Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles

Abstract

1. Beef heart mitochondria have a cytochrome c 1 : c : aa 3 ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have a ratio of 0.65 : 0.4 : 1.0. More than 50% of the submitochondrial particle membrane is in the ‘inverted’ configuration, shielding the catalytically active cytochrome c. The ‘endogenous’ cytochrome c of particles turns over at a maximal rate between 450 and 550 s −1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300–400 s −1, at 28° – 30°C, pH 7.4. 2. Ascorbate plus N, N, N′, N′-tetramethyl- p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c 1 and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively. 3. Cytochrome c 1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate+ N, N, N′, N′-tetramethyl- p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c 1 and c in the aerobic steady state, with a rate constant for the c 1 → c reduction step greater than 10 3 s −1. 4. The greater apparent response of the c aa 3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c 1 c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa 3 units in situ. Cytochrome c 1 can either reduce the cytochrome c-cytochrome aa 3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.