Journal of Biological Chemistry
Volume 278, Issue 49, 5 December 2003, Pages 48914-48920
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Enzyme Catalysis and Regulation
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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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 μmversus 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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This work was supported in part by the Biotechnology and Biological Research Council (BBSRC) and Department of Trade and Industry (DTI) under the aegis of a LINK program and GlaxoSmithKline (GSK), Pfizer, Ultrafine Chemicals, and the Edinburgh Protein Interaction Centre (Wellcome Trust). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.