Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Biol Chem. 2011 Nov 11;286(45):39224-35. doi: 10.1074/jbc.M111.286351. Epub 2011 Sep 14.
Nitric-oxide synthases (NOS) are heme-thiolate enzymes that generate nitric oxide (NO) from L-arginine. Mammalian and bacterial NOSs contain a conserved tryptophan (Trp) that hydrogen bonds with the heme-thiolate ligand. We mutated Trp(66) to His and Phe (W66H, W66F) in B. subtilis NOS to investigate how heme-thiolate electronic properties control enzyme catalysis. The mutations had opposite effects on heme midpoint potential (-302, -361, and -427 mV for W66H, wild-type (WT), and W66F, respectively). These changes were associated with rank order (W66H < WT < W66F) changes in the rates of oxygen activation and product formation in Arg hydroxylation and N-hydroxyarginine (NOHA) oxidation single turnover reactions, and in the O(2) reactivity of the ferrous heme-NO product complex. However, enzyme ferrous heme-O(2) autoxidation showed an opposite rank order. Tetrahydrofolate supported NO synthesis by WT and the mutant NOS. All three proteins showed similar extents of product formation (L-Arg → NOHA or NOHA → citrulline) in single turnover studies, but the W66F mutant showed a 2.5 times lower activity when the reactions were supported by flavoproteins and NADPH. We conclude that Trp(66) controls several catalytic parameters by tuning the electron density of the heme-thiolate bond. A greater electron density (as in W66F) improves oxygen activation and reactivity toward substrate, but decreases heme-dioxy stability and lowers the driving force for heme reduction. In the WT enzyme the Trp(66) residue balances these opposing effects for optimal catalysis.
一氧化氮合酶（NOS）是一种血红素硫醇酶，它从 L-精氨酸生成一氧化氮（NO）。哺乳动物和细菌 NOS 都含有一个保守的色氨酸（Trp），它与血红素硫醇配体形成氢键。我们在枯草芽孢杆菌 NOS 中突变色氨酸（Trp）66 为组氨酸（His）和苯丙氨酸（Phe）（W66H，W66F），以研究血红素硫醇电子性质如何控制酶催化。突变对血红素中点电势（W66H、野生型（WT）和 W66F 分别为-302、-361 和-427 mV）产生相反的影响。这些变化与 Arg 羟化和 N-羟基精氨酸（NOHA）氧化单轮反应中氧活化和产物形成的速率（W66H < WT < W66F）的顺序变化以及亚铁血红素-NO 产物络合物的 O2 反应性相关。然而，酶亚铁血红素-O2 自氧化显示出相反的顺序。四氢叶酸支持 WT 和突变体 NOS 的 NO 合成。在单轮研究中，所有三种蛋白质在产物形成（L-Arg→NOHA 或 NOHA→瓜氨酸）方面都表现出相似的程度，但当反应由黄素蛋白和 NADPH 支持时，W66F 突变体的活性降低了 2.5 倍。我们得出结论，色氨酸（Trp）66 通过调节血红素硫醇键的电子密度来控制几个催化参数。更高的电子密度（如在 W66F 中）可以改善氧的活化和对底物的反应性，但降低血红素二氧基的稳定性并降低血红素还原的驱动力。在 WT 酶中，色氨酸（Trp）66 残基平衡了这些相反的影响，以实现最佳催化。
