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

We have employed gamma -irradiation at cryogenic temperatures (77 K and als o similar to6 K) of the ternary complexes of camphor, dioxygen, and ferro-c ytochrome P450cam to inject the "second" electron of the catalytic process. We have used EPR and ENDOR spectroscopies to characterize the primary prod uct of reduction as well as subsequent states created by annealing reduced oxyP450, both the WT enzyme and the D251N and T252A mutants, at progressive ly 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 st ep 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 a round 200 K causes 5B to convert to a product state, 7A, in which the produ ct 5-exo-hydroxycamphor is coordinated to the ferriheme in a nonequilibrium configuration. Chemical and EPR quantitations indicate the reaction pathwa y involving 5B yields 5-exo-hydroxycamphor quantitatively. Analogous (but l ess extensive) results are seen for the alternate substrate, adamantane. (i v) Although the T252A mutation does net interfere with the formation of 5B, the cryoreduced oxyT252A does not yield product, which suggests that 5B is a key intermediate at or near the branch-point that leads,either to produc t formation or to nonproductive "uncoupling" and H2O2 production. The D251N mutation appears to perturb multiple stages in the catalytic cycle. (v) Th ere is no spectroscopic evidence for the buildup of a high-valence oxyferry l/porphyrin pi -cation radical intermediate, 6. However, ENDOR spectroscopy of 7A in H2O and D2O buffers shows that 7A contains hydroxycamphor, rather than water, bound to Fe3+, and that the proton removed from the C(5) carbo n of substrate during hydroxylation is trapped as the hydroxyl proton. This demonstrates that hydroxylation of substrates by P450cam in fact occurs by the formation and reaction of 6. (vi) Annealing at greater than or equal t o 220 K converts the initial product state 7A to the equilibrium product st ate 7, with the transition occurring via a second nonequilibrium product st ate, 7B, in the D251N mutant; in states 7B and 7 the hydroxycamphor hydroxy l proton no longer is trapped. (vii) The present results are discussed in t he context of other efforts to detect intermediates in the P450 catalytic c ycle.