Neurogenetics (2006) 7: 51–57 DOI 10.1007/s10048-005-0015-z ORIGINAL ARTICLE Simona Lucioli . Klaus Hoffmeier . Rosalba Carrozzo . Alessandra Tessa . Bernd Ludwig . Filippo M. Santorelli Introducing a novel human mtDNA mutation into the Paracoccus denitrificans COX I gene explains functional deficits in a patient Received: 16 February 2005 / Accepted: 22 August 2005 / Published online: 12 November 2005 # Springer-Verlag 2005 Abstract We identified a novel mutation (S142F) in the human mtDNA CO I gene in a patient with a clinical phenotype resembling mitochondrial cardioencephalomyo- pathy. To substantiate pathogenicity, we modeled the iden- tified mutation in the homologous gene in Paracoccus denitrificans and analyzed the biochemical consequences. We observed a deleterious effect on enzyme activity, with a lack of heme a 3 . Taking advantage of the extensive structural homology between the bacterial enzyme and the mammalian core complex, we conclude that the novel S142F mutation is disease-related. This approach can be used in other cases to support the pathogenicity of novel variants in the mitochondrial genome. Keywords Cytochrome c oxidase . MELAS . mtDNA mutation . Bacterial model . Paracoccus denitrificans Introduction Cytochrome c oxidase (COX), the terminal component of the mitochondrial respiratory chain, catalyzes the exergon- ic reduction of molecular oxygen to water. Mammalian COX is a heteromeric complex consisting of three catalytic subunits (CO I to III), encoded by mitochondrial DNA (mtDNA), and ten “accessory” subunits, encoded by nu- clear genes. These “accessory” subunits are possibly in- volved in the structural stabilization of the complex, and probably in the modulation of its catalytic activity, whereas CO I and CO II contain all the redox-active prosthetic groups. The function of CO III remains largely unknown. The redox centers involved in the electron transfer are two heme A moieties (a and a 3 ) and two copper centers (Cu A and Cu B )[1–3]. Different clinical phenotypes associated with COX defi- ciency have been described, generally characterized by early onset of symptoms and fatal outcome. Most of the syndromes are inherited as autosomal recessive traits. To date, no mutation in any of the nucleus-encoded COX subunits has been identified. Instead, all the genetically defined cases so far reported are due to mutations in genes encoding factors involved in the biogenesis and assembly of the complex, including SURF1, COX10, SCO1, SCO2, and COX15 [4–8]. Mutations in the SURF1 gene are by far the most prevalent cause of infantile COX deficiency, whereas mutations in the other nuclear genes involved in COX biogenesis are considerably less common. Maternally in- herited mutations in mtDNA-encoded COX subunits are rarer, often occur in adults, and are associated with multi- system diseases, including mitochondrial encephalo- myopathy with lactic acidosis and stroke-like episodes (MELAS) [9–19]. In several cases of COX deficiency, however, the responsible gene remains unknown. New mutations in the mitochondrial genome are gen- erally considered disease-related when they fulfill canoni- cal criteria for pathogenicity, which include a high degree of conservation throughout evolution of the mutated resi- due, heteroplasmy, segregation of the mutation with the disease phenotype in the family, and absence in controls. Nonetheless, pathogenicity remains provisional until the functional significance of the mutation has been estab- lished or additional patients have been identified. The three subunits forming the catalytic core of COX are highly conserved among species and the determination of high-resolution structures of the enzyme from beef heart and from Paracoccus denitrificans has confirmed the high degree of structural similarity between the core subunits of the mammalian and bacterial enzymes [20, 21]. We employed a composite strategy to demonstrate the func- tional significance of a novel mutation (S142F) in CO I in a patient affected by a severe cardioencephalomyopathy resembling MELAS. As consensus criteria for pathogenic S. Lucioli . R. Carrozzo . A. Tessa . F. M. Santorelli (*) Molecular Medicine, IRCCS, “Bambino Gesù” Children’ s Hospital, Rome, Italy e-mail: fms3@na.flashnet.it Fax: +39-06-68592024 K. Hoffmeier . B. Ludwig Molecular Genetics, Institute of Biochemistry, J.W. Goethe Universität, Frankfurt, Germany