EPR spectroscopic characterization of neuronal NO synthase

Chris Galli, Ryan MacArthur, Husam M. Abu-Soud, Pamela Clark, Dennis J. Stuehr, Gary W Brudvig

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exists as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 Hz at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH·, was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substrate analogue N(ω)-nitro-L-arginine methylester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, 4.15, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D = 5.2 cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain is partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.

Original languageEnglish
Pages (from-to)2804-2810
Number of pages7
JournalBiochemistry
Volume35
Issue number8
DOIs
Publication statusPublished - Feb 27 1996

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Electron Spin Resonance Spectroscopy
Heme
Nitric Oxide Synthase
Paramagnetic resonance
Arginine
Oxygenases
Substrates
Iron
Oxidoreductases
Binding Sites
Flavins
Electrons
Oxygen
Flavin Mononucleotide
Flavin-Adenine Dinucleotide
4,6-dinitro-o-cresol
Calmodulin
Dissolved oxygen
Microwaves
NADP

ASJC Scopus subject areas

  • Biochemistry

Cite this

Galli, C., MacArthur, R., Abu-Soud, H. M., Clark, P., Stuehr, D. J., & Brudvig, G. W. (1996). EPR spectroscopic characterization of neuronal NO synthase. Biochemistry, 35(8), 2804-2810. https://doi.org/10.1021/bi9520444

EPR spectroscopic characterization of neuronal NO synthase. / Galli, Chris; MacArthur, Ryan; Abu-Soud, Husam M.; Clark, Pamela; Stuehr, Dennis J.; Brudvig, Gary W.

In: Biochemistry, Vol. 35, No. 8, 27.02.1996, p. 2804-2810.

Research output: Contribution to journalArticle

Galli, C, MacArthur, R, Abu-Soud, HM, Clark, P, Stuehr, DJ & Brudvig, GW 1996, 'EPR spectroscopic characterization of neuronal NO synthase', Biochemistry, vol. 35, no. 8, pp. 2804-2810. https://doi.org/10.1021/bi9520444
Galli C, MacArthur R, Abu-Soud HM, Clark P, Stuehr DJ, Brudvig GW. EPR spectroscopic characterization of neuronal NO synthase. Biochemistry. 1996 Feb 27;35(8):2804-2810. https://doi.org/10.1021/bi9520444
Galli, Chris ; MacArthur, Ryan ; Abu-Soud, Husam M. ; Clark, Pamela ; Stuehr, Dennis J. ; Brudvig, Gary W. / EPR spectroscopic characterization of neuronal NO synthase. In: Biochemistry. 1996 ; Vol. 35, No. 8. pp. 2804-2810.
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