PHOTOINDUCED ELECTRON-TRANSFER BETWEEN CYTOCHROME-C PEROXIDASE AND YEAST CYTOCHROME-C LABELED AT CYS-102 WITH (4-BROMOMETHYL-4'-METHYLBIPYRIDINE)[BIS(BIPYRIDINE)]RUTHENIUM2+

The synthesis of (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]ruthenium(II) hexafluorophosphate is described. This new reagent was found to selectively label the single sulfhydryl group at Cys-102 on yeast iso-1-cytochrome c to form the (dimethylbipyridine-Cys-102-cytochrome c)[bis(bipyridine)]ruthenium derivative (Ru-102-cyt c). Excitation of Ru-102-cyt c with a short light flash resulted in formation of excited-state Ru(II*), which rapidly transferred an electron to the ferric heme group to form Fe(II). When the cytochrome c peroxidase compound I (CMPI) was present in the solution, electron transfer from photoreduced Fe(II) in Ru-102-cyt c to the radical site in CMPI was observed. At high ionic strength (100 mM sodium phosphate and 25 mM EDTA, pH 7), second-order kinetics were observed with a rate constant of (7.5 +/- 0.7) x 10(7) M-1 s-1. The second-order rate constant for native iso-1-cytochrome c was (6.7 +/- 0.7) x 10(7) M-1 s-1 under these conditions. The second-order rate constant for electron transfer from Ru-102-cyt c to the radical site in CMPI increased as the ionic strength was decreased, reaching a value of (4.8 +/- 0.5) x 10(8) M-1 s-1 in 40 mM EDTA, pH 7. At lower ionic strength, a complex was formed between Ru-102-cyt c and CMPI, and the rate for intracomplex electron transfer to the radical site was found to be greater than 50 000 s-1. As a series of light flashes were used to titrate CMPI to CMPII, the reaction between Ru-102-cyt c Fe(II) and the Fe(IV) site in CMPII was observed. The intracomplex rate constant for this reaction was 250 +/- 50 s-1 at low ionic strength (5 mM EDTA and 5 mM sodium phosphate, pH 7) and increased to 1000 +/- 200 s-1 in 10 mM EDTA and 5 mM sodium phosphate. At still higher ionic strengths this reaction became second order, but it was slower than electron transfer to the radical site in CMPI under all conditions.