Role of the linker region connecting the reductase and heme domains in cytochrome P450BM-3.
Cytochrome P450BM-3 is a fatty acid monooxygenase that contains the catalytic P450 heme domain covalently attached to a diflavin P450 reductase domain. The function of the linker region connecting the C-terminal end of the heme domain to the N-terminal end of the reductase domain has been studied by deleting parts of the linker and changing the sequence of the linker. Deleting three or six residues or changing an Arg-Lys-Lys stretch in the middle of the linker to Ala-Ala-Ala does not alter the functional properties of either domain. The mutants retain full cytochrome c and ferricyanide reductase activities characteristic of the P450 reductase domain. The heme domain in the mutants retains its ability to bind a fatty acid substrate giving the full low-to-high spin shift and exhibits the normal 450 nm absorption band characteristic of the reduced carbon monoxide complex. However, the six amino acid deletion mutant exhibit nearly undetectable levels of fatty acid hydroxylase activity, the three amino acid deletion mutant about 10% activity, and the three Ala substitution mutant about 50% activity. The mutants also exhibit slower rates of reductase-to-heme electron transfer rates that correlate with the loss in fatty acid hydroxylase activity. These results indicate that the length of the linker and, to a much less extent, the sequence are important for correctly orienting the reductase and heme domains, which apparently is necessary to achieve efficient reductase-to-heme electron transfer rates.
Govindaraj
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Department of Molecular Biology and Biochemistry, University of California, Irvine 92717, USA.
Poulos
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D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D009227:Myristic Acids; D019814:Myristic Acid; 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