A proximal tryptophan in NO synthase controls activity by a novel mechanism

The heme of neuronal nitric oxide synthase (nNOS) participates in O-2 activation but also binds self-generated NO, resulting in reversible feedback inhibition. We utilized mutagenesis to investigate if a conserved tryptophan residue (Trp409), which engages in pi -stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis rind regulation by NO. Mutants W409F and W409Y were hyperactive regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxy-arginine oxidation. or Arg and tetrahydrobiopterin binding. In the absence of Arg electron flux through the heme was slower in the W409 mutants than in wild-type. However, less NO complex accumulated during NO synthesis by the mutants. To understand the mechanism, we compared the kinetics of heme-NO complex formation, rate of heme reduction, k(cat) prior to and after NO complex formation, NO binding affinity, NO complex stability, and its reaction with O-2. During the initial phase of NO synthesis, heme-NO complex formation was three and five times slower in W409F and W409Y. which corresponded to a slower heme reduction. NO complex formation inhibited wild-type turnover 7-fold but reduced mutant turnover less than 2-fold, giving mutants higher steady-state activities. NO binding kinetics were similar among mutants and wild type, although mutants also formed a 417 nm ferrous-NO complex. Oxidation of ferrous-NO complex was seven times faster in mutants than in wild type. We conclude that mutant hyperactivity primarily derives from slower heme reduction and faster oxidation of the heme-NO complex by O-2. In this way Trp409 mutations minimize NO feedback inhibition by limiting buildup of the ferrous-NO complex during the steady state. Conservation of W409 among NOS suggests that this proximal Trp may regulate NO feedback inhibition and is important for enzyme physiologic function. (C) 2001 Elsevier Science B.V. All rights reserved.