Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM-3 using site-directed mutagenesis.
Cytochrome P450BM-3 is a catalytically self-sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 34:11221-11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin-to-heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six-proline substitution, did not change significantly relative to wild-type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin-to-heme electron transfer.
Govindaraj
S
S
Department of Molecular Biology & Biochemistry, University of California at Irvine 92717, USA.
Poulos
T L
TL
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000737:chemistry*
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; C014227:ferricyanide reductase