*4.3. Complex IV*

Complex IV or cytochrome oxidase (COX) is an integral mitochondrial inner membrane protein complex that catalyzes the oxidation of cytochrome *c* and the reduction of molecular oxygen to water. This reaction is coupled to the translocation of protons from the matrix to the inter-membrane space [117]. In both human and yeast mitochondria, the COX catalytic core is composed of three proteins encoded in the mitochondrial DNA. They are Cox1, Cox2, and Cox3 in yeast, and MTCO1, MTCO2, and MTCO3 in humans. All the redox centers of COX are located in the Cox1 and Cox2 subunits. Yeast but not human Cox2 is synthesized with a cleavable N-terminal presequence that is required for correct insertion of the protein into the membrane [118]. Cox3 does not contain redox centers. It is thought to stabilize the catalytic core and to enhance the uptake of protons from the mitochondrial matrix [119]. One of the redox centers of Cox1, corresponding to cytochrome *a*, contains heme *a*. The second center, corresponding to cytochrome *a*3, consists of a binuclear heme *a*-CuB. The third center, located on Cox2, is the binuclear CuA. In addition to the catalytic core, COX is composed of several other structural subunits, all encoded in nuclear DNA (Table 4). Recently NDUFA4/COXFA4, a subunit previously thought to be part of Complex I, has been shown to be a subunit of COX [120].

COX catalyzes the consecutive transfer of 4 electrons from cytochrome *c* to a molecule of oxygen bound to the heme *a* of cytochrome *a*<sup>3</sup> with the formation of water. Each electron of cytochrome *c* first reduces the Cu<sup>A</sup> center of Cox2, from which it is then transferred to the heme *a* of cytochrome *a* and finally to the binuclear heme *a* –Cu center of cytochrome *a*<sup>3</sup> [121,122].

The heme *a* differs from heme *b* at two positions of the porphyrin ring. While heme *b* is present in hemoglobin and most heme containing enzymes, heme *a* appears only in COX. The biosynthesis of heme *a* is initiated by the addition of a farnesyl group to the C-2 position of the heme *b* porphyrin ring by farnesyl transferase, encoded by *COX10* [123]. The resulting heme *o* is then converted to heme *a* by the oxidation of a methyl to a formyl group on C-8 of the porphyrin ring in a reaction that requires Cox15, mitochondrial ferredoxin, and ferredoxin reductase [124,125]. Some other factors, such as Shy1 (human SURF1) and Pet117, have been shown to be involved in the hemylation of Cox1 [126,127]. Additionally, several proteins with human homologs, including Cox17, Sco1, Sco2, Cox11, Cox19, Cox23, Cox16, and Cmc1 have been implicated in the trafficking of copper and maturation of the Cu<sup>A</sup> and heme *a*-Cu<sup>B</sup> centers, as reviewed elsewhere [128].

Pathological mutations have been reported in both mitochondrial and nuclear encoded COX subunits, as well as in proteins involved in the biogenesis of the complex. As of today, mutations in more than 20 genes can lead to COX deficiency with a broad spectrum of clinical phenotypes (Table 5).

Furthermore, COX interacts with other complexes of the electron transport chain in entities called supercomplexes or respirasomes that are thought to provide a kinetic advantage by allowing for a more efficient transfer of electrons between the respiratory complexes and their intermediary carriers cytochrome *c* and coenzyme Q [129,130]. In humans, the respirasome is composed of Complex I, III, and IV in variable stoichiometry, while in yeast it is composed of Complex III and IV in strict 2:2 and 2:1 ratios [61,131,132]. Mutations resulting in COX deficiency affect respirasome biogenesis, which could ultimately lead to complex secondary phenotypes, as discussed elsewhere [133].


**Table 4.** Yeast complex IV subunits and their human homologs. The table also shows Complex IV assembly factors that are associated with diseases.

<sup>1</sup> Yeast subunit 5b is a paralog of subunit 5a and under standard conditions of growth is present at low concentrations [134].

4.3.1. Mutations in Complex IV Catalytic Subunits

Generally, when compared to nuclear structural subunits and factors, mitochondrial COX genes are associated with milder and late onset clinical phenotypes [135]. As of today, there are 42 pathogenic mutations reported for MTCO1, 26 for MTCO2, and 24 for MTCO3 [69]. The phenotypes associated with mutations in these subunits are briefly summarized in the paragraphs bellow and are cited from the MITOMAP [69].

The most frequent homoplasmic pathogenic mutations in *MTCO1* are associated with prostate cancer, LHON, SNHL (sensorineural hearing loss) and DEAF (maternally-inherited deafness). The most frequent diseases caused by homoplasmic variants are dilated cardiomyopathy and maternally inherited epilepsy and ataxia. Clinical phenotypes associated with heteroplasmic variants include epilepsy partialis continua, Leigh syndrome, asthenozoospermic infertility, MELAS, myoglobinuria, motor neuron disease, Rhabdomyolysis and acquired idiopathic sideroblastic anemia. Additionally, both homoplasmic and heteroplasmic variants can lead to exercise intolerance.

Homoplasmic pathogenic *MTCO2* variants are mostly associated with progressive encephalomyopathy, possible susceptibility to hypertrophic cardiomyopathy (HCM), SNHL, DEAF, and LHON. For example, there are 147 sequences containing the m.7859G>A substitution that causes progressive encephalomyopathy. Less frequent mutations can cause Alpers-Huttenlocher-like, Asthenozoospermia, developmental delay, ataxia, seizure, hypotonia, hepatic failure, myopathy, MELAS, cerebellar and pyramidal syndrome with cognitive impairment, pseudoexfoliation glaucoma, multisystem disorder, Rhabdomyolysis, biliary atresia, and MIDD (maternally-inherited diabetes and deafness).

The most frequent pathogenic *MTCO3* variants are homoplasmic and lead to LHON. Other clinical phenotypes associated with mutations in *MTCO3* include Alzheimer's disease, MELAS, Leigh syndrome, cardiomyopathy, exercise intolerance, myoglobinuria, myopathy, asthenozoospermia, failure to thrive, cognitive impairment, optic atrophy, encephalopathy, rhabdomyolysis, and sporadic bilateral optic neuropathy.
