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

1. Beef heart mitochondria have a cytochrome c₁ : c : aa₃ 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 c₁ and cytochrome c steady states at 552.5–547 nm and 550–556.5 nm, respectively.3. Cytochrome c₁ 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 c₁ and c in the aerobic steady state, with a rate constant for the c₁ → c reduction step greater than 10³ s^?1.4. The greater apparent response of the $\text{c}\text{aa}{}_3$ 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 $\text{c}{}_1\text{c}$ step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa₃ units in situ. Cytochrome c₁ can either reduce the cytochrome c-cytochrome aa₃ complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.