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Gating NO Release from Nitric Oxide Synthase

Whited, Charlotte A. and Warren, Jeffrey J. and Lavoie, Katherine D. and Weinert, Emily E. and Agapie, Theodor and Winkler, Jay R. and Gray, Harry B. (2012) Gating NO Release from Nitric Oxide Synthase. Journal of the American Chemical Society, 134 (1). pp. 27-30. ISSN 0002-7863. PMCID PMC3257399. doi:10.1021/ja2069533.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Warren, Jeffrey J.0000-0002-1747-3029
Agapie, Theodor0000-0002-9692-7614
Winkler, Jay R.0000-0002-4453-9716
Gray, Harry B.0000-0002-7937-7876
Additional Information:© 2011 American Chemical Society. Received: July 29, 2011. Publication Date (Web): December 7, 2011. C.A.W. thanks Michael Winter for valuable discussions and Dr. Josh Woodward for a spectrum of ferric-NO NOS. Supported by an NSF graduate fellowship (C.A.W.), the NIH (DK019038 to H.B.G. and J.R.W.; GM068461 to J.R.W.; GM095037 postdoctoral fellowship to J.J.W.), and the Arnold and Mabel Beckman Foundation.
Funding AgencyGrant Number
NSF Graduate Research FellowshipUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
Issue or Number:1
PubMed Central ID:PMC3257399
Record Number:CaltechAUTHORS:20120403-112410644
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Official Citation:Gating NO Release from Nitric Oxide Synthase Charlotte A. Whited, Jeffrey J. Warren, Katherine D. Lavoie, Emily E. Weinert, Theodor Agapie, Jay R. Winkler, and Harry B. Gray Journal of the American Chemical Society 2012 134 (1), 27-30
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:29957
Deposited By: Ruth Sustaita
Deposited On:03 Apr 2012 18:41
Last Modified:09 Nov 2021 19:33

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