title: Cytochrome P450 is present in both ferrous and ferric forms in the resting state within intact Escherichia coli and hepatocytes.
abstract: Cytochrome P450 enzymes (P450s) are exceptionally versatile monooxygenases, mediating hydroxylations of unactivated C-H bonds, epoxidations, dealkylations, and N- and S-oxidations as well as other less common reactions. In the conventional view of the catalytic cycle, based upon studies of P450s in vitro, substrate binding to the Fe(III) resting state facilitates the first 1-electron reduction of the heme. However, the resting state of P450s in vivo has not been examined. In the present study, whole cell difference spectroscopy of bacterial (CYP101A1 and CYP176A1, i.e. P450cam and P450cin) and mammalian (CYP1A2, CYP2C9, CYP2A6, CYP2C19, and CYP3A4) P450s expressed within intact Escherichia coli revealed that both Fe(III) and Fe(II) forms of the enzyme are present in the absence of substrates. The relevance of this finding was supported by similar observations of Fe(II) P450 heme in intact rat hepatocytes. Electron paramagnetic resonance (EPR) spectroscopy of the bacterial forms in intact cells showed that a proportion of the P450 in cells was in an EPR-silent form in the native state consistent with the presence of Fe(II) P450. Coexpression of suitable cognate electron donors increased the degree of endogenous reduction to over 80%. A significant proportion of intracellular P450 remained in the Fe(II) form after vigorous aeration of cells. The addition of substrates increased the proportion of Fe(II) heme, suggesting a kinetic gate to heme reduction in the absence of substrate. In summary, these observations suggest that the resting state of P450s should be regarded as a mixture of Fe(III) and Fe(II) forms in both aerobic and oxygen-limited conditions.
authors: School of Biomedical Sciences, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
authors: Wayne A
authors: WA
authors: Johnston
authors: Dominic J B
authors: DJ
authors: Hunter
authors: Christopher J
authors: CJ
authors: Noble
authors: Graeme R
authors: GR
authors: Hanson
authors: Jeanette E
authors: JE
authors: Stok
authors: Martin A
authors: MA
authors: Hayes
authors: James J
authors: JJ
authors: De Voss
authors: Elizabeth M J
authors: EM
authors: Gillam
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008297:Male; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D008024:Ligands; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D010100:Oxygen