Hydroxylation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes.

TLDR: Chemical and EPR quantitations indicate the reaction pathway involving 5B yields 5-exo-hydroxycamphor quantitatively, and gamma-irradiation at cryogenic temperatures of the ternary complexes of camphor, dioxygen, and ferro-cytochrome P450cam uses EPR and ENDOR spectroscopies to characterize the primary product of reduction as well as subsequent states created by annealing reduced oxyP450.

Abstract: We have employed γ-irradiation at cryogenic temperatures (77 K and also ∼6 K) of the ternary complexes of camphor, dioxygen, and ferro-cytochrome P450cam to inject the “second” electron of the catalytic process. We have used EPR and ENDOR spectroscopies to characterize the primary product of reduction as well as subsequent states created by annealing reduced oxyP450, both the WT enzyme and the D251N and T252A mutants, at progressively higher temperatures. (i) The primary product upon reduction of oxyP450 4 is the end-on, “H-bonded peroxo” intermediate 5A. (ii) This converts even at cryogenic temperatures to the hydroperoxo-ferriheme species, 5B, in a step that is sensitive to these mutations.Yields of 5B are as high as 40%. (iii) In WT and D251N P450s, brief annealing in a narrow temperature range around 200 K causes 5B to convert to a product state, 7A, in which the product 5-exo-hydroxycamphor is coordinated to the ferriheme in a nonequilibrium configuration. Chemical and EPR quantitations indicate the ...