INTRACOMPLEX ELECTRON-TRANSFER BETWEEN RUTHENIUM-CYTOCHROME-C DERIVATIVES AND CYTOCHROME-C-OXIDASE

The reactions of bovine cytochrome c oxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine) ruthenium(II) were studied by laser flash photolysis. All of the derivatives form complexes with cytochrome c oxidase at low ionic strength (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in rapid electron transfer to the ferric heme group of cytochrome c, followed by electron transfer to cytochrome c oxidase. The photoreduced heme Fe(II) in the cytochrome c derivative modified at lysine 25 on the periphery of the heme crevice domain transferred an electron to Cu(A) with a rate constant of 1.1 x 10(4) s-1. Cu(A) then transferred an electron to cytochrome a with a rate constant of 2.3 x 10(4) s-1. The derivatives modified at lysines 7, 39, 55, and 60 remote from the heme crevice domain of cytochrome c have nearly the same kinetics. The rate constant for electron transfer from the cytochrome c heme to Cu(A) is greater than 10(5) s-1, and the rate constant for electron transfer from Cu(A) to cytochrome a is 2 x 10(4) s-1. The cytochrome c derivatives modified at lysines 13 and 27 in the heme crevice domain react much more slowly than the other derivatives, with intracomplex rate constants for oxidation of cytochrome c ranging from 1000 to 6000 s-1. The bulky ruthenium group at the heme crevice domain of these derivatives apparently alters the binding orientation, leading to smaller electron-transfer rates. At 200 mM ionic strength the complexes of all the derivatives are fully dissociated, and second-order kinetics are observed. The derivatives modified at lysines 7, 25, 39, 55, and 60 have nearly the same second-order rate constants as native cytochrome, while the rate constants of the derivatives modified at lysines 13 and 27 are considerably smaller.