Molecular basis for hyperactivity in tryptophan 409 mutants of neuronal NO synthase.
A ferrous heme-NO complex builds up in rat neuronal NO synthase during catalysis and lowers its activity. Mutation of a tryptophan located directly below the heme (Trp(409)) to Phe or Tyr causes hyperactive NO synthesis and less heme-NO complex buildup in the steady state (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907-26911). To understand the mechanism, we used conventional and stopped flow spectroscopy to compare kinetics of heme-NO complex formation, enzyme activity prior to and after complex formation, NO binding affinity, NO complex stability, and its reaction with O(2) in mutants and wild type nNOS. During the initial phase of NO synthesis, heme-NO complex formation was 3 and 5 times slower in W409F and W409Y, and their rates of NADPH oxidation were 50 and 30% that of wild type, probably due to slower heme reduction. NO complex formation slowed NADPH oxidation in the wild type by 7-fold but reduced mutant activities less than 2-fold, giving mutants higher final activities. NO binding kinetics were similar among mutants and wild type, although in ferrous W409Y (and to a lesser extent W409F) the 436-nm NO complex converted to a 417-nm NO complex with time. Oxidation of the ferrous heme-NO complex to ferric enzyme was 7 times faster in Trp(409) mutants than in wild type. Thus, mutant hyperactivity derives from slower formation and faster decay of the heme-NO complex. Together, these minimize partitioning into the NO-bound form.
Adak
S
S
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA. adaks@ccf.org
Wang
Q
Q
Stuehr
D J
DJ
D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014364:Tryptophan; D014443:Tyrosine
D011994:Recombinant Proteins; D009569:Nitric Oxide; D014443:Tyrosine; D006418:Heme; D014364:Tryptophan; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat