Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis.
Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.
Shen
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McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Porter
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Wilson
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Kasper
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004252:DNA Mutational Analysis; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013057:Spectrum Analysis; D014443:Tyrosine
D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D009249:NADP; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D009245:NADH Dehydrogenase