EPR spectroscopic characterization of neuronal NO synthase

Biochemistry
C GalliG Brudvig

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 exits 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 substate analogue Nomega-nitro-L-arginine methyl ester (NAME) had no effect on the flavin spin relaxation kinetics. The observed ...Continue Reading

References

Jun 5, 1987·Journal of Molecular Biology·T L PoulosA J Howard

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Citations

Mar 18, 2009·Biochemistry·Robielyn P IlaganDennis J Stuehr
Aug 11, 2010·Journal of the American Chemical Society·Andrei V AstashkinChangjian Feng
May 2, 2009·Journal of the American Chemical Society·Joseph SempombeChangjian Feng
Jun 7, 2012·The Journal of Physical Chemistry. a·Andrei V AstashkinChangjian Feng
Aug 20, 2009·Dalton Transactions : an International Journal of Inorganic Chemistry·Changjian Feng, Gordon Tollin
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Jul 19, 2014·Biochemistry·Miles D Bradshaw, Betty J Gaffney
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Feb 20, 2008·The Journal of Biological Chemistry·Chin-Chuan WeiDennis J Stuehr
Apr 11, 2002·Chemical Reviews·Gerald M RosenSovitj Pou

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