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M309630200v1
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Papers In Press, published online ahead of print September 27, 2003
J. Biol. Chem, 10.1074/jbc.M309630200
Submitted on August 29, 2003
Revised on September 22, 2003
Accepted on September 27, 2003

A self-sufficient cytochrome P450 with a primary structural organisation that includes a flavin domain and a [2Fe2S] redox center

Gareth A. Roberts, Ayhan Celik, Dominic J.B. Hunter, Tobias W.B. Ost, John H. White, Stephen K. Chapman, Nicholas J. Turner, and Sabine L. Flitsch

School of Chemistry, University of Edinburgh, Edinburgh, Scotland EH9 3JJ

Corresponding Author: s.flitsch{at}ed.ac.uk

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 encode 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 paper 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 cytochromes P450. Pre-steady-state kinetic analysis indicates that P450 RhF shows a 500-fold preference for NAPDH over NADH in terms of Kd value (6.6 µM 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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