Photoinduced Oxidation of Horseradish Peroxidase

Photoexcited [Ru(bpy) 3 ] 2+ (bpy = 2,2‘-bipyridine) can be quenched with [Co(NH 3 ) 5 Cl] 2+ to give [Ru(bpy) 3 ] 3+; this photogenerated oxidant ( E 0 = 1.25 V vs NHE) reacts with horseradish peroxidase isoenzyme c (HRPc) to produce oxidized protein species. Spectra and kinetics measured by laser-flash transient spectroscopy show that oxidation of the trivalent resting state, [PFe III ] + (P = porphyrin dianion), to ferryl compound II, [PFe IV O], is preceded by generation of a π-cation porphyrin radical intermediate, [PFe III ] • 2+ . In the interval 7.8 < pH < 9.8, the rate-limiting step for the transformation of the radical 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 step 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 (10.3 < pH < 10.8) in which the heme iron changes from a five-coordinate, high-spin species to a six-coordinate, low-spin complex (p K a = 10.9); this burst is attributed to very rapid conversion of a hydroxo-ligated ferric π-cation radical porphyrin to a ferryl species. The rate constant for the porphyrin-centered oxidation of compound II to compound I ([PFe IV O] to [PFe IV O] • +; k = 1.1 × 10 8 M - 1 s - 1 ) is slightly larger than that for the oxidation of [PFe III ] + to [PFe III ] • 2+ ( k = 2.5 × 10 7 M - 1 s - 1 ) at pH 10.3; both porphyrin-centered oxidations are much faster than the conversion of [PFe III ] + to [PFe IV O] below pH 9.8.