Neuronal nitric-oxide synthase mutant (Ser-1412 → Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis
Neuronal nitric-oxide synthase mutant (Ser-1412 → Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis
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DOI：
10.1074/jbc.m006857200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Stuehr, DJ
中科院分区：
文献类型：
--
作者：
Adak, S;Santolini, J;Stuehr, DJ
大鼠神经元型一氧化氮合酶（nNOS）在其还原酶结构域中含有Akt依赖的磷酸化基序。我们突变的目标残基在该网站（Ser-1412天冬氨酸），以模拟磷酸化，然后其特征在于突变体使用常规和停流光谱。与野生型相比，S1412 D nNOS催化细胞色素c和铁氰化物还原的速度更快，但稳态时NO合成的速度更慢，NADPH氧化的解偶联程度更高。为了理解这些行为如何与黄素和血红素还原速率相关，我们利用了三种大豆钙调蛋白（CaMs），其支持突变体和野生型nNOS中的一系列较慢的黄素和血红素还原速率，还原酶活性和两个催化参数（血红素-NO复合物形成的速度和量）直接与黄素和血红素还原的速度相关。相反，稳态NO合成增加，达到一个平台，然后下降，在最高速率的血红素还原，得到S1412 D nNOS +钙调素。用大豆钙调素替代减缓血红素还原和增加稳态NO合成的突变体。我们得出以下结论。1)S1412 D突变加速了还原酶结构域的电子转移。2)更快的血红素还原速度内在的NO合成，但减少NO释放在稳定状态。3)血红素减少显示在稳态期间关于NO释放的最佳值。S1412 D nNOS的独特行为揭示了血红素还原速率在控制稳态活性中的重要性，并表明nNOS已经具有接近最佳的血红素还原速率。
Rat neuronal NO synthase (nNOS) contains an Akt-dependent phosphorylation motif in its reductase domain. We mutated a target residue in that site (Ser-1412 to Asp) to mimic phosphorylation and then characterized the mutant using conventional and stopped-flow spectroscopies. Compared with wild-type, S1412D nNOS catalyzed faster cytochrome c and ferricyanide reduction but displayed slower steady-state NO synthesis with greater uncoupling of NADPH oxidation, Paradoxically, the mutant had faster heme reduction, faster heme-NO complex formation, and greater heme-NO complex accumulation at steady state. To understand how these behaviors related to flavin and heme reduction rates, we utilized three soybean calmodulins (CaMs) that supported a range of slower flavin and heme reduction rates in mutant and wild-type nNOS, Reductase activity and two catalytic parameters (speed and amount of heme-NO complex formation) related directly to the speed of flavin and heme reduction. In contrast, steady-state NO synthesis increased, reached a plateau, and then fell at the highest rate of heme reduction that was obtained with S1412D nNOS + CaM. Substituting with soybean CaM slowed heme reduction and increased steady-state NO synthesis by the mutant. We conclude the following. 1) The S1412D mutation speeds electron transfer out of the reductase domain. 2) Faster heme reduction speeds intrinsic NO synthesis but diminishes NO release in the steady state. 3) Heme reduction displays an optimum regarding NO release during steady state. The unique behavior of S1412D nNOS reveals the importance of heme reduction rate in controlling steady-state activity and suggests that nNOS already has a near-optimal rate of heme reduction.