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

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

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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 K-d value (6.6 muM 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).

    Original languageEnglish
    Pages (from-to)48914-48920
    Number of pages7
    JournalJournal of Biological Chemistry
    Issue number49
    Publication statusPublished - 5 Dec 2003


    • P450BM3

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