Rq. Liu et al., DESIGN OF RUTHENIUM-CYTOCHROME-C DERIVATIVES TO MEASURE ELECTRON-TRANSFER TO CYTOCHROME-C PEROXIDASE, Biochimie, 77(7-8), 1995, pp. 549-561
A new technique has been introduced to measure interprotein electron t
ransfer which involves photoexcitation of a tris(bipyridine)ruthenium
(Ru) complex covalently attached to one of the proteins. Four differen
t strategies have been developed to specifically attach Ru to protein
lysine amino groups, histidine imidazole groups, and cysteine sulfhydr
yl groups. These strategies have been used to prepare more than 20 dif
ferent singly labeled Ru-cytochrome c derivatives. The new ruthenium p
hotoexcitation technique has been used to study the mechanism for elec
tron transfer between cytochrome c and cytochrome c peroxidase. Laser
excitation of a complex between Ru-cytochrome c and cytochrome c perox
idase compound I results in formation of Ru(II) which is a strong red
ucing agent, and rapidly transfers an electron to heme c Fe(III) to fo
rm 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 i
s 6 x 10(4) s(-1) for a horse Ru-cytochrome c derivative labeled at ly
sine 27, and greater than 10(6) s(-1) for yeast Ru-cytochrome c deriva
tives. A second laser flash results in electron transfer from heme c t
o the oxyferryl heme in cytochrome c peroxidase compound II with a rat
e constant of 350 s(-1). The ruthenium photoreduction technique has be
en 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 oxy
ferryl heme.