Important role of tetrahydrobiopterin in no complex formation and interdomain electron transfer in neuronal nitric-oxide synthase.
Neuronal nitric-oxide synthase (nNOS) is composed of a heme oxygenase domain and a flavin-bound reductase domain. Ca(2+)/calmodulin (CaM) is essential for interdomain electron transfer during catalysis, whereas the role of the catalytically important cofactor, tetrahydrobiopterin (H4B) remains elusive. The product NO appears to bind to the heme and works as a feedback inhibitor. The present study shows that the Fe(3+)-NO complex is reduced to the Fe(2+)-NO complex by NADPH in the presence of both l-Arg and H4B even in the absence of Ca(2+)/CaM. The complex could not be fully reduced in the absence of H4B under any circumstances. However, dihydrobiopterin and N(G)-hydroxy-l-Arg could be substituted for H4B and l-Arg, respectively. No direct correlation could be found between redox potentials of the nNOS heme and the observed reduction of the Fe(3+)-NO complex. Thus, our data indicate the importance of the pterin binding to the active site structure during the reduction of the NO-heme complex by NADPH during catalytic turnover.
Noguchi
T
T
Institute for Chemical Reaction Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan.
Sagami
I
I
Daff
S
S
Shimizu
T
T
D000693:Anaerobiosis; D000818:Animals; D000975:Antioxidants / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D007501:Iron / Q000737:chemistry; D046911:Macromolecular Substances; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry / Q000494:pharmacology; D009569:Nitric Oxide / Q000737:chemistry*; D020030:Nitric Oxide Donors / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D013053:Spectrophotometry
D000975:Antioxidants; D002147:Calmodulin; D046911:Macromolecular Substances; D020030:Nitric Oxide Donors; D001708:Biopterins; D009569:Nitric Oxide; D009249:NADP; D001120:Arginine; D007501:Iron; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C003402:sapropterin; D002118:Calcium