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首页> 外文期刊>Biochemistry >Interactions between substrate analogues and heme ligands in nitric oxide synthase.
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Interactions between substrate analogues and heme ligands in nitric oxide synthase.

机译：一氧化氮合酶中底物类似物和血红素配体之间的相互作用。

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The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N(omega)-hydroxy L-arginine as well as many substrate analogues and inhibitors. Resonance Raman spectra of carbon monoxide-bound NOS were measured to determine how these substrates and analogues interact with heme, the prosthetic group which activates oxygen for the catalytic generation of NO and citrulline from arginine in the enzyme. Two distinct conformations of the Fe-C-O moiety were detected in the resonance Raman spectra, although in the optical absorption spectra the two species are indistinguishable. In one, termed the beta-form, the Fe-CO stretching frequency and the C-O stretching frequency, located at approximately 487 and approximately 1949 cm(-1), respectively, demonstrate that the Fe-C-O group adopts a linear conformation perpendicular to the heme plane ("open" structure). In the other, termed the alpha-form, frequencies of approximately 502 and approximately 1929 cm(-1),respectively, indicate that the binding properties of bound CO are significantly affected by its immediate environment thereby leading to a "closed" structure. In the presence of L-arginine or N(omega)-OH-L-arginine all of the molecules exhibit the closed structure, indicating that the substrates exert a strong polar (and/or steric) effect on the heme-bound ligand. In the absence of any substrate or inhibitor only half of the heme population adopts the open structure whereas the rest of the heme content retains the closed conformation. It is proposed that in this population with the closed structure tetrahydrobiopterin, a cofactor of NO synthase, may reside in close proximity to the heme-bound ligand and interact with it in a similar manner as do substrates. The inverse correlation between the Fe-CO and C-O stretching modes suggests that in NOS the bonding of the cysteine to the heme iron may be weaker, as found in chloroperoxidase, than in cytochrome P-450 enzymes. This work continually proves resonance Raman spectroscopy as a powerful probe for the interactions between substrate/inhibitor and the heme active site of proteins.
机译：一氧化氮合酶（NOS）中的底物结合位点可以容纳生理底物L-精氨酸和N（ω）-羟基L-精氨酸以及许多底物类似物和抑制剂。测量了与一氧化碳结合的NOS的共振拉曼光谱，以确定这些底物和类似物如何与血红素相互作用，血红素是激活氧以从酶中的精氨酸催化生成NO和瓜氨酸的辅基。在共振拉曼光谱中检测到Fe-C-O部分的两种不同构象，尽管在光吸收光谱中这两种没有区别。在一个称为β-形式的Fe-CO拉伸频率和CO拉伸频率分别位于约487 cm（-1）和1949 cm（-1）处，表明Fe-CO基团采用垂直于血红素平面（“开放”结构）。在另一种称为“α-形式”的频率中，大约502和大约1929 cm（-1）的频率分别表示所结合的CO的结合特性受其直接环境的影响很大，从而导致“封闭”结构。在存在L-精氨酸或N（ω）-OH-L-精氨酸的情况下，所有分子均显示出封闭结构，这表明底物对血红素结合的配体施加强极性（和/或空间）作用。在没有任何底物或抑制剂的情况下，只有一半的血红素群体采用开放结构，而其余的血红素含量则保持封闭构象。有人提出，在具有闭合结构的四氢生物蝶呤（NO合酶的辅助因子）的这一种群中，它可能紧邻血红素结合的配体并以与底物相似的方式与其相互作用。 Fe-CO和C-O拉伸模式之间的负相关关系表明，在NOS中，如在氯过氧化物酶中发现的，半胱氨酸与血红素铁的键合可能比在细胞色素P-450酶中的弱。这项工作不断证明共振拉曼光谱法是检测底物/抑制剂与蛋白质血红素活性位点之间相互作用的有力探针。

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