Wang Zhi-Qiang, Wei Chin-Chuan, Sharma Manisha, Pant Kartikeya, Crane Brian R, Stuehr Dennis J
Department of Immunology, the Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
J Biol Chem. 2004 Apr 30;279(18):19018-25. doi: 10.1074/jbc.M311663200. Epub 2004 Feb 19.
Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.
一氧化氮合酶（NOSs）释放一氧化氮（NO）在很大程度上取决于酶铁血红素-NO产物复合物（Fe(III)NO）的解离。尽管来自枯草芽孢杆菌的类NOS蛋白（bsNOS）从反应中间体N-羟基-L-精氨酸（NOHA）生成Fe(III)NO，但其NO解离速度比哺乳动物NOSs慢约20倍。晶体结构表明，bsNOS血红素口袋附近保守的缬氨酸到异亮氨酸的转换可能决定其动力学特征。为了验证这一点，我们在小鼠诱导型NOS加氧酶结构域（iNOSoxy，V346I）和bsNOS（I224V）中产生了互补突变，并表征了它们从NOHA合成NO的动力学和程度以及它们的NO结合动力学。这些突变并没有显著改变精氨酸、（6R）-四氢生物蝶呤的结合，也没有改变血红素或各种血红素-配体复合物的电子性质。采用停流光谱法研究单周转NOHA反应过程中的血红素转变。I224V bsNOS显示出三种血红素转变，涉及四种物种，这在野生型NOS中通常会出现，即起始的亚铁酶、亚铁-双氧（Fe(II)O(2)）中间体、Fe(III)NO和终末的高铁酶。相对于野生型bsNOS，每种转变的速率都有所增加，其中Fe(III)NO解离速度快3.6倍。在V346I iNOSoxy中，我们依次观察到起始的亚铁、Fe(II)O(2)、Fe(III)NO和高铁血红素物种的混合物以及终末的高铁酶。相对于野生型iNOSoxy，每种转变的速率都降低了，其中Fe(III)NO解离速度慢3倍。对NO结合动力学的独立测量证实，V346I iNOSoxy的NO结合和解离比野生型慢。两个突变体的瓜氨酸生成仅略低于野生型酶，表明偶联良好。我们的数据表明，缬氨酸/异亮氨酸转换导致的血红素口袋更大程度的屏蔽会减慢NOS中的NO合成和NO释放，从而确定了调节这些动力学变量的结构基础。
