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首页> 外文期刊>Biochemistry >EPR SPECTROSCOPIC CHARACTERIZATION OF NEURONAL NO SYNTHASE
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EPR SPECTROSCOPIC CHARACTERIZATION OF NEURONAL NO SYNTHASE

机译：EPR光谱学表征神经元合酶

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摘要

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-omega-nitro-L-arginine methyl ester (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 indic;lte 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.
机译：神经元NO合酶（nNOS）由结合FAD，FMN，NADPH和钙调蛋白的还原酶结构域，以及结合血红素，四氢生物蝶呤和底物L-精氨酸的加氧酶结构域组成。静止的nNOS中的一种黄素以空气稳定的半醌自由基形式存在。在NO合成过程中，黄素与血红素铁之间发生电子转移。我们已经通过电子顺磁共振（EPR）光谱表征了nNOS血红素铁和黄素半醌自由基。在厌氧条件下，黄素自由基的自旋弛豫非常慢（在22 K时为8 Hz），并通过自旋-自旋耦合通过溶解的双氧而增强了13倍。黄素自由基，可能是半醌FMNH（。），通过在厌氧条件下逐步进行的微波功率饱和和EPR饱和恢复显示与位于nNOS加氧酶域中的血红素铁自旋旋转偶联。对不含血红素但含有黄素自由基的nNOS制剂的分析显示，自旋-自旋偶联将黄素自由基的松弛速率提高了15倍。结合的底物（L-精氨酸）或底物类似物N- ω-硝基-L-精氨酸甲酯（NAME）对黄素自旋弛豫动力学没有影响。观察到的nNOS血红素的g值为7.68、4.15和1.81，并且由于L-精氨酸或NAME占据了底物结合位点而没有变化。基质对血红素零场分裂参数D = 5.2 cm（-1）也没有影响。总之，数据表明黄素和血红素氧化还原中心在nNOS中彼此靠近，这与它们参与域间电子转移一致。黄素自由基受溶解氧的影响，表明其还原酶结构域内的结合位点部分暴露于溶剂，但当底物与加氧酶结构域结合时不受影响。底物结合似乎也发生在nNOS血红素铁的第一个配位壳之外。

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