Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron.

ABSTRACT: 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 ?s and decays rapidly to CYP101Fe3+, with a k440 nm of 9.6 × 104 s-1 All processes are complete within 40 ?s 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 ?G‡ 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 ?s. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time.

SUBMITTER: Erdogan H 

PROVIDER: S-EPMC5766938 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature