DESIGN OF A RUTHENIUM-CYTOCHROME-C DERIVATIVE TO MEASURE ELECTRON-TRANSFER TO THE RADICAL-CATION AND OXYFERRYL HEME IN CYTOCHROME-C PEROXIDASE

A new ruthenium-labeled cytochrome c derivative was designed to measur e the actual rate of electron transfer to the Trp-191 radical cation a nd the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(Fe-I V=O,R(.+))}. The H39C,C102T variant of yeast iso-1-cytochrome c was la beled at the single cysteine residue with a tris(bipyridyl)ruthenium(I I) reagent to form Ru-39-Cc. This derivative has the same reactivity w ith CMPI as native yCc measured by stopped-flow spectroscopy, indicati ng that the ruthenium group does not interfere with the interaction be tween the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI comp lex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in el ectron transfer from Ru-II+ to heme c Fe-III with a rate constant of 5 x 10(5) s(-1), followed by electron transfer from heme c Fe-II to the Trp-191 indolyl radical cation in CMPI(Fe-IV=O,R(.+)) with a rate con stant of k(eta) = 2 x 10(6) s(-1). A subsequent laser flash led to ele ctron transfer from heme c to the oxyferryl heme in CMPII-(Fe-IV=O,R) with a rate constant of k(etb) = 5000 s(-1). The location of the bindi ng domain was determined using a series of surface charge mutants of C cP, The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Sci ence 258, 1748-1755], A mechanism is proposed for reduction of the oxy ferryl heme in which internal electron transfer in CMPII(Fe-IV=O,R) le ads to the regeneration of the radical cation in CMPII-(Fe-III,R(.+)), which is then reduced by yCc(II). Thus, both steps in the complete re duction of CMPI involve electron transfer from yCc(II) to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that th e electron transfer pathway identified in the crystalline yCc-CcP comp lex is very efficient. Stopped-flow studies indicate that native yCc(I I) initially reduces the Trp-191 radical cation in CMPI with a second- order rate constant k(a), which increases from 1.8 x 10(8) M(-1) s(-1) at 310 mM ionic strength to >3 x 10(9) M(-1) s(-1) at ionic strengths below 100 mM. A second molecule of yCc(II) then reduces the oxyferryl heme in CMPII with a second-order rate constant k(b) which increases from 2.7 x 10(7) M(-1) s(-1) at 310 mM ionic strength to 2.5 x 10(8) M (-1) s(-1) at 160 mM ionic strength, As the ionic strength is decrease d below 100 mM the rate constant for reduction of the oxyferryl heme b ecomes progressively slower as the reaction is limited by release of t he product yCc(III) from the yCc(III)-CMPII complex. Both ruthenium ph otoreduction studies and stopped-flow studies demonstrate that the Trp -191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.