A fixed mutation in the respiratory complex I impairs mitochondrial bioenergetics in the endangered Apennine brown bear.

Effective conservation genomics of endangered species requires realistic understanding of the fitness consequences caused by the accumulation of deleterious mutations in declining populations. We experimentally investigated three mutations which have been bioinformatically predicted to be deleterious in the mitochondrial ND5 subunit of respiratory complex I and are fixed in the Apennine brown bear, an inbred population of about 50 individuals isolated in Central Italy. Functional assays in transfected cell models and fibroblasts demonstrated that the G527E substitution significantly reduces mitochondrial transmembrane potential and calcium uptake by ca. 40 and 25% of the control level, respectively, while increasing reactive oxygen species production by ca. 45%. While further confirming these findings, experiments with bear fibroblasts highlighted lower oxygen consumption and impaired mitochondrial turnover in the Apennine bear. Molecular dynamics simulations uncovered structural effects of the G527E substitution, including increased rigidity of the ND5 and associated NDUFB8 subunits and altered hydration dynamics in key aqueous channels of the complex I which are essential for proton pumping. These findings validate previous bioinformatic predictions of the negative fitness effects for one out of three mtDNA mutations, and elucidate the molecular mechanisms behind compromised bioenergetics in this endangered bear population produced by the G527E substitution. By linking genotype to phenotype via advanced molecular biology tools in a non-model species of conservation concern, this study highlights the need to fully integrate genomics and molecular experimental approaches in modern conservation biology.