Laser Photoexcitation of NAD(P)H Induces Reduction of P450 BM3 Heme Domain on the Microsecond Time Scale
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- 作者：Hazel M. Girvan ; Derren J. Heyes ; Nigel S. Scrutton ; Andrew W. Munro
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：May 23, 2007
- 年：2007
- 卷：129
- 期：20
- 页码：6647 - 6653
- 全文大小：132K
- 年卷期：v.129,no.20(May 23, 2007)
- ISSN：1520-5126
文摘
We demonstrate that photoexcitation of NAD(P)H at 355 nm using a Nd:YAG laser leads torapid reduction of the heme domain of the Bacillus megaterium fatty acid hydroxylase flavocytochromeP450 BM3. An aqueous electron derived from photoexcited NAD(P)H is rapidly transferred to the hemedomain, enabling the formation of a carbon monoxy complex of the ferrous P450 (FeII-CO) on themicrosecond time scale. Using this approach we have determined the limiting rate constant (1770 s-1 forsubstrate-free heme domain) for formation of the FeII-CO complex. We find no dependence of the observedrate of FeII-CO complex formation on NAD(P)H concentration but demonstrate a hyperbolic dependenceon carbon monoxide concentration. The apparent dissociation constant for the complex of carbon monoxidebound noncovalently to the ferric form of the BM3 heme domain (and with NADH as reductant) is 323 
M.Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide andthe ferric BM3 heme domain (Kd increased to 1404 M) but enhanced the rate of formation of the FeII-COcomplex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)Hphotoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route toprobing the P450 catalytic cycle and its transient intermediates.
M.Binding of a P450 substrate (N-palmitoylglycine) weakened the complex between carbon monoxide andthe ferric BM3 heme domain (Kd increased to 1404 M) but enhanced the rate of formation of the FeII-COcomplex (3036 s-1 for substrate-free heme domain). This study demonstrates the applicability of NAD(P)Hphotoexcitation as a method for rapid electron delivery to P450 enzymes and provides a new route toprobing the P450 catalytic cycle and its transient intermediates.