Deuterium Isotope Effects in Norcamphor Metabolism by Cytochrome P-450cam: Kinetic Evidence for the Two-Electron Reduction of a High-Valent Iron-Oxo Intermediate

William M. Atkins, Stephen Sligar

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1610-1616
Number of pages7
JournalBiochemistry
Volume27
Issue number5
DOIs
StatePublished - Mar 1 1988

ASJC Scopus subject areas

  • Biochemistry

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