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.
Atkins
W M
WM
Department of Biochemistry, University of Illinois, Urbana 61801.
Sligar
S G
SG
D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D004579:Electron Transport; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009636:Norbornanes / Q000138:chemical synthesis / Q000378:metabolism*; D010084:Oxidation-Reduction; D011865:Radioisotope Dilution Technique
D009636:Norbornanes; D009243:NAD; C055059:norcamphor; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase