Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.