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J Biol Chem, Vol. 274, Issue 38, 26907-26911, September 17, 1999
From the The heme of neuronal nitric-oxide synthase
participates in oxygen activation but also binds self-generated NO
during catalysis resulting in reversible feedback inhibition. We
utilized point mutagenesis to investigate if a conserved tryptophan
residue (Trp-409), which engages in
Tryptophan 409 Controls the Activity of Neuronal Nitric-oxide
Synthase by Regulating Nitric Oxide Feedback Inhibition
,,,
Department of Immunology, Lerner Research
Institute, Cleveland Clinic, Cleveland, Ohio 44195 and the
§ Department of Molecular Biology and the Skaggs Institute
for Chemical Biology, The Scripps Research Institute, La Jolla,
California 92037
-stacking with the heme and
hydrogen bonds to its axial cysteine ligand, helps control catalysis
and regulation by NO. Surprisingly, mutants W409F and W409Y were
hyperactive compared with the wild type regarding NO synthesis without
affecting cytochrome c reduction, reductase-independent
N-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin
binding. In the absence of Arg, NADPH oxidation measurements showed
that electron flux through the heme was actually slower in the Trp-409
mutants than in wild-type nNOS. However, little or no NO complex
accumulated during NO synthesis by the mutants, as opposed to the wild
type. This difference was potentially related to mutants forming
unstable 6-coordinate ferrous-NO complexes under anaerobic conditions
even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an
inactive ferrous-NO complex during the steady state. This overcomes the
negative effect of the mutation on electron flux and results in
hyperactivity. Conservation of Trp-409 among different NOS suggests
that the ability of this residue to regulate heme reduction and NO
complex formation is important for enzyme physiologic function.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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