Effect of subunit III removal on control of cytochrome c oxidase activity by pH.
Studies were undertaken to assess the postulated involvement of subunit III in the proton-linked functions of cytochrome c oxidase. The effect of pH on the steady-state kinetic [corrected] parameters of subunit III containing and subunit III depleted cytochrome oxidase was determined by using beef heart and rat liver enzymes reconstituted into phospholipid vesicles. The TNmax and Km values for the III-containing enzyme increase with decreasing pH in a manner quantitatively similar to that reported by Thornstrom et al. [(1984) Chem. Scr. 24, 230-235], giving three apparent pKa values of less than 5.0, 6.2, and 7.8. The maximal activities of the subunit III depleted enzymes (beef heart and rat liver) show a similar dependence on pH, but the Km values are consistently higher than those of the III-containing enzyme, an effect that is accentuated at low pH. The pH dependence of TNmax/Km for both forms of the enzyme (+/- subunit III) indicates that protonation of a group with an apparent pKa of 5.7 lowers the affinity for substrate (cytochrome c) independently of a continued increase in maximal velocity. N,N'-Dicyclohexylcarbodiimide (DCCD) decreases the pH responsiveness of the electron-transfer activity to the same extent in both III-containing and III-depleted enzymes, indicating that this effect is mediated by a peptide other than subunit III. Control of intramolecular electron transfer by a transmembrane pH gradient (or alkaline intravesicular pH) is shown to occur in cytochrome oxidase vesicles with cytochrome c as the electron donor, in agreement with results of Moroney et al. [(1984) Biochemistry 23, 4991-4997] using hexaammineruthenium(II) as the reductant.(ABSTRACT TRUNCATED AT 250 WORDS)
Gregory
L C
LC
Department of Biochemistry, Michigan State University, East Lansing 48824.
Ferguson-Miller
S
S
D000818:Animals; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology
D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D003576:Electron Transport Complex IV