Gating NO release from nitric oxide synthase

Charlotte A. Whited, Jeffrey J. Warren, Katherine D. Lavoie, Emily E. Weinert, Theodor Agapie, Jay R. Winkler, Harry B. Gray

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work indicated that NO release was gated at position 223 in mammalian enzymes; our kinetics experiments include mutants at that position along with measurements on the wild type enzyme. Employing stopped-flow UV-vis methods, reactions were triggered by mixing a reduced enzyme/N-hydroxy-l-arginine complex with an aerated buffer solution. NO release kinetics were obtained for wt NOS and three mutants (H134S, I223V, H134S/I223V). We have confirmed that wt gsNOS has the lowest NO release rate of known NOS enzymes, whether bacterial or mammalian. We also have found that steric clashes at positions 223 and 134 hinder NO escape, as judged by enhanced rates in the single mutants. The empirical rate of NO release from the gsNOS double mutant (H134/I223V) is nearly as rapid as that of the fastest mammalian enzymes, demonstrating that both positions 223 and 134 function as gates for escape of the product diatomic molecule.

Original languageEnglish (US)
Pages (from-to)27-30
Number of pages4
JournalJournal of the American Chemical Society
Volume134
Issue number1
DOIs
StatePublished - Jan 11 2012

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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    Whited, C. A., Warren, J. J., Lavoie, K. D., Weinert, E. E., Agapie, T., Winkler, J. R., & Gray, H. B. (2012). Gating NO release from nitric oxide synthase. Journal of the American Chemical Society, 134(1), 27-30. https://doi.org/10.1021/ja2069533