Functional and Protein Processing Insights from a Truncation Mutation in Subunit III of Cytochrome c Oxidase from Rhodobacter sphaeroides

Abstract

A human mitochondrial DNA mutation in the Cytochrome c Oxidase (COX) subunit III (SIII) gene causes a truncation after its three n-terminal transmembrane helices and results phenotypically in severe lactic acidosis episodes. We created the equivalent mutation at position I115 in SIII of R. sphaeroides COX (I115stop). Truncated SIII enzyme was expressed and purified, and SDS-PAGE showed an absence of full length SIII and a doublet band of lower molecular weight which was immunoreactive to a SIII site-specific antibody. MALDI-TOF determined these peptide masses to be 12919 m/z and 11462 m/z, results consistent with a I115 truncation in SIII (12915 m/z) and subsequent proteolytic processing after F101 (11461 m/z). Wildtype COX subunit II is known to undergo protease processing in vivo, yielding different forms of the subunit (IIA, IIC). SDS-PAGE and MALDI-TOF showed higher levels of the less-processed IIC form in I115stop mutant preparations as compared to wildtype, which had higher levels of the IIA form. Functional assays show the I115stop mutant has a maximal electron transfer activity that is approximately 30% of wildtype (480±90 e-/s∗mol versus 1670±180 e-/s∗mol) and exhibits suicide inactivation similar to a form of the enzyme lacking SIII altogether (I/IIOX). The first three helices of SIII putatively contain conserved lipid binding sites, so the electron assays were then conducted in the presence of exogenous lipids. The I115stop mutant exhibited a greater stimulation of activity due to lipid than I/IIOX (23% versus 5%). Additionally, protection from suicide inactivation by lipid was 2.4 fold greater in the I115stop mutant than I/IIOX. Taken together, the results indicate that the truncation mutation alters native subunit II c-terminal processing, and they support the hypothesis that SIII is involved in functional lipid binding.