A self-sufficient cytochrome p450 with a primary structural organization that includes a flavin domain and a [2Fe-2S] redox center.
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).
D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D005415:Flavins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System