Cytochrome c as an electron shuttle between the outer and inner mitochondrial membranes.

Addition of exogenous NADH to rotenone- and antimycin A-treated mitochondria, in 125 mM KCl, results in rates of oxygen uptake of 0.5-1 and 10-12 nanoatoms of oxygen X mg protein-1 X min-1 in the absence and presence of cytochrome c, respectively. During oxidation of exogenous NADH there is a fast and complete reduction of cytochrome b5 while endogenous or added exogenous cytochrome c become 10-15% and 100% reduced, respectively. The reoxidation of cytochrome b5, after exhaustion of NADH, precedes that of cytochrome c. NADH oxidation is blocked by mersalyl, an inhibitor of NADH-cytochrome b5 reductase. These observations support the view of an electron transfer from the outer to the inner membrane of intact mitochondria. Both the rate of exogenous NADH oxidation and the steady state level of cytochrome c reduction increase with the increase of ionic strength, while the rate of succinate oxidation undergoes a parallel depression. These observations suggest that the functions of cytochrome c as an electron carrier in the inner membrane and as an electron shuttle in the intermembrane space are alternative. It is concluded that aerobic oxidation of exogenous NADH involves the following pathway: NADH leads to NADH-cytochrome b5 reductase leads to cytochrome b5 leads to intermembrane cytochrome c leads to cytochrome oxidase leads to oxygen. It is suggested that the communication between the outer and inner membranes mediated by cytochrome c may affect the oxidation-reduction level of cytosolic NADH and the related oxidation-reduction reactions.