Important Role of Tetrahydrobiopterin in NO Complex Formation and Interdomain Electron Transfer in Neuronal Nitric-Oxide Synthase

Abstract

Neuronal nitric-oxide synthase (nNOS) is composed of a heme oxygenase domain and a flavin-bound reductase domain. Ca2+/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 Fe3+–NO complex is reduced to the Fe2+–NO complex by NADPH in the presence of both l-Arg and H4B even in the absence of Ca2+/CaM. The complex could not be fully reduced in the absence of H4B under any circumstances. However, dihydrobiopterin and NG-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 Fe3+–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.