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.