A self-sufficient cytochrome p450 with a primary structural organization that includes a flavin domain and a [2Fe-2S] redox center

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

Research output: Contribution to journalArticlepeer-review

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).

Original languageEnglish
Pages (from-to)48914-20
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number49
DOIs
Publication statusPublished - 5 Dec 2003

Keywords

  • Base Sequence
  • Cytochrome P-450 Enzyme System/chemistry
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Flavins/chemistry
  • Iron-Sulfur Proteins/chemistry
  • Oxidation-Reduction
  • Protein Conformation
  • Recombinant Proteins/chemistry
  • Rhodococcus/enzymology
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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