Cytochrome c oxidase

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Cytochrome c oxidase
The 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 homo dimer in this structure)
Identifiers
EC number	1.9.3.1
CAS number	9001-16-5
Databases
IntEnz	IntEnz view
BRENDA	BRENDA entry
ExPASy	NiceZyme view
KEGG	KEGG entry
MetaCyc	metabolic pathway
PRIAM	profile
PDB structures	RCSB PDB PDBe PDBsum
Gene Ontology	AmiGO / EGO

The enzyme cytochrome c oxidase or Complex IV, EC 1.9.3.1) is a large transmembrane protein complex found in bacteria and the mitochondrion of eukaryotes.

It is the last enzyme in the respiratory electron transport chain of mitochondria (or bacteria) located in the mitochondrial (or bacterial) membrane. It receives an electron from each of four cytochrome c molecules, and transfers them to one oxygen molecule, converting molecular oxygen to two molecules of water. In the process, it binds four protons from the inner aqueous phase to make water, and in addition translocates four protons across the membrane, helping to establish a transmembrane difference of proton electrochemical potential that the ATP synthase then uses to synthesize ATP.

Structure

Subunit I and II of Complex IV excluding all other subunits, PDB 2EIK

The complex is a large integral membrane protein composed of several metal prosthetic sites and 14 ^[1] 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 Cu_A and Cu_B centers.^[2] In fact, the cytochrome a₃ and Cu_B 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 Cu_A binuclear center and passes an electron to it, being oxidized back to cytochrome c containing Fe³⁺. The reduced Cu_A binuclear center now passes an electron on to cytochrome a, which in turn passes an electron on to the cytochrome a₃-Cu_B 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- Cu_B binuclear center to accept four electrons in reducing molecular oxygen 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.^[3]

Assembly

Site of assembly is believed to occur near TOM/TIM, where complex intermediates are accessible to bind with subunits imported from cytosol. Hemes and cofactors are inserted into subunits I & II. Subunits I and IV initiate assembly. Other subunits may form sub-complex intermediates that later bind to others to form COX complex. In post-assembly modifications, the enzyme is dimerized, which is required for active/efficient enzyme action. Dimers are connected by a cardiolipin molecule.^[4][5]

Table of conserved subunits of cytochrome oxidase c complex^[6][7]

No.	Subunit name	Human protein	Protein description from UniProt	Pfam family with Human protein
1	Cox1	COX1_HUMAN	Cytochrome c oxidase subunit 1	Pfam PF00115
2	Cox2	COX2_HUMAN	Cytochrome c oxidase subunit 2	Pfam PF02790, Pfam PF00116
3	Cox3	COX3_HUMAN	Cytochrome c oxidase subunit 3	Pfam PF00510
4	Cox4i1	COX41_HUMAN	Cytochrome c oxidase subunit 4 isoform 1, mitochondrial	Pfam PF02936
5	Cox4a2	COX42_HUMAN	Cytochrome c oxidase subunit 4 isoform 2, mitochondrial	Pfam PF02936
6	Cox5a	COX5A_HUMAN	Cytochrome c oxidase subunit 5A, mitochondrial	Pfam PF02284
7	Cox5b	COX5B_HUMAN	Cytochrome c oxidase subunit 5B, mitochondrial	Pfam PF01215
8	Cox6a1	CX6A1_HUMAN	Cytochrome c oxidase subunit 6A1, mitochondrial	Pfam PF02046
9	Cox6a2	CX6A2_HUMAN	Cytochrome c oxidase subunit 6A2, mitochondrial	Pfam PF02046
10	Cox6b1	CX6B1_HUMAN	Cytochrome c oxidase subunit 6B1	Pfam PF02297
11	Cox6b2	CX6B2_HUMAN	Cytochrome c oxidase subunit 6B2	Pfam PF02297
12	Cox6c	COX6C_HUMAN	Cytochrome c oxidase subunit 6C	Pfam PF02937
13	Cox7a1	CX7A1_HUMAN	Cytochrome c oxidase subunit 7A1, mitochondrial	Pfam PF02238
14	Cox7a2	CX7A2_HUMAN	Cytochrome c oxidase subunit 7A2, mitochondrial	Pfam PF02238
15	Cox7a3	COX7S_HUMAN	Putative cytochrome c oxidase subunit 7A3, mitochondrial	Pfam PF02238
16	Cox7b	COX7B_HUMAN	Cytochrome c oxidase subunit 7B, mitochondrial	Pfam PF05392
17	Cox7c	COX7C_HUMAN	Cytochrome c oxidase subunit 7C, mitochondrial	Pfam PF02935
18	Cox7r	COX7R_HUMAN	Cytochrome c oxidase subunit 7A-related protein, mitochondrial	Pfam PF02238
19	Cox8a	COX8A_HUMAN	Cytochrome c oxidase subunit 8A, mitochondrial P	Pfam PF02285
20	Cox8c	COX8C_HUMAN	Cytochrome c oxidase subunit 8C, mitochondrial	Pfam PF02285
Assembly subunits[8][9][10]
1	Coa1	COA1_HUMAN	Cytochrome c oxidase assembly factor 1 homolog	Pfam PF08695
2	Coa3	COA3_HUMAN	Cytochrome c oxidase assembly factor 3 homolog, mitochondrial	Pfam PF09813
3	Coa4	COA4_HUMAN	Cytochrome c oxidase assembly factor 4 homolog, mitochondrial	Pfam PF06747
4	Coa5	COA5_HUMAN	Cytochrome c oxidase assembly factor 5	Pfam PF10203
5	Coa6	COA6_HUMAN	Cytochrome c oxidase assembly factor 6 homolog	Pfam PF02297
6	Coa7	COA7_HUMAN	Cytochrome c oxidase assembly factor 7,	Pfam PF08238
7	Cox11	COX11_HUMAN	Cytochrome c oxidase assembly protein COX11 mitochondrial	Pfam PF04442
8	Cox14	COX14_HUMAN	Cytochrome c oxidase assembly protein	Pfam PF14880
9	Cox15	COX15_HUMAN	Cytochrome c oxidase assembly protein COX15 homolog	Pfam PF02628
10	Cox16	COX16_HUMAN	Cytochrome c oxidase assembly protein COX16 homolog mitochondrial	Pfam PF14138
11	Cox17	COX17_HUMAN	Cytochrome c oxidase copper chaperone	Pfam PF05051
12	Cox18[11]	COX18_HUMAN	Mitochondrial inner membrane protein (Cytochrome c oxidase assembly protein 18)	Pfam PF02096
13	Cox19	COX19_HUMAN	Cytochrome c oxidase assembly protein	Pfam PF06747
14	Cox20	COX20_HUMAN	Cytochrome c oxidase protein 20 homolog	Pfam PF12597

Biochemistry

Summary reaction:

4 Fe²⁺-cytochrome c + 8 H⁺_in + O₂ → 4 Fe³⁺-cytochrome c + 2 H₂O + 4 H⁺_out

Two electrons are passed from two cytochrome c's, through the Cu_A and cytochrome a sites to the cytochrome a₃- Cu_B 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 Cu_B 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 arising from another cytochrome c is passed through the first two electron carriers to the cytochrome a₃- Cu_B binuclear center, and this electron and two protons convert the tyrosyl radical back to Tyr, and the hydroxide bound to Cu_B²⁺ to a water molecule. The fourth electron from another cytochrome c flows through Cu_A and cytochrome a to the cytochrome a₃- Cu_B 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- Cu_B center as it was at the start of this cycle. The net process is that four reduced cytochrome c's are used, along with 4 protons, to reduce O₂ to two water molecules.

Inhibition

Cyanide, sulfide, azide, and carbon monoxide^[12] all bind to cytochrome c oxidase, thus competitively inhibiting the protein from functioning, which results in chemical asphyxiation of cells. Methanol in methylated spirits is converted into formic acid, which also inhibits the same oxidase system.

Subcellular Localization and Presence at Extramitochondrial Sites

Cytochrome c oxidase has 3 subunits which are encoded by mitochondrial DNA. 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.^[13] 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.,^[14][15] This raises the possibility about existence of yet unidentified specific mechanisms for protein translocation from mitochondria to other cellular destinations.^[13][15][16]

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 affect predominantly tissues with high energy demands (brain, heart, muscle). Among the many classified mitochondrial diseases, those involving dysfunctional COX assembly are thought to be the most severe.^[17]

The vast majority of COX disorders are linked to mutations in nuclear-encoded proteins referred to as assembly factors, or assembly proteins. These assembly factors contribute to COX structure and functionality, and are involved in several essential processes, including transcription and translation of mitochondrion-encoded subunits, processing of preproteins and membrane insertion, and cofactor biosynthesis and incorporation.^[18]

Currently, mutations have been identified in seven COX assembly factors: SURF1, SCO1, SCO2, COX10, COX15, COX20, COA5 and LRPPRC. Mutations in these proteins can result in altered functionality of sub-complex assembly, copper transport, or translational regulation. Each gene mutation is associated with the etiology of a specific disease, with some having implications in multiple disorders. Disorders involving dysfunctional COX assembly via gene mutations include Leigh syndrome, cardiomyopathy, leukodystrophy, anemia, and sensorineural deafness.

Histochemistry

COX histochemistry is used for mapping regional brain metabolism in animals, since there is a direct relation between enzyme activity and neuronal activity.^[19] Such brain mapping has been accomplished in spontaneous mutant mice with cerebellar disease such as reeler^[20] and a transgenic model of Alzheimer's disease.^[21] This technique has also been used to map learning activity in animal brain.^[22]

Additional images

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  ETC
  
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  Complex IV