Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase and subunit III-deficient enzyme: Analysis of respiratory control and proton translocating activities

Abstract

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) or subunit III ( M r 29884)-deficient enzyme (COV-III) were characterized for electron transfer and proton translocating activities in order to investigate the relationship between the respiratory control ratio (RCR) and the apparent proton translocated to electron transferred stoichiometry ( H + e − ratio) in these preparations. We did not observe a quantitative correlation between the RCR value and the H + e − ratio in the preparations. Significant deviation between these two parameters was observed in COV-III and also in COV. However, a new parameter, RCR val, did show a linear relationship with the H + e − ratio of each preparation. Subunit III (SIII)-deficient cytochrome c oxidase isolated by either native gel electrophoresis or chymotrypsin treatment and incorporated into COV-III exhibited H + e − ratios of 0.34 ± 0.10, compared to 0.63 ± 0.09 for COV, emphasizing that the 50% decrease of proton translocating activity is independent of the method of removal of SIII from the enzyme. COV and COV-III also showed similar rates of alkalinization of the extravesicular media after the initial proton translocation reaction (0.07–0.09 neq OH −/s), suggesting that these two preparations had similar endogenous proton permeabilities. In contrast, cytochrome c oxidase (COX) treated with Triton X-100 (3 mg/mg COX) and incorporated into phospholipid vesicles [COV (+TX)]exhibited slower rates of alkalinization (0.04 neq OH −/s), while having a H + e − ratio similar to that of COV (0.66 ± 0.10). The passive proton permeabilities of these preparations were tested by valinomycin-induced K + H + exchange activity. COV (+TX) and COV-III exhibited similar pseudo-first-order rate constants (10 peq OH −/s), while COV had a 20-fold higher rate constant. These results taken together suggest that the different preparations of COX-containing phospholipid vesicles have different biophysical properties. In addition, the decrease in proton-pumping activity observed in COV-III is due to removal of SIII from COX, suggesting that SIII may act either as a passive proton-conducting channel or as a regulator of COX conformation and/or functional activities.