A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity
A conserved flavin-shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity
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DOI：
10.1073/pnas.192283399
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发表时间：
2002-10-15
影响因子：
11.1
通讯作者：
Stuehr, DJ
中科院分区：
文献类型：
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作者：
Adak, S;Sharma, M;Stuehr, DJ
一氧化氮合酶（nos）是一种黄素酶，含有铁氧还蛋白：NADP(+)-还原酶（FNR）模块，用于结合NADPH和FAD，并且不常见，因为它们的电子转移反应由Ca2+结合蛋白钙调蛋白控制。NOS的FNR模块中有一个保守的芳香残基，可以屏蔽FAD的异alloxazine环，并调节NADPH与相关黄蛋白的结合亲和力和特异性。我们将神经元NOS中的ph -1395 （F1395）突变为Tyr和Ser，并测试了它们在钙调素缺失或存在时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的芳香侧链控制着NADH的结合特异性，但对NADPH的亲和力没有很大影响。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合成的测量结果表明，在无钙调素状态下，F1395的芳香侧链是抑制神经元NOS黄素电子进出的必要条件，也是钙调素完全解除这种抑制所必需的。我们推测F1395的苯基侧链是构象触发机制的一部分，根据钙调素的存在负或正控制NOS电子转移。这种对保守芳香残基的利用拓宽了我们对黄蛋白结构和调控的理解。
Nitric oxide synthases (NOSs) are flavoheme 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 Ca2+-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, ferricyanide 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.