Cytochrome c oxidase

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.^[1][2]

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 ^[3] 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.^[4] 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.^[5]

Assembly

The 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 on, bind to others sub-complexes to form holo COX. In post-assembly modifications, the enzyme is dimerized, which is required for active/efficient enzyme action.^[6][7]

Complex subunits

Conserved subunits of the cytochrome oxidase c complex:^[8][9]

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^[14] 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. High levels of ATP can allosterically inhibit cytochrome c oxidase, binding from within the mitochondrial matrix.^[15]

Subcellular distribution

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.^[16] 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.^[17][18] This raises the possibility about existence of yet unidentified specific mechanisms for protein translocation from mitochondria to other cellular destinations.^[16][18][19]

Clinical significance

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.^[20]

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.^[21]

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.^[22] Such brain mapping has been accomplished in spontaneous mutant mice with cerebellar disease such as reeler^[23] and a transgenic model of Alzheimer's disease.^[24] This technique has also been used to map learning activity in animal brain.^[25]

Additional images

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  ETC

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  Complex IV

See also

• Cytochrome c oxidase subunit I
• Cytochrome c oxidase subunit II
• Cytochrome c oxidase subunit III
• Heme a

References

External links

• The Cytochrome Oxidase home page at Rice University
• Interactive Molecular model of cytochrome c oxidase (Requires MDL Chime)
• UMich Orientation of Proteins in Membranes families/superfamily-4
• Cytochrome-c Oxidase at the US National Library of Medicine Medical Subject Headings (MeSH)