4.3.3. Mutations in Complex IV Assembly Factors

*SURF1*, the human homolog of yeast *SHY1*, has been implicated in the maturation of the heme *a* centers of Complex IV [138]. Mutations in this gene are the most frequent cause of Leigh syndrome stemming from COX deficiency [139]. The first cases of Leigh syndrome caused by *SURF1* mutations were described in 1998 [140,141]. Since then, many other cases have been reported. A systematic review by Wedatilake et al. [139] lists 43 records describing 129 cases of Leigh syndrome with SURF1 deficiency caused by 83 different mutations. The authors also performed a study that included about 50 patients, in which the most frequently occurring mutation was L105\* (16 homozygous and 11 compound heterozygous) and no specific correlation of genotype to phenotype

was established [139]. Besides Leigh syndrome, there are also reports of *SURF1* mutations associated with Charcot–Marie–Tooth disease [142].

As already mentioned, Cox10 and Cox15 are required for the conversion of heme *b* to the heme *a* of the two redox centers in Cox1 (MTCO1). In *S. cerevisiae*, *cox10* and *cox15* mutants have no Complex IV activity [143,144]. Yeast *cox10* mutants can be functionally complemented by the human homolog of *COX10* [145]. Pathogenic mutations in *COX10* and *COX15* are associated with Leigh syndrome, cardiomyopathy, and encephalopathy, among others (Table 6), and, typically, such patients have an early fatal outcome due to respiratory failure. However, a single adult patient, a 37-year old woman, was identified with isolated COX deficiency associated with a relatively mild clinical phenotype (myopathy, demyelinating neuropathy, premature ovarian failure, short stature, hearing loss, pigmentary maculopathy, and renal tubular dysfunction) due to compound heterozygous mutations resulting in D336V and R339W substitutions in *COX10* [146]. Surprisingly, no COX was detected in blue native gels on mitochondria extracted from the patient's muscle cells. The mutations were introduced into yeast both individually and in combination, all resulting in the loss of the respiratory capacity, which supported the pathogenicity of the mutations [146].


**Table 5.** Pathologies resulting from mutations in genes encoding Complex IV subunits and assembly factors, and their yeast homologs.


#### **Table 5.** *Cont.*


**Table 5.** *Cont.*

<sup>1</sup> Review describing many mutations. <sup>2</sup> In different patients. <sup>3</sup> Splicing mutation.

There is also a single case of a long surviving Leigh syndrome patient resulting from compound heterozygous mutations leading to S152\* and S344P substitutions in Cox15. The 16 year old patient presented 42% and 22% of residual COX activity in skeletal muscle cells and fibroblasts, respectively. A normal amount of assembled COX holoenzyme was present in cultured fibroblasts, which could account for the slower clinical progression of the disease [147].

Human SCO1 and SCO2 are copper proteins involved in the metalation of MTCO2 [148]. Although both proteins have homologs in yeast, only Sco1 is needed for metalation of Cox2 [149]. A role of Sco1 in assembly of COX is supported by its presence in a Cox2 assembly intermediate [150]. Pathological mutations in *SCO1* and *SCO2* have been described to result in cardioencephalomyopathy. Gurgel-Giannetti et al. [151] reviewed about 40 patients with mutations in *SCO2*, the majority presenting with cardioencephalomyopathy while two patients suffered from Leigh syndrome. With the exception of one patient that had a homozygous G193S substitution, all other patients presented the E140K substitution which was found either in homozygosis or in association with a second mutation [151]. Furthermore, mutations in *SCO2* have also been associated with Charcot–Marie–Tooth disease [152] and with high degree myopia [153,154].


**Table 6.** Human ATP Synthase subunits and theirs yeast homologs.


**Table 6.** *Cont.*

Additionally, in the past 10 years, pathogenic mutations have been identified in COX assembly factors, products of *FAM36A*, *COX14*, *PET117*, *COA5*, *PET100*, *COA6*, and *COA3* with a variety of clinical phenotypes (Table 5). The pathologies were found in infants with either homozygous or compound heterozygous mutations.

#### *4.4. ATP Synthase*

The F1Fo-ATP synthase or Complex V is a large multimeric protein complex located in the inner membrane of mitochondria. Its principal function is to phosphorylate ADP to ATP using the energy of the proton gradient formed during oxidation of NADH and succinate [188]. This means of making ATP, historically referred to as oxidative phosphorylation, is reversible, allowing the energy released when the ATP synthase hydrolyzes ATP to be stored as a proton gradient capable of driving other energy-demanding chemical, transport, and physical processes. The mitochondrial ATP synthase consists of three distinct structural components: the F<sup>1</sup> ATPase, the peripheral stalk, and a membrane-embedded unit referred to as F<sup>o</sup> [189]. In both mammalian and yeast cells, the F<sup>1</sup> ATPase is composed of five distinct subunits: three α and three β subunits, that form a hexameric barrel structure, and the monomeric γ, δ, and ε subunits that constitute the central stalk [190]. The peripheral stalk is composed of four nuclear encoded subunits: b, d, OSCP, and h (yeast) or F6 (human). The membrane-embedded domain is comprised of eight subunits, three of which (Atp6, Atp8, and Atp9) are encoded in the mitochondrial genome of *S. cerevisiae* [191], but only two (MT-ATP6 and MT-ATP8) in mammalian mitochondria [192]. Atp9 is present in multiple copies in a ring that rotates during catalysis. The number of Atp9 molecules per ring differs depending on the organism. The yeast and human rings consist of ten and eight subunits of Atp9, respectively [193–195].

#### 4.4.1. Mutations in ATP Synthase Mitochondrially Encoded Subunits

The first mitochondrial disease caused by an ATP synthase dysfunction was found in patients with mutations in *MT-ATP6*, a mitochondrial gene encoding a key component of the F<sup>o</sup> proton channel [196]. The clinical phenotypes of these patients depend largely on the relative levels of heteroplasmy. For instance, patients harboring an m.T8993G substitution with a 70–90% mutation load often exhibit 'milder' syndromes, such as NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) or FBSN (familial bilateral striated necrosis) [196]. Patients with the same m.T8993G substitution, but with a mutation load exceeding 90–95%, present with a more severe neurological disorder: maternally inherited Leigh syndrome characterized by fatal infantile encephalopathy [196].

The m.T8993G pathogenic mutation in *MT-ATP6* was the first reported mitochondrial disease associated with an ATP synthase defect [197]. Since then, more than 40 pathogenic variants have been reported [69]. Four of these, m.T8993G, m.T8993C, m.T9185C, and m.T9185C, constitute most of all known cases, while the remaining variants mostly appear as isolated cases [198]. The clinical presentations of these patients are heterogeneous, with Leigh syndrome and NARP being the most frequently reported (Table 7). Recurrent phenotypes also include Charcot–Marie–Tooth peripheral neuropathy, spinocerebellar ataxia, and familiar upper motor neuron disease. Isolated cases of MLASA (mitochondrial lactic acidosis and sideroblastic anemia), HCM, primary lactic acidosis, 3-methylglutaconic aciduria and optic neuropathy have also been reported [198].

Due to the heteroplasmy accompanying mitochondrial deficiencies caused by *MT-ATP6* mutations, the pathogenic mechanisms underlying these disorders have been challenging to elucidate. In this respect, homoplasmic *S. cerevisiae* clones carrying the pathogenic *ATP6* mutations have served as a good model for characterizing the etiology of *MT-ATP6* diseases. The first modeling in yeast of a pathogenic mutation in *ATP6* was done by Rak et al. [199]. These authors have introduced in yeast the equivalent of the T8993G mutation responsible for NARP. Although the ATP synthase was correctly assembled and present at 80% of wild-type levels, the yeast mutant showed poor respiratory growth and mitochondrial ATP synthesis was only 10% of that of the wild-type. The mutant also had lower steady state levels of COX, suggesting a co-regulation of ATP synthase activity and COX expression [199]. Additionally, there is more evidence that the biogenesis of these two enzymes may be coupled. It was recently showed that Atco, an assembly intermediate composed of COX and ATP synthase subunits, namely Cox6 and Atp9, is a precursor and the sole Atp9 source for ATP synthase assembly [200].

Another study showed that a Leigh syndrome causing m.T9191C mutation with an L242P substitution in *ATP6* reduced both assembly of the yeast ATP synthase and the efficiency of ATP synthesis by 90% [201]. Based on the biochemical phenotypes of the mutant and of revertants with amino acid substitutions at this position, the proline was proposed to disrupt the terminal α-helix causing a displacement of other neighboring helices involved in proton transfer at the Atp6 and Atp9 interface [202]. Additionally, the conformational change induced by the mutations promoted proteolysis of Atp6, thereby accounting for the reduction of assembled ATP synthase. Revertants with replacements of the helix-disrupting proline by threonine or serine completely restored the assembly defect, but only partially the efficiency of proton translocation. These observations, combined with modeling of the interface between the two subunits, suggested that the suppressor mutations did not completely compensate for the displacement of residues involved in release of protons from the Atp9 ring and their transfer to a neighboring aspartic acid residue in Atp6 [202]. These findings are consistent with previous models of the disease, suggesting that certain pathogenic *ATP6* mutations do not completely block the proton translocation mechanisms of the F<sup>o</sup> domain, but rather disrupt proton transport during rotation of the ring. As the conformational changes in the F<sup>1</sup> moiety are induced by rotation of the Atp9 ring, the uncoupling leads to the inability of ATP synthase to harness the energy normally released from the translocation of protons for ATP synthesis [203].

The different biochemical anomalies discerned in the numerous *MT-ATP6* pathogenic variants suggest several pathophysiological mechanisms responsible for diseases associated with defects in the ATP synthase [198]. These include decreased holoenzyme assembly, destabilization of the proton pore resulting in mitochondrial membrane potential buildup, impairment of the proton pump leading to decreased membrane potential, reduced ATP synthesis and abnormal sensitivity to the ATP synthase inhibitor oligomycin [198].

A smaller subset of mitochondrial mutations causing ATP synthase deficiencies has been ascribed to the second mitochondrial gene, *MT-ATP8,* encoding a subunit of Fo. The first pathogenic mutation in *MT-ATP8* was identified in a 16-year-old patient with apical HCM and neuropathy, with a marked reduction in ATP synthase activity in fibroblasts and muscle tissue [204]. Since then, several other patients have been identified with pathogenic *MT-ATP8* mutations. One of the patients was a seven-month-old infant diagnosed with tetralogy of Fallot, the most common type of congenital heart defect characterized by ventricular septal defect, pulmonary stenosis, right ventricular hypertrophy and aortic dextroposition [205].

*MT-ATP6* and *MT-ATP8* genes overlap 46 nucleotides that span 16 codons [206]. On the MITOMAP [69] are presently listed four known pathogenic mutations in this region that contribute to an amino acid change in both genes, and three other variants that have a hypothesized deleterious effect. An ATP synthase deficiency has been associated with an m.C8561G mutation in two siblings with cerebral ataxia and loss of neuromuscular function. The mutation, resulting in P12R and P66A changes in *MT-ATP6* and *MT-ATP8*, respectively, when expressed in homoplasmic myoblasts, reduced cellular ATP by 15% but had only a marginal effect on steady state concentrations of the ATP synthase [207]. Even though there was no obvious decrease in the steady-state concentration of ATP synthase, a substantial increase of an uncharacterized assembly intermediate was noted. Another study reported an m.C8561T mutation with other amino acid substitutions in a patient with a similar biochemical but more severe clinical presentation [208]. Neither studies examined the effect of the singular or combined *ATP8* and *ATP6* mutations on the yeast ATP synthase.
