CO binding to the isolated oxygenase domain of neuronal nitric oxide synthase: effects of inhibitors and mutations at the substrate-binding site.
In order to understand the heme distal structure of neuronal nitric oxide synthase (nNOS), we studied the binding affinity of CO for the ferrous wild type enzyme and the Glu592Ala and Tyr588His substrate binding-site mutants (generated in the oxygenase domain, nNOSox) in the presence of substrates and inhibitors. The CO binding affinities (K(d) = 10-21 mM) of nNOSox in the presence of the substrates, L-Arg and NHA, or inhibitors such as NAME and agmatine were more than two-fold lower than in their absence (K(d) = 5 mM). The presence of NIL strongly inhibited CO binding and gave a K(d) of more than 100 mM. These effects were not observed for the Glu592Ala mutant. The trend in CO binding affinities observed for the Tyr588His mutant was similar to that of the wild type enzyme. The presence of the isolated reductase domain did not affect CO binding. We discuss the role of substrate and inhibitor binding in CO complexation as well as in catalysis.
Bengea
Simona
S
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan.
Sagami
Ikuko
I
Shimizu
Toru
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010088:Oxidoreductases / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D002147:Calmodulin; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D001120:Arginine; D010088:Oxidoreductases; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I