Analysis of the domain properties of the novel cytochrome P450 RhF

• Published in: FEBS letters Vol. 579; no. 10; pp. 2215 - 2220
• Main Authors: Hunter, Dominic J.B., Roberts, Gareth A., Ost, Tobias W.B., White, John H., Müller, Steffen, Turner, Nicholas J., Flitsch, Sabine L., Chapman, Stephen K.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 11.04.2005
• Subjects: 2Fe–2S; 5-amino-4-oxo-pentanoic acid; ALA; Base Sequence; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; DNA Primers; Electron Spin Resonance Spectroscopy; FAD; Flavin; flavin adenine dinucleotide; flavin mononucleotide; FMN; high performance liquid chromatography–mass spectrometry; HPLC–MS; IPTG; Isoenzymes - chemistry; Isoenzymes - metabolism; isopropyl β-d-1-thiogalactopyranoside; Luria–Bertani medium; Monooxygenase; NAD(P)H; nicotinamide adenine dinucleotide (phosphate), reduced form; optically transparent thin layer electrochemistry; OTTLE; parts per million; phenylmethylsulfonyl fluoride; PMSF; PPM; Reduction potential; Rhodococcus; Rhodococcus - enzymology; SHE; standard hydrogen electrode; Thermodynamics; FMN; Cytochrome P450; FAD; OTTLE; Rhodococcus; Reduction potential; PPM; Monooxygenase; HPLC–MS; 2Fe–2S; SHE; Flavin; PMSF; LB; ALA; IPTG; NAD(P)H
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2005.03.016

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are −240 and −270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe–2S ferredoxin with a reduction potential of −214 mV.