UNCOUPLING OXYGEN-TRANSFER AND ELECTRON-TRANSFER IN THE OXYGENATION OF CAMPHOR ANALOGS 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 almo st 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 t o decreases in the rate of electron uptake (up to 10-fold) and the rat e of oxygenated product formation (up to 210-fold). Two modes of uncou pling are seen: (a) two-electron uncoupling in which the decrease in o xygenated product formation is balanced by increases in H2O2 formation and (b) four-electron ''oxidase'' uncoupling where the NADH/O-2 ratio has changed from one to nearly two and relatively little H2O2 is form ed, Both enantiomers of 5-methylenylcamphor are two-electron uncoupler s, while (1R)- and (1S)-5,5-difluorocamphor and (1R)-9,9,9-d(3)-5,5-di fluorocamphor are four-electron uncouplers. An intermolecular isotope effect of 11.7 is observed for oxygenation of C-9 in (1R)-5,5-difluoro camphor. With this substrate, the significant decrease in the rate of oxygenated product formation combined with the large isotope effect su ggest that the rate-limiting step has switched from electron to oxygen transfer.