Separate Oxidase and Reductase Reaction Sites on Cytochrome c Demonstrated with Purified Site-specific Antibodies

Abstract

Two of the four antibody populations elicited in rabbits by human cytochrome c were isolated in relatively pure form. One is the antibody population which is present in considerable excess over the other three; it was found to block the reaction of human cytochrome c with bovine cytochrome c oxidase, one combining site blocking the oxidation of 1 molecule of cytochrome c. The reaction of cytochrome c with the succinate-cytochrome c reductase is not blocked by this antibody. The other antibody population which has been purified is that controlled by the presence of isoleucyl residue 58 in human cytochrome c, which is replaced by a threonine in the otherwise identical Macaca mulatta protein. This antibody does not block the reactions with either the cytochrome c oxidase or the cytochrome c reductase. Mixtures of Fab fragments derived from all four antibody populations block both enzymic reactions. Thus, separate areas on the surface of cytochrome c are involved in its interaction with the oxidase and reductase segments of the respiratory chain. Antibodies or Fab fragments bound at sites other than those which completely prevent the enzymic reactions cause decreases in rate, probably due to the increase in the size of the reactant from a molecular weight of 12,500 for free cytochrome c to 5 to about 13 times that much for different cytochrome c-Fab fragment or antibody complexes. In addition, in the presence of low molar ratios of unfractionated total Fab fragments to cytochrome c, increases in the rate constant for the oxidase reaction were observed. These may be due to the blockage by an antibody population of an area on the cytochrome c surface which would otherwise bind to the oxidase in a nonreactive combination. No such rate increases were detected in the reductase reaction, which followed simple Michaelis-Menten kinetics, contrary to the complex situation with the oxidase reaction.