, with two electrons coming from the fe2+-cytochrome a3, which is converted to the ferryl oxo form (fe4+=o). the oxygen atom close to cub picks up one electron from cu+, and a second electron and a proton from the hydroxyl of tyr(244), which becomes a tyrosyl radical. the second oxygen is converted to a hydroxide ion by picking up two electrons and a proton. a third electron from another cytochrome c is passed through the first two electron carriers to the cytochrome a3–cub binuclear center, and this electron and two protons convert the tyrosyl radical back to tyr, and the hydroxide bound to cub2+ to a water molecule. the fourth electron from another cytochrome c flows through cua and cytochrome a to the cytochrome a3–cub binuclear center, reducing the fe4+=o to fe3+, with the oxygen atom picking up a proton simultaneously, regenerating this oxygen as a hydroxide ion coordinated in the middle of the cytochrome a3–cub center as it was at the start of this cycle. overall, four reduced cytochrome c's are oxidized while o2 and four protons are reduced to two water molecules.: 841–5 inhibition cox exists in three conformational states: fully oxidized (pulsed), partially reduced, and fully reduced. each inhibitor has a high affinity to a different state. in the pulsed state, both the heme a3 and the cub nuclear centers are oxidized; this is the conformation of the enzyme that has the highest activity. a two-electron reduction initiates a conformational change that allows oxygen to bind at the active site to the partially-reduced enzyme. four electrons bind to cox to fully reduce the enzyme. its fully reduced state, which consists of a reduced fe2+ at the cytochrome a3 heme group and a reduced cub+ binuclear center, is considered the inactive or resting state of the enzyme. cyanide, azide, and carbon monoxide all bind to cytochrome c oxidase, inhibiting the protein from functioning and leading to the chemical asphyxiation of cells. higher concentrations of molecular oxygen are needed to compensate for increasing inhibitor concentrations, leading to an overall decrease in metabolic activity in the cell in the presence of an inhibitor. other ligands, such as nitric oxide and hydrogen sulfide, can also inhibit cox by binding to regulatory sites on the enzyme, reducing the rate of cellular respiration. cyanide is a non-competitive inhibitor for cox, binding with high affinity to the partially-reduced state of the enzyme and hindering further reduction of the enzyme. in the pulsed state, cyanide binds slowly, but with high affinity. the ligand is posited to electrostatically stabilize both metals at once by positioning itself between them. a high nitric oxide concentration, such as one added exogenously to the enzyme, reverses cyanide inhibition of cox. nitric oxide can reversibly bind to either metal ion in the binuclear center to be oxidized to nitrite. no and cn− will compete with oxygen to bind at the site, reducing the rate of cellular respiration. endogenous no, however, which is produced at lower levels, augments cn− inhibition. higher levels of no, which correlate with the existence of more enzyme in the reduced state, lead to a greater inhibition of cyanide. at these basal concentrations, no inhibition of complex iv is known to have beneficial effects, such as increasing oxygen levels in blood vessel tissues. the inability of the enzyme to reduce oxygen to water results in a buildup of oxygen, which can diffuse deeper into surrounding tissues. no inhibition of complex iv has a larger effect at lower oxygen concentrations, increasing its utility as a vasodilator in tissues of need. hydrogen sulfide will bind cox in a noncompetitive fashion at a regulatory site on the enzyme, similar to carbon monoxide. sulfide has the highest affinity to either the pulsed or partially reduced states of the enzyme, and is capable of partially reducing the enzyme at the heme a3 center. it is unclear whether endogenous h2s levels are sufficient to inhibit the enzyme. there is no interaction between hydrogen sulfide and the fully reduced conformation of cox. methanol in methylated spirits is converted into formic acid, which also inhibits the same oxidase system. high levels of atp can allosterically inhibit cytochrome c oxidase, binding from within the mitochondrial matrix. extramitochondrial and subcellular localizations location of the 3 cytochrome c oxidase subunit genes in the human mitochondrial genome: coxi, coxii, and coxiii (orange boxes). cytochrome c oxidase has 3 subunits which are encoded by mitochondrial dna (cytochrome c oxidase subunit i, subunit ii, and subunit iii). of these 3 subunits encoded by mitochondrial dna, two have been identified in extramitochondrial locations. in pancreatic acinar tissue, these subunits were found in zymogen granules. additionally, in the anterior pituitary, relatively high amounts of these subunits were found in growth hormone secretory granules. the extramitochondrial function of these cytochrome c oxidase subunits has not yet been characterized. besides cytochrome c oxidase subunits, extramitochondrial localization has also been observed for large numbers of other mitochondrial proteins. this raises the possibility about existence of yet unidentified specific mechanisms for protein translocation from mitochondria to other cellular destinations. genetic defects and disorders defects involving genetic mutations altering cytochrome c oxidase (cox) functionality or structure can result in severe, often fatal metabolic disorders. such disorders usually manifest in early childhood and