Biochemical and biophysical studies on cytochrome aa3 I. Steady-state kinetics of cytochrome aa3

Abstract

1. 1. The steady-state kinetics of the reaction in the system: ascorbate → cytochrome c → cytochrome aa 3 → O 2 were studied, measuring both the degree of reduction of cytochrome c and the rates of O 2 consumption. 2. 2. In disagreement with some earlier reports it could be shown that the sole reaction of ascorbate is the reduction of ferricytochrome c. From the kinetics of the overall reaction, the rate constant for the reaction between ferricytochrome c and ascorbate could be calculated to be 23 M −1·sec −1, in fair agreement with the value of 50 M −1·sec −1 obtained from direct stopped-flow measurements. 3. 3. Comparison of the rates of O 2 uptake at infinite cytochrome c and ascorbate concentrations for heart-muscle preparation and isolated cytochrome aa 3 revealed the presence of an inhibitor in the latter preparation. A mechanism of inhibition based on Minnaert's Mechanism IV ▪, in which the inhibitor reacts with E, ES and EP forming inactive complexes with about the same K i , is in agreement with the experimental data. 4. 4. The K D for ES and EP, calculated from the kinetics of the overall reaction, was found to be 30 and 30–40 μM for the isolated and particulate cytochrome aa 3, respectively. By making use of the data of Gibson et al. ( J. Biol. Chem., 240 (1965) 888) on the reaction between ferrocytochrome c and ferricytochrome a, values for k −1 and k 2 of 1200 and 300 sec −1, respectively, were calculated. 5. 5. Minnaert's Mechanism IV ( Biochim. Biophys. Acta, 50 (1961) 23) gives the simplest explanation of the observed steady-state kinetics.