The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain
The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain
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DOI：
10.1074/jbc.m400872200
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发表时间：
2004-08-20
影响因子：
4.8
通讯作者：
Stuehr, DJ
中科院分区：
文献类型：
--
作者：
Konas, DW;Zhu, K;Stuehr, DJ
Phe(1395) 与神经元一氧化氮合酶 (nNOS) 中的 FAD 异咯嗪环平行堆叠，是结构相关黄素蛋白中保守芳香族氨基酸的代表。该实验室之前表明，需要 Phe(1395) 才能获得通常在野生型 nNOS 中观察到的电子转移特性和钙调蛋白 (CaM) 响应。在这里，我们表征了 nNOS 黄素蛋白结构域 (nNOSr) 的 F1395S 突变体的物理特性、NADP(+) 结合特征、黄素还原动力学、稳态和稳态前细胞色素 c 还原动力学，以及响应 CaM 或 NADP(H) 结合屏蔽其 FMN 辅因子的能力。 F1395S nNOSr 将 NADP(+) 与 65% 以上的烟酰胺环结合，与 FAD 形成有效构象，用于氢化物转移，并且 NADP(+) 解离速率减慢 8 倍。 CaM 刺激野生型 nNOSr 中 NADPH 依赖性黄素减少的速率，但不刺激 F1395S 突变体，该突变体的黄素减少动力学与不含 CaM 的野生型 nNOSr 相似。无 CaM 的 F1395S nNOSr 缺乏通常在 nNOSr 中观察到的细胞色素 c 还原酶活性的抑制。前稳态和 EPR 实验的综合结果表明，与野生型相比，这与 NADP(+) 结合状态下的 FMN 屏蔽程度较低有关。我们得出结论，Phe(1395) 通过两种方式调节 nNOSr 催化。它促进 NADP(+) 释放以防止该步骤受到限速，并且使 NADP(H) 能够正确调节涉及 FMN 子域的构象平衡，该子域控制 FMN 辅助因子在电子转移中的反应性。
Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP(+) with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP(+) dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP(+)-bound state as compared with wild type. We conclude that Phe(1395) regulates nNOSr catalysis in two ways. It facilitates NADP(+) release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.