文摘
Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins,putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This studyaddresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and usedto activate O2 at the heme of P450cam. It is shown that kcat/Km(O2) is independent of the PdR concentrationand hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450camor PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a changein the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx(high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transportof electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.