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.

Dimers are connected by a cardiolipin molecule,[11][14][15] 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.

[11] 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.

A two-electron reduction initiates a conformational change that allows oxygen to bind at the active site to the partially-reduced enzyme.

[19] Cyanide, azide, and carbon monoxide[20] all bind to cytochrome c oxidase, inhibiting the protein from functioning and leading to the chemical asphyxiation of cells.

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.

A high nitric oxide concentration, such as one added exogenously to the enzyme, reverses cyanide inhibition of COX.

Higher levels of NO, which correlate with the existence of more enzyme in the reduced state, lead to a greater inhibition of cyanide.

[19] At these basal concentrations, NO inhibition of Complex IV is known to have beneficial effects, such as increasing oxygen levels in blood vessel tissues.

[25] NO inhibition of Complex IV has a larger effect at lower oxygen concentrations, increasing its utility as a vasodilator in tissues of need.

[25] Hydrogen sulfide will bind COX in a noncompetitive fashion at a regulatory site on the enzyme, similar to carbon monoxide.

Additionally, in the anterior pituitary, relatively high amounts of these subunits were found in growth hormone secretory granules.

[27][29][30] 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).

Mutations in these proteins can result in altered functionality of sub-complex assembly, copper transport, or translational regulation.

Disorders involving dysfunctional COX assembly via gene mutations include Leigh syndrome, cardiomyopathy, leukodystrophy, anemia, and sensorineural deafness.