Characterization of tetrahydrobiopterin-free nitric oxide synthase

Indncible nitric oxide synthase (XOS) purified from marine in aero p liages i-. homodnneric. catalyzes the oxidation of I.-arginine to citrtilline and nitric ox ide and contains the following cofactors bound to each 130 kDa Mibunit: iion protoporphyrin IX. FAD. KMX. and o(R)-telrahydro-L-biopterin (H4B). iNOS expressed in E. coli may be purified in the absence of pterin (apoNOS ) or in the presence of t he redox stable analog, 5-deaza-6( R,S )-methyl let rah vdropter m ( DZPIL]). The former exhibits a low-spin henie Soret and the latter a high-spin herne Suret. In either case XOS activity with arginine or Xr'-hydroxyarginine is absolutely dependent on the addition of H-H. ('ytochrome te is reduced ,il rates comparable to wild-type. Rates of N A DPIl oxidation in the presence or absence of substrate are slowed. In the presence of NADPII the fhnin.s exhibit the characteristic extinction decrease upon reduction. However, a re du ted heme-CO lomplex f 115 urn) forms only with the addition of dithionit>l An immediate .shift in the heme Soret from -121 to 398 nin results upon the addition of DZPH, to apoNOS with a K- of approximately ou /AI. DXl'lij. which has a high redox [>olential relative to H4B. does not substitute for H(M in either maintaining NOS oligorneric structure or supporting catalysis- Re dox activity of the pterin is apparently necessary for the proper functioning of NOS. Ait hough assignation of specific tasks to II-B is not yet possible t IM cofartor is crucial in the finely balanced, interdependent network of cofartors that catalyses nil he oxide formation.