Abstract
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.
Highlights
The ferrous nitric oxide (NO) complex of Neuronal nitric-oxide synthase (nNOS) is stable under inert atmosphere when L-Arg and H4B are bound, it decays in the presence of O2 to form ferric NOS, which can again participate in NO synthesis
A majority of wild type nNOS converted to the ferrous NO complex as judged by buildup of heme Soret absorbance at 436 nm, a single broad visible band near 560 nm, and loss of absorbance at 395 nm, consistent with previous reports [14, 15]
A majority of mutant enzymes remained ferric as judged by continued strong absorbance at 395 nm in both cases. These levels of NO complex were maintained for at least 150 s of the reaction in all cases. This confirms that the Trp409 mutants generate less heme-NO complex during steady state NO synthesis than wild type nNOS
Summary
Materials—Oxygen and NO gas were purchased from Liquid Carbonic Company and Matheson Inc., respectively. Reaction of Ferrous NO Complexes with Oxygen—Anaerobic solutions of full-length nNOS enzymes (4 mM) containing saturating L-Arg and H4B were reduced with a minimum amount of dithionite, and saturated NO solution was added to give an NO concentration of 0.1 mM This solution was transferred to the stopped flow reservoir and rapidly mixed with air-saturated buffer solutions at 10 °C. Formation and Reduction of the W409Y Ferric NO Complex—An anaerobic buffer solution containing 3 mM W409Y oxygenase domain (W409Yox), L-Arg, and H4B in a cuvette had NO-saturated solution added to form the ferric NO complex This was transferred to the stopped flow instrument and rapid mixed at 10 °C with a 0.1 mM dithionite solution.
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