Deuterium isotope effects in norcamphor metabolism by cytochrome P-450cam: kinetic evidence for the two-electron reduction of a high-valent iron-oxo intermediate

The kinetics of NADH consumption, oxygen uptake, and hydrogen peroxide production have been studied for norcamphor metabolism by cytochrome P-450cam. The kinetic deuterium isotope effects on these processes, with specifically deuteriated norcamphor, are 0.77, 1.22, and 1.16, respectively. Steady-state UV-visible spectroscopy indicates that transfer of the second electron to the dioxy ferrous P-450 is the rate-limiting step, as it is when camphor is the substrate. The inverse deuterium isotope effect for NADH consumption is consistent with an isotope-dependent branching between monooxygenase and oxidase activity, where these reactivities differ in their NADH:oxygen stoichiometries. However, no isotope-dependent redistribution of steady-state intermediates was detected by isotopic difference UV-visible spectroscopy in the presence of norcamphor. The kinetic isotope effects and steady-state spectral results suggest that the high-valent iron-oxo hydroxylating intermediate FeO]3+ is reduced by NADH and the physiological electron-transfer proteins to afford water.