Photoexcited [Ru(bpy)3](2+) (bpy = 2,2'-bipyridine) can be quenched wi
th [Co(NH3)(5)Cl](2+) to give [RU(bpy)(3)](3+); this photogenerated ox
idant (E(0) = 1.25 V vs NHE) reacts with horseradish peroxidase isoenz
yme c (HRPc) to produce oxidized protein species. Spectra and kinetics
measured by laser-flash transient spectroscopy show that oxidation of
the trivalent resting state, [PFeIII](+) (P = porphyrin dianion), to
ferryl compound II, [PFeIV = O], is preceded by generation of a;R-cati
on porphyrin radical intermediate, [PFeIII](. 2+). In the interval 7.8
< pH < 9.8, the rate-limiting step for the transformation of the radi
cal intermediate to compound II is the binding of a water molecule to
the five-coordinate heme iron, k(obsd) = (4.1 +/- 0.9) s(-1); this ste
p is followed by fast proton and electron transfer to give the ferryl
species. There is a burst in compound II formation in the pH region (1
0.3 < pH < 10.8) in which the heme iron changes from a five-coordinate
, high-spin species to a six-coordinate, low-spin complex (pK(a) = 10.
9); this burst is attributed to very rapid conversion of a hydroxo-lig
ated ferric pi-cation radical porphyrin to a ferryl species. The rate
constant for the porphyrin-centered oxidation of compound II to compou
nd I ([PFeIV = O] to [PFeIV = O](.+); k = 1.1 x 10(8) M(-1) s(-1)) is
slightly larger than that for the oxidation of [PFeIII](+) to [PFeIII]
(. 2+) (k = 2.5 x 10(7) M(-1) s(-1)) at pH 10.3; both porphyrin-center
ed oxidations are much faster than the conversion of [PFeIII](+) to [P
FeIV = O] below pH 9.8.