Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase.
A new technique has been introduced to measure interprotein electron transfer which involves photoexcitation of a tris(bipyridine)ruthenium (Ru) complex covalently attached to one of the proteins. Four different strategies have been developed to specifically attach Ru to protein lysine amino groups, histidine imidazole groups, and cysteine sulhydryl groups. These strategies have been used to prepare more than 20 different singly-labeled Ru-cytochrome c derivatives. The new ruthenium photoexcitation technique has been used to study the mechanism for electron transfer between cytochrome c and cytochrome c peroxidase. Laser excitation of a complex between Ru-cytochrome c and cytochrome c peroxidase compound I results in formation of Ru(II*) which is a strong reducing agent, and rapidly transfers an electron to heme c Fe(III) to form Fe(II). The heme c Fe(II) then rapidly transfers an electron to the Trp-191 radical cation in CMPI. The rate constant for this reaction is 6 x 10(4) s-1 for a horse Ru-cytochrome c derivative labeled at lysine 27, and greater than 10(6) s-1 for yeast Ru-cytochrome c derivatives. A second laser flash results in electron transfer from heme c to the oxyferryl heme in cytochrome c peroxidase compound II with a rate constant of 350 s-1. The ruthenium photoreduction technique has been used to study the interaction domain between the two proteins, the pathway for electron transfer to the radical cation and the oxyferryl heme, and the specific residues in the heme crevice which control the electron transfer properties of the Trp-191 radical cation and the oxyferryl heme.
Liu
R Q
RQ
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701, USA.
Geren
L
L
Anderson
P
P
Fairris
J L
JL
Peffer
N
N
McKee
A
A
Durham
B
B
Millet
F
F
D000818:Animals; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004579:Electron Transport; D005609:Free Radicals; D006736:Horses; D007834:Lasers; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D012428:Ruthenium / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D003574:Cytochrome c Group; D005609:Free Radicals; D012428:Ruthenium; D014364:Tryptophan; D003578:Cytochrome-c Peroxidase