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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity
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A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity

机译：保守的黄素保护残基调节NO合酶的电子转移和烟酰胺辅酶的特异性

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摘要

Nitric oxide synthases (NOSs) are f lavoheme enzymes that contain a ferredoxin:NADP~+-reductase (FNR) module for binding NADPH and FAD and are unusual because their electron transfer reactions are controlled by the Ca~(2+)-binding protein calmodulin. A conserved aromatic residue in the FNR module of NOS shields the isoalloxazine ring of FAD and is known to regulate NADPH binding affinity and specificity in related flavoproteins. We mutated Phe-1395 (F1395) in neuronal NOS to Tyr and Ser and tested their effects on nucleotide coenzyme specificity, catalytic activities, and electron transfer in the absence or presence of calmodulin. We found that the aromatic side chain of F1395 controls binding specificity with respect to NADH but does not greatly affect affinity for NADPH. Measures of flavin and heme reduction kinetics, ferricya-nide and cytochrome c reduction, and NO synthesis established that the aromatic side chain of F1395 is required to repress electron transfer into and out of the flavins of neuronal NOS in the calmodulin-free state, and is also required for calmodulin to fully relieve this repression. We speculate that the phenyl side chain of F1395 is part of a conformational trigger mechanism that negatively or positively controls NOS electron transfer depending on the presence of calmodulin. Such use of the conserved aromatic residue broadens our understanding of flavoprotein structure and regulation.
机译：一氧化氮合酶（NOSs）是一种黄素血红素酶，其包含铁氧还蛋白：NADP〜+-还原酶（FNR）模块以结合NADPH和FAD，并且由于它们的电子转移反应受Ca〜（2+）结合蛋白控制而异常。钙调蛋白。 NOS的FNR模块中的保守芳香族残基保护FAD的异恶嗪环，并已知其调节相关黄素蛋白中的NADPH结合亲和力和特异性。我们将神经元NOS中的Phe-1395（F1395）突变为Tyr和Ser，并测试了它们在不存在或存在钙调蛋白的情况下对核苷酸辅酶特异性，催化活性和电子转移的影响。我们发现，F1395的芳香族侧链控制着相对于NADH的结合特异性，但对NADPH的亲和力没有太大影响。黄素和血红素还原动力学，铁氰化物和细胞色素c还原以及NO合成的测量方法确定，需要F1395的芳香族侧链来抑制电子在无钙调蛋白状态下进出神经元NOS的黄素的转移，以及还需要钙调蛋白来完全缓解这种抑制。我们推测F1395的苯基侧链是构象触发机制的一部分，该构象根据钙调蛋白的存在而负向或正向控制NOS电子转移。保守芳香族残基的这种使用拓宽了我们对黄素蛋白结构和调节的理解。

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