Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron

Cytochrome P450cam (CYP101Fe³⁺) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe³⁺ by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO₂]²⁺ Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH]²⁺ (compound 0) accumulates within 5 μs and decays rapidly to CYP101Fe³⁺, with a k_{440 nm} of 9.6 × 10⁴ s^-1 All processes are complete within 40 μs at 4 °C. Importantly, no transient absorbance bands could be assigned to CYP101[FeO²⁺por^•+] (compound 1) or CYP101[FeO²⁺] (compound 2). However, indirect evidence for the involvement of compound 1 was obtained from the kinetics of formation and decay of a tyrosyl radical. 5-Hydroxycamphor was formed quantitatively, and the catalytic activity of the enzyme was not impaired by exposure to radiation during the pulse radiolysis experiment. The rapid decay of compound 0 enabled calculation of the limits for the Gibbs activation energies for the conversions of compound 0 → compound 1 → compound 2 → CYP101Fe³⁺, yielding a ΔG^‡ of 45, 39, and 39 kJ/mol, respectively. At 37 °C, the steps from compound 0 to the iron(III) state would take only 4 μs. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time.