title: Ferrous and ferric state of cytochromes P450 in intact Escherichia coli cells: a possible role of cytochrome P450-flavodoxin interactions.
abstract: OBJECTIVE
  Cytochromes P450 (CYPs) are heme enzymes oxygenating a broad range of substrates. Their activity is dependent on the presence of a suitable electron donor (eukaryotic NADPH:CYP oxidoreductase or cytochrome b5). The Escherichia naturally contain no CYPs and no NADPH:CYP oxidoreductase, however it was reported that some CYPs heterologously expressed in E. coli may exist in the ferrous form. A small bacterial flavoprotein, flavodoxin is considered to be responsible for reduction some of these CYPs.
  
  
  METHODS
  The reduction state of several human CYPs expressed in the intact living E. coli cells was examined. In addition, molecular dynamics and steered molecular dynamics simulations were performed to predict and compare affinity of flavodoxin toward selected CYPs.
  
  
  RESULTS
  We determined the reduction state of five human CYPs heterologously expressed in E. coli. The computationally predicted stabilities of CYP-flavodoxin complexes correlate with the percentage of reduced CYPs in bacterial cells. The mean electron transfer distance within optimized complexes was also related to the percentage of reduced CYPs.
  
  
  CONCLUSIONS
  Depending on the resting state, the CYPs heterologously expressed in E. coli could be divided into two groups; CYP2C8, 2C9, 3A4 are in E. coli present mainly in the oxidized form; while CYP1A1, 1A2, 2A6, 2A13, 2B6, 2D6 are found predominantly in the reduced form. We found a significant correlation between the stability of CYP-flavodoxin complexes and the percentage of reduced CYPs in bacteria. Hence, the naturally expressed flavodoxin is probably responsible for reduction of a larger group of human CYPs in bacterial cells.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Culka
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Milichovsky
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Jerabek
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborova
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Vaclav
authors.initials: V
authors.lastname: Martinek
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6 / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6 / Q000378:metabolism; D065727:Cytochrome P-450 CYP2C8 / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9 / Q000378:metabolism; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004926:Escherichia coli; D005290:Ferric Compounds / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D010084:Oxidation-Reduction
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D005418:Flavodoxin; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C569463:CYP1A1 protein, human; C504573:CYP1A2 protein, human; C414206:CYP2A13 protein, human; C585710:CYP2A6 protein, human; C585599:CYP2B6 protein, human; C450259:CYP2C8 protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D065727:Cytochrome P-450 CYP2C8; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase