We have employed gamma-irradiation at cryogenic temperatures (77 K and also approximately 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 reaction pathway involving 5B yields 5-exo-hydroxycamphor quantitatively. Analogous (but less extensive) results are seen for the alternate substrate, adamantane. (iv) Although the T252A mutation does not 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 product formation or to nonproductive "uncoupling" and H(2)O(2) production. The D251N mutation appears to perturb multiple stages in the catalytic cycle. (v) There is no spectroscopic evidence for the buildup of a high-valence oxyferryl/porphyrin pi-cation radical intermediate, 6. However, ENDOR spectroscopy of 7A in H(2)O and D(2)O buffers shows that 7A contains hydroxycamphor, rather than water, bound to Fe(3+), and that the proton removed from the C(5) carbon 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 > or = 220 K converts the initial product state 7A to the equilibrium product state 7, with the transition occurring via a second nonequilibrium product state, 7B, in the D251N mutant; in states 7B and 7 the hydroxycamphor hydroxyl proton no longer is trapped. (vii) The present results are discussed in the context of other efforts to detect intermediates in the P450 catalytic cycle.

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In the first measurement of enzymatic proton transfer at liquid helium temperatures, we examine protonation of the peroxo-ferriheme state of heme oxygenase (HO) produced by in situ radiolytic cryoreduction of oxy-HO in H2O and D2O solvents at ca. 4 K and above, and compare these findings with analogous measurements for oxy-P450cam and for oxy-Mb. Proton transfer in HO occurs at helium temperatures in both solvents; it occurs in P450cam at approximately 50 K and higher; in Mb it does not occur until T > 170 K. For Mb, this transfer at 180 K is biphasic, and the majority phase shows a solvent kinetic isotope effect of 3.8. We discuss these results in the context of the picture of environmentally coupled tunneling, which links proton transfer to two classes of protein motions: environmental reorganization (lambda in Marcus-like equations) and protein fluctuations ("active dynamics"; gating) which modulate the distance of proton transfer.