Title |
A Self-sufficient Cytochrome P450 with a Primary Structural Organization That Includes a Flavin Domain and a [2Fe-2S] Redox Center*
|
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Published in |
Journal of Biological Chemistry, September 2003
|
DOI | 10.1074/jbc.m309630200 |
Pubmed ID | |
Authors |
Gareth A. Roberts, Ayhan Çelik, Dominic J.B. Hunter, Tobias W.B. Ost, John H. White, Stephen K. Chapman, Nicholas J. Turner, Sabine L. Flitsch |
Abstract |
p450 RhF from Rhodococcus sp. NCIMB 9784 is the first example of a new class of cytochrome p450 in which electrons are supplied by a novel, FMN- and Fe/S-containing, reductase partner in a fused arrangement. We have previously cloned the gene encoding the enzyme and shown it to comprise an N-terminal p450 domain fused to a reductase domain that displays similarity to the phthalate family of oxygenase reductase proteins. A reductase of this type had never previously been reported to interact with a cytochrome p450. In this report we describe the purification and partial characterization of p450 RhF. We show that the enzyme is self-sufficient in catalyzing the O-dealkylation of 7-ethoxycoumarin. The p450 RhF catalyzed O-dealkylation of 7-ethoxycoumarin is inhibited by several compounds that are known inhibitors of cytochrome p450. Presteady state kinetic analysis indicates that p450 RhF shows a 500-fold preference for NAPDH over NADH in terms of Kd value (6.6 microm versus 3.7 mm, respectively). Potentiometric studies show reduction potentials of -243 mV for the two-electron reduction of the FMN and -423 mV for the heme (in the absence of substrate). |
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