PHOTOOXIDATION OF TRP-191 IN CYTOCHROME-C PEROXIDASE BY RUTHENIUM CYTOCHROME-C DERIVATIVES

A novel photoinduced electron-transfer reaction is reported in complex es between resting ferric state cytochrome c peroxidase (CcP) and seve ral horse cytochrome c derivatives labeled at single lysine amino grou ps with bis(bipyridine)](dicarboxybipyridine)ruthenium(II) (Ru-CC). Ph otoexcitation of Ru(II) in the 1:1 Ru-27-CC:CcP complex results in for mation of a metal-to-ligand charge-transfer state, Ru(II), which is a strong reducing agent and rapidly transfers an electron to the CC hem e Fe(III) with rate constant k(1) = 2.3 x 10(7) s(-1). The resulting R u(III) is a strong oxidizing agent with a redox potential of 1.3 V, an d it oxidizes the indole ring of Trp-191 with rate constant k(3) = 7 x 10(6) s(-1). The cycle is completed by electron transfer from Fe(II) in CC to the Trp-191 radical in CcP with rate constant k(4) = 6.1 x 10 (4) s(-1). The Ru group is located close to the interaction domain in the Ru-27-CC:CcP complex, allowing rapid electron transfer with both t he heme in CC and Trp-191 in CcP. The electron-transfer reaction was n ot observed in CcP compound I, where Trp-191 is already oxidized to th e radical, or in the W191F mutant, where the indole group is replaced with a phenyl group. The electron-transfer reaction was observed in Cc P mutants modified at residues in the heme crevice, R48K, R48L, H52L, M230I, and M231I, but not in D235N which destabilizes the radical on T rp-191. Increasing the ionic strength results in an increase in the eq uilibrium dissociation constant K of the Ru-27-CC:CcP complex and an i ncrease in the rate constant k(5) for dissociation of the transient in termediate containing Fe(II) CC and the radical form of CcP. Both K an d k(5) were also increased significantly by the mutations D34N, E290N, and A193F involving residues located in the interaction domain of the crystalline complex between yeast CC and CcP [Pelletier & Kraut (1992 ) Science 258, 1748-1755]. This new method allows the study of the ele ctron-transfer reaction between CC and the radical on Trp-191 in the c omplete absence of hydrogen peroxide, and it opens the possibility of measurements at low temperatures in frozen glasses or in crystals.