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.(ABSTRACT TRUNCATED AT 250 WORDS)
Geren
L
L
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Hahm
S
S
Durham
B
B
Millett
F
F
D003545:Cysteine / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010777:Photochemistry; D011725:Pyridines / Q000737:chemistry; D012428:Ruthenium / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013057:Spectrum Analysis
D003574:Cytochrome c Group; D011725:Pyridines; D012428:Ruthenium; D003578:Cytochrome-c Peroxidase; D003545:Cysteine