title: Cytochrome P450 BM3, NO binding and real-time NO detection.
abstract: Nitric oxide is known to coordinate to ferrous heme proteins very tightly, following which it is susceptible to reaction with molecular oxygen or free NO. Its coordination to ferric heme is generally weaker but the resultant complexes are more stable in the presence of oxygen. Here we report determination of the binding constants of Cytochrome P450 BM3 for nitric oxide in the ferric state in the presence and absence of substrate. Compared to other 5-coordinate heme proteins, the K(d) values are particularly low at 16 and 40 nM in the presence and absence of substrate respectively. This most likely reflects the high hydrophobicity of the active site of this enzyme. The binding of NO is tight enough to enable P450 BM3 oxygenase domain to be used to determine NO concentrations and in real-time NO detection assays, which would be particularly useful under conditions of low oxygen concentration, where current methods break down.
authors.affiliation: EASTChem School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
authors.forename: Tobias W B
authors.initials: TW
authors.lastname: Ost
authors.forename: Sidong
authors.initials: S
authors.lastname: Liu
authors.forename: Simon
authors.initials: S
authors.lastname: Daff
mesh_terms: D000818:Animals; D001410:Bacillus megaterium / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004229:Dithiothreitol; D057075:Enzyme Assays / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism; D005822:Genetic Vectors; D057927:Hydrophobic and Hydrophilic Interactions; D007093:Imidazoles / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005290:Ferric Compounds; D007093:Imidazoles; D009569:Nitric Oxide; C029899:imidazole; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D010100:Oxygen; D004229:Dithiothreitol