Heme distortion modulated by ligand-protein interactions in inducible nitric-oxide synthase
Heme distortion modulated by ligand-protein interactions in inducible nitric-oxide synthase
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DOI：
10.1074/jbc.m400968200
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发表时间：
2004-06-18
影响因子：
4.8
通讯作者：
Rousseau, DL
中科院分区：
文献类型：
--
作者：
Li, D;Stuehr, DJ;Rousseau, DL
一氧化氮合酶（NOS）的催化中心由巯基配位的血红素大环、四氢生物蝶呤（H4 B）辅因子和L-精氨酸（L-Arg）/N-羟基精氨酸底物结合位点组成。为了确定辅因子、底物和容纳血红素的蛋白质基质之间的相互作用如何调节NOS的酶活性，用共振拉曼光谱检查了NOS的诱导型同种型（iNOS（oxy））的加氧酶结构域的CO-、NO-和CN-结合的加合物。CO结合的亚铁蛋白的拉曼数据表明，L-Arg的存在导致Fe-C-O部分由于H-键合相互作用而采用弯曲结构，而H4 B结合不产生影响。在CN-结合铁蛋白和一氧化氮（NO）-结合亚铁蛋白中发现了类似的行为。相比之下，在NO结合的铁络合物中，单独添加L-Arg不影响Fe-N-O部分的结构性质，但H4 B结合迫使其采用弯曲结构，这通过随后添加L-Arg进一步增强。不同的血红素配体和蛋白质基质之间的差分相互作用，响应于L-Arg和/或H4 B结合耦合到血红素扭曲，反映了在低频拉曼光谱的各种面外血红素模式的发展。血红素变形的程度和对称性由配体、底物和辅因子结合调节，可能对酶的催化和自抑制特性提供重要的控制。
The catalytic center of nitric-oxide synthase ( NOS) consists of a thiolate-coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor, and an L-arginine (L-Arg)/N-hydroxyarginine substrate binding site. To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO-, NO-, and CN--bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOS(oxy)) were examined with resonance Raman spectroscopy. The Raman data of the CO- bound ferrous protein demonstrated that the presence of L-Arg causes the Fe-C-O moiety to adopt a bent structure because of an H-bonding interaction whereas H4B binding exerts no effect. Similar behavior was found in the CN--bound ferric protein and in the nitric oxide (NO)-bound ferrous protein. In contrast, in the NO- bound ferric complexes, the addition of L-Arg alone does not affect the structural properties of the Fe-N-O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of L-Arg. The differential interactions between the various heme ligands and the protein matrix in response to L-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra. The extent and symmetry of heme deformation modulated by ligand, substrate, and cofactor binding may provide important control over the catalytic and autoinhibitory properties of the enzyme.