Molecular basis for hyperactivity in tryptophan 409 mutants of neuronal NOsynthase

A ferrous heme-NO complex builds up in rat neuronal NO synthase during cata lysis and lowers its activity. Mutation of a tryptophan located directly be low the heme (Trp(409)) to phe or Tyr causes hyperactive NO synthesis and l ess 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 conve ntional and stopped flow spectroscopy to compare kinetics of heme-NO comple x formation, enzyme activity prior to and after complex formation, NO bindi ng 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 N ADPH oxidation were 50 and 30% that of wild type, probably due to slower he me reduction. NO complex formation slowed NADPH oxidation in the wild type by 7-fold but reduced mutant activities less than 2-fold, giving mutants hi gher final activities. NO binding kinetics were similar among mutants and w ild type, although in ferrous W409Y land 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) mut ants than in wild type. Thus, mutant hyperactivity derives from slower form ation and faster decay of the heme-NO complex. Together, these minimize par titioning into the NO-bound form.