Structural and Functional Effects of C-Terminal Truncations in Cytochrome c Oxidase Subunit III from Rhodobacter Sphaeroides

Abstract

Cytochrome c oxidase (COX) catalyzes the oxidation of ferrocytochrome c and reduction of oxygen to water while pumping protons across the inner mitochondrial or bacterial membrane. In this work, two R. sphaeroides COX subunit III (SIII) truncation mutations which mimic those observed in human mitohcondria were characterized. One truncation created was after the third helix of SIII (Δ114 SIII), while the other was after the sixth helix (Δ238 SIII). Isolated and purified Δ114 SIII and the Δ228 SIII retained at least 70% and 30%, respectively of its SIII content. MALDI-TOF showed that Δ114 SIII and I/II COX (where SIII was genetically deleted) had altered in vivo processing of subunits II and IV. Electron transfer assays showed that Δ114 SIII and Δ238 SIII were inhibited by 50% and 70%, compared to control COX. The pH dependencies of the electron transfer rate in both mutations and I/II COX were similar (pKa= 7.4), as compared to wild-type (pKa= 8.7). Both mutations underwent turnover induced inactivation; however, when Δ114 SIII was supplemented with 1 mg/ml asolectin in the assay buffer, the catalytic lifetime of increased two fold when compared to I/II COX. Δ114 SIII and Δ238 SIII exhibited proton-pumping stoichiometries of 0.40 and 0.32, as compared to 0.76 wild-type COX and 0.38 for I-II COX. These results indicate that the five C-terminal helices in SIII play a critical role in the processing of subunits II and IV and in the assembly of COX. Also, structural lipids within the v-shaped cleft of SIII are necessary for providing protection against turnover induced inactivation, and that perturbation in the structure of SIII results in a lower efficiency of vectorial proton pumping by modifying the conformation of subunit I.