Uncoupling oxygen transfer and electron transfer in the oxygenation of camphor analogues by cytochrome P450-CAM. Direct observation of an intermolecular isotope effect for substrate C-H activation.
The hydroxylation of (1R)-camphor by cytochrome P450-CAM involves almost complete coupling of electron to oxygen transfer. Modifications at C-5 of camphor, the normal site of hydroxylation by P450-CAM, lead to as much as 98% uncoupling of electron and oxygen transfer as well as to decreases in the rate of electron uptake (up to 10-fold) and the rate of oxygenated product formation (up to 210-fold). Two modes of uncoupling are seen: (a) two-electron uncoupling in which the decrease in oxygenated product formation is balanced by increases in H2O2 formation and (b) four-electron "oxidase" uncoupling where the NADH/O2 ratio has changed from one to nearly two and relatively little H2O2 is formed. Both enantiomers of 5-methylenylcamphor are two-electron uncouplers, while (1R)- and (1S)-5,5-difluorocamphor and (1R)-9,9,9-d3-5,5-difluorocamphor are four-electron uncouplers. An intermolecular isotope effect of 11.7 is observed for oxygenation of C-9 in (1R)-5,5-difluorocamphor. With this substrate, the significant decrease in the rate of oxygenated product formation combined with the large isotope effect suggest that the rate-limiting step has switched from electron to oxygen transfer.
D002164:Camphor / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D015394:Molecular Structure; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity
D002164:Camphor; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase; D010100:Oxygen