Metabolic Switching in Cytochrome P-450cam: Deuterium Isotope Effects on Regiospecificity and the Monooxygenase/Oxidase Ratio

William M. Atkins, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

Cytochrome P-450cam, isolated and purified to homogeneity from the soil bacterium Pseudomonas putida, has been shown to catalyze the hydroxylation of the substrate analogue norcamphor to form three distinct products and yields: 5-exo-hydroxynorcamphor (45%), 6-exo-hydroxynorcamphor (47%), and 3-exo-hydroxynorcamphor (8%). Specific deuteriation of the norcamphor skeleton at the 5-, 6-, and 3-positions drastically alters this product distribution, indicating a substantial deuterium isotope effect. When the sum total of all oxygenated products formed in the presence of norcamphor is compared to the number of reducing equivalents consumed in the reaction (NADH), a striking unaccountability of electrons is observed. These are shown to reside in excess water produced by the four-electron reduction of atmospheric dioxygen by P-450cam. Metabolism of specifically deuteriated norcamphor demonstrates a deuterium isotope effect on the branching ratio of substrate hydroxylation to excess water production and suggests that this oxidase activity of P-450camresults from the two-electron reduction of a single oxygen-iron intermediate, [FeO]3+.

Original languageEnglish (US)
Pages (from-to)3754-3760
Number of pages7
JournalJournal of the American Chemical Society
Volume109
Issue number12
DOIs
StatePublished - Jun 1 1987

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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