Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3.
Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.
Kitazume
Tatsuya
T
Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, USA.
Haines
Donovan C
DC
Estabrook
Ronald W
RW
Chen
Baozhi
B
Peterson
Julian A
JA
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002021:Buffers; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010710:Phosphates / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D002021:Buffers; D010710:Phosphates; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases