Steady-State Kinetic Investigation of Cytochrome P450cam: Interaction with Redox Partners and Reaction with Molecular Oxygen

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• 作者：Matthew M. Purdy ; Laura S. Koo ; Paul R. Ortiz de Montellano ; and Judith P. Klinman
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：January 13, 2004
• 年：2004
• 卷：43
• 期：1
• 页码：271 - 281
• 全文大小：151K
• 年卷期：v.43,no.1(January 13, 2004)
• ISSN：1520-4995

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 k_cat/K_m(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.