Pathogenic variants in cytochrome c oxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygous COA5 missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in who we have identified the same COA5 variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygous COA5 knockout U2OS cell line with similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role for COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role for the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.

The authors have declared no competing interest.

RWT is supported by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Diseases (MR/S005021/1), the UK NIHR Biomedical Research Centre in Age and Age-Related Diseases award to the Newcastle upon Tyne Hospitals NHS Foundation, the Lily Foundation, LifeArc and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders. RWT and MW are supported by the Pathology Society; RWT, MW and AP are supported by Mito Foundation. JXT was supported by a PhD studentship from the Lily Foundation and a Newcastle University Overseas Research Studentship award. CBJ is supported by funding from the Academy of Finland (decision #336455), the Magnus Ehrnroot Foundation and the Jane and Aatos Erkko Foundation (#230004). The Proteomics Core Facility, University of Oslo/Oslo University Hospital is supported by the Core Facilities program of the South-Eastern Norway Regional Health Authority, and is a member of the National Network of Advanced Proteomics Infrastructure (NAPI) which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910). IW is supported by the Deutsche Forschungsgemeinschaft (DFG): SFB1531-S01, project number 456687919 and WI 3728/3-1, project number 515944830.

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All clinical investigations were evaluated according to the Declaration of Helsinki. Ethical approval was granted by the Newcastle and North Tyneside Researach Ethics Committee (REC reference: 16/NE/0267).

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The mass spectrometry proteomics data for label-free whole cell proteomics and complexome profiling produced in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al, 2022) and assigned the dataset identifier PXD050891 and PXD053461 respectively.