Ancient Nuclear Genome Duplication Events in Vertebrates Facilitated the Functional Diversification of Ohnologous Cytochrome c : Insights from Buergeria Frogs

In vertebrates, a nuclear gene encoding mitochondrial cytochrome c (CYC) was duplicated and diverged into one with ubiquitous expression (somatic-type) and the other with testis-preferred expression (testis-type). In this study, synteny based phylogenetic analyses suggest that cyc ohnologs originated through whole genome duplication (WGD) events in the common ancestor of vertebrates and have been conserved for approximately 450-540 million years. Molecular evolutionary analyses revealed pervasive purifying selection and an approximate Phe/Tyr dichotomy at position 46 between somatic- and testis-type CYC proteins. This was the case in two closely related amphibians, Buergeria buergeri and its hot-spring-dwelling species, Buergeria japonica. The recombinant testis-type CYC proteins from B. buergeri (Bb-CYCT) and B. japonica (Bj-CYCT) exhibited significantly higher resistance to hydrogen peroxide (H2O2) than somatic-type counterpart proteins (Bb-CYCS and Bj-CYCS). Structural analysis showed that the Tyr-46 residues in the testis-type CYC proteins face the heme, suggesting a protective role against H2O2 through an oxidation of its hydroxyl group compared to the spatially corresponding Phe-46 residue in the somatic-type counterparts. When the Tyr-46 residue in Bj-CYCT was replaced with the Phe residue, the H2O2 resistance was significantly reduced, confirming the functional importance of Tyr-46. These results demonstrate that functional divergence between somatic- and testis-type Buergeria CYC proteins is at least partly attributable to the Phe-to-Tyr substitution, suggesting that ancient nuclear genome duplication contributed to tissue-specific divergence of mitochondrial function.