Assembly of Cytochrome-c Oxidase in the Absence of Assembly Protein Surf1p Leads to Loss of the Active Site Heme

Surf1p is a protein of the inner membrane of mitochondria that functions in the assembly of cytochrome-c oxidase. The specifics of the role of Surf1p have remained unresolved. Numerous mutations in human Surf1p lead to severe mitochondrial disease. A homolog of human Surf1p is encoded by the genome of the α-proteobacterium Rhodobacter sphaeroides, which synthesizes a mitochondrial-like aa3-type cytochrome-c oxidase. The gene for Surf1p was deleted from the genome of R. sphaeroides. The resulting aa3-type oxidase was purified and analyzed by biochemical methods plus optical and EPR spectroscopy. The oxidase that assembled in the absence of Surf1p was composed of three subpopulations with structurally distinct heme a3-Cu active sites. 50% of the oxidase lacked heme a3, 10–15% contained heme a3 but lacked CuBB, and 35–40% had a normal heme a3 -CuB active site with normal activity. CuA assembly was unaffected. All of the oxidase contained low-spin heme a, but the environment of the heme a center was slightly altered in the 50% of the enzyme that lacked heme a3. Introduction of a normal copy of the gene for Surf1p on an exogenous plasmid resulted in a single population of normally assembled, highly active enzyme. The data indicate that Surf1p plays a role in facilitating the insertion of heme a3 into the active site of cytochrome-c oxidase. The results suggest that maturation of the heme a3-CuB center is a step that limits the association of subunits I and II in the assembly of mitochondrial cytochrome oxidase.