DESIGN OF RUTHENIUM-CYTOCHROME-C DERIVATIVES TO MEASURE ELECTRON-TRANSFER TO CYTOCHROME-C PEROXIDASE

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.