Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase

Robielyn P. Ilagan, Jesús Tejero, Kulwant S. Aulak, Sougata Sinha Ray, Craig Hemann, Zhi Qiang Wang, Mahinda Gangoda, Jay L. Zweier, Dennis J. Stuehr

Research output: Contribution to journalArticle

Abstract

Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of L-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS(nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.

Original languageEnglish (US)
Pages (from-to)3864-3876
Number of pages13
JournalBiochemistry®
Volume48
Issue number18
DOIs
StatePublished - May 12 2009
Externally publishedYes

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Flavin Mononucleotide
Nitric Oxide Synthase Type I
Oxygenases
Flavin-Adenine Dinucleotide
NADP
Electrons
Nitric Oxide Synthase
Citrulline
Electron Spin Resonance Spectroscopy
Enzymes
Calmodulin
Catalysis
Heme
Shielding
Oxidation-Reduction
Paramagnetic resonance
Arginine
Conformations
Rats
Nitric Oxide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ilagan, R. P., Tejero, J., Aulak, K. S., Ray, S. S., Hemann, C., Wang, Z. Q., ... Stuehr, D. J. (2009). Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase. Biochemistry®, 48(18), 3864-3876. https://doi.org/10.1021/bi8021087

Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase. / Ilagan, Robielyn P.; Tejero, Jesús; Aulak, Kulwant S.; Ray, Sougata Sinha; Hemann, Craig; Wang, Zhi Qiang; Gangoda, Mahinda; Zweier, Jay L.; Stuehr, Dennis J.

In: Biochemistry®, Vol. 48, No. 18, 12.05.2009, p. 3864-3876.

Research output: Contribution to journalArticle

Ilagan, RP, Tejero, J, Aulak, KS, Ray, SS, Hemann, C, Wang, ZQ, Gangoda, M, Zweier, JL & Stuehr, DJ 2009, 'Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase', Biochemistry®, vol. 48, no. 18, pp. 3864-3876. https://doi.org/10.1021/bi8021087
Ilagan RP, Tejero J, Aulak KS, Ray SS, Hemann C, Wang ZQ et al. Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase. Biochemistry®. 2009 May 12;48(18):3864-3876. https://doi.org/10.1021/bi8021087
Ilagan, Robielyn P. ; Tejero, Jesús ; Aulak, Kulwant S. ; Ray, Sougata Sinha ; Hemann, Craig ; Wang, Zhi Qiang ; Gangoda, Mahinda ; Zweier, Jay L. ; Stuehr, Dennis J. / Regulation of FMN subdomain interactions and function in neuronal nitric oxide synthase. In: Biochemistry®. 2009 ; Vol. 48, No. 18. pp. 3864-3876.
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AU - Tejero, Jesús

AU - Aulak, Kulwant S.

AU - Ray, Sougata Sinha

AU - Hemann, Craig

AU - Wang, Zhi Qiang

AU - Gangoda, Mahinda

AU - Zweier, Jay L.

AU - Stuehr, Dennis J.

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AB - Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of L-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS(nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.

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