In an attempt to establish the relative importance of diffusional and chemical control in the reactivity of the two substrates, ubiquinol and cytochrome c, we have undertaken an extensive characterization of the steady-state kinetics of ubiquinol-cytochrome c reductase (EC 1.10.2.2) when present in open submitochondrial particles from bovine heart. The kinetic pattern follows a Ping Pong mechanism; contrary to the situation found with the isolated enzyme [Speck and Margoliash (1984) J. Biol. Chem. 259, 1064-1072, and confirmed in our laboratory], no substrate inhibition by oxidized cytochrome c was observed with the membrane-bound enzyme. Endogenous oxidized ubiquinone-10 is unable to exert product inhibition under the conditions employed. In the Ping Pong mechanism for this enzyme, the reaction scheme can be clearly divided into two parts, and the K(min.) (k(cat.)/K(m)) value for one substrate is independent of the rate constant for the second substrate. Both ubiquinol-1 and ubiquinol-2 can be used as electron donors reacting with the enzyme from within the lipid bilayer [Fato, Castelluccio, Palmer and Lenaz (1988) Biochim. Biophys. Acta 932, 216-222]; the k(min.) values for ubiquinols, when calculated on the basis of their membranous concentrations, are significantly lower than the k(min.) for cytochrome c. The temperature-dependence of the kinetic parameters was investigated by titrating each of the substrates under quasi-saturating concentrations of the second substrate. Arrhenius plots of V(max.) extrapolated from both cytochrome c and ubiquinol titrations were linear, when care was taken to verify the quasi-saturating concentrations of the fixed co-substrate. The Arrhenius plots for the k(min.) values for both ubiquinol and cytochrome c were linear, but the activation energy was much higher for the former, particularly when calculated for ubiquinol dissolved in the lipid phase; the very low value of activation energy of the k(min.) for cytochrome c is strong support for diffusion control being present in the reaction of cytochrome c with the membranous enzyme. In contrast to the soluble enzyme, ubiquinone titrations of submitochondrial particles at low cytochrome c concentrations deviated from hyperbolic behaviour. Changing the medium viscosity with either poly(ethylene glycol) or sucrose had a strong effect on the cytochrome c k(min.), whereas the change in the ubiquinol k(min.) was much smaller. From the viscosity studies the extent of diffusional control could be calculated, revealing that the reaction with cytochrome c was mostly diffusion-limited. The viscosity of the membrane was changed by incorporating cholesterol; no significant effect on the ubiquinol k(min.) ascribable to diffusion control could be recognized. The bulk of the studies reported here strongly suggest that ubiquinol-cytochrome c reductase is subject to diffusion control of the interaction of cytochrome c from the aqueous medium, but not of the interaction of the short-chain ubiquinols from the membrane interior.
