Effect of pH on the Steady State Kinetics of Bovine Heart NADH: Coenzyme Q Oxidoreductase

Complete initial steady state kinetics of NADH-decylubiquinone (DQ) oxidoreductase reaction between pH 6.5 and 9.0 show an ordered sequential mechanism in which the order of substrate bindings and product releases is NADH-DQ–DQH₂-NAD⁺. NADH binding to the free enzyme is accelerated by protonation of an amino acid (possibly a histidine) residue. The NADH release is negligibly slow under the turnover conditions. The rate of DQ binding to the NADH-bound enzyme and the maximal rate at the saturating concentrations of the two substrates, which is determined by the rates of DQH₂ formation in the active site and releases of DQH₂ and NAD⁺ from the enzyme, are insensitive to pH, in contrast to clear pH dependencies of the maximal rates of cytochrome c oxidase and cytochrome bc ₁ complex. Physiological significances of these results are discussed.