Photoinduced Electron-Transfer between Cytochrome-C Peroxidase and Yeast Cytochrome-C Labeled at Cys-102 with (4-Bromomethyl-4'-Methylbipyridine)[Bis(Bipyridine)]Ruthenium2+

by Geren, Lois; Hahm, Seung; Durham, Bill; Millett, Francis

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.

Journal
Biochemistry
Volume
30
Issue
39
Year
1991
Start Page
9450-9457
URL
https://dx.doi.org/10.1021/bi00103a009
ISBN/ISSN
1520-4995; 0006-2960
DOI
10.1021/bi00103a009