Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pKa of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious autooxidation of tyrosine and tryptophan residues in the enzyme’s framework. Here we report the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥ 90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and X-ray absorption spectroscopies, we determine a pKa value for this compound of 11.9. Marcus theory analysis indicates that this elevated pKa results in a >10,000 fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

No abstract

No abstract