Ilagan Robielyn P, Tejero Jesús, Aulak Kulwant S, Ray Sougata Sinha, Hemann Craig, Wang Zhi-Qiang, Gangoda Mahinda, Zweier Jay L, Stuehr Dennis J
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Biochemistry. 2009 May 12;48(18):3864-76. doi: 10.1021/bi8021087.
Nitric oxide synthases (NOS) are modular, calmodulin- (CaM-) dependent, flavoheme enzymes that catalyze oxidation of l-arginine to generate nitric oxide (NO) and citrulline. During catalysis, the FMN subdomain cycles between interaction with an NADPH-FAD subdomain to receive electrons and interaction with an oxygenase domain to deliver electrons to the NOS heme. This process can be described by a three-state, two-equilibrium model for the conformation of the FMN subdomain, in which it exists in two distinct bound states (FMN-shielded) and one common unbound state (FMN-deshielded). We studied how each partner subdomain, the FMN redox state, and CaM binding may regulate the conformational equilibria of the FMN module in rat neuronal NOS (nNOS). We utilized four nNOS protein constructs of different subdomain composition, including the isolated FMN subdomain, and determined changes in the conformational state by measuring the degree of FMN shielding by fluorescence, electron paramagnetic resonance, or stopped-flow spectroscopic techniques. Our results suggest the following: (i) The NADPH-FAD subdomain has a far greater capacity to interact with the FMN subdomain than does the oxygenase domain. (ii) CaM binding has no direct effects on the FMN subdomain. (iii) CaM destabilizes interaction of the FMN subdomain with the NADPH-FAD subdomain but does not measurably increase its interaction with the oxygenase domain. Our results imply that a different set point and CaM regulation exists for either conformational equilibrium of the FMN subdomain. This helps to explain the unique electron transfer and catalytic behaviors of nNOS, relative to other dual-flavin enzymes.
一氧化氮合酶（NOS）是模块化的、钙调蛋白（CaM）依赖性的黄素血红素酶，可催化L-精氨酸氧化生成一氧化氮（NO）和瓜氨酸。在催化过程中，FMN亚结构域在与NADPH-FAD亚结构域相互作用以接收电子和与加氧酶结构域相互作用以将电子传递至NOS血红素之间循环。此过程可用FMN亚结构域构象的三态双平衡模型来描述，其中它以两种不同的结合状态（FMN屏蔽）和一种常见的未结合状态（FMN去屏蔽）存在。我们研究了每个伙伴亚结构域、FMN氧化还原状态和CaM结合如何调节大鼠神经元NOS（nNOS）中FMN模块的构象平衡。我们利用了四种不同亚结构域组成的nNOS蛋白构建体，包括分离的FMN亚结构域，并通过荧光、电子顺磁共振或停流光谱技术测量FMN屏蔽程度来确定构象状态的变化。我们的结果表明：（i）NADPH-FAD亚结构域与FMN亚结构域相互作用的能力比加氧酶结构域大得多。（ii）CaM结合对FMN亚结构域没有直接影响。（iii）CaM使FMN亚结构域与NADPH-FAD亚结构域的相互作用不稳定，但并未显著增加其与加氧酶结构域的相互作用。我们的结果表明，FMN亚结构域的构象平衡存在不同的设定点和CaM调节。这有助于解释nNOS相对于其他双黄素酶独特的电子转移和催化行为。
