, Faculty of Chemistry, Jagiellonian University, 30-387 Krakow, Poland.
Polish Academy of Sciences, Jerzy Haber Institute of Catalysis and Surface Chemistry, 30-239 Krakow, Poland.
Inorg Chem. 2023 Apr 10;62(14):5630-5643. doi: 10.1021/acs.inorgchem.3c00180. Epub 2023 Mar 30.
Conversion of NO to stable -nitrosothiols is perceived as a biologically important strategy of NO storage and a signal transduction mechanism. Transition-metal ions and metalloproteins are competent electron acceptors that may promote the formation of -nitrosothiols from NO. We selected -acetylmicroperoxidase (AcMP-11), a model of protein heme centers, to study NO incorporation to three biologically relevant thiols (glutathione, cysteine, and -acetylcysteine). The efficient formation of -nitrosothiols under anaerobic conditions was confirmed with spectrofluorimetric and electrochemical assays. AcMP-11-assisted incorporation of NO to thiols occurs via an intermediate characterized as an -coordinated -nitrosothiol, (AcMP-11)Fe(N(O)SR), which is efficiently converted to (AcMP-11)Fe(NO) in the presence of NO excess. Two possible mechanisms of -nitrosothiol formation at the heme-iron were considered: a nucleophilic attack on (AcMP-11)Fe(NO) by a thiolate and a reaction of (AcMP-11)Fe(RS) with NO. Kinetic studies, performed under anaerobic conditions, revealed that the reversible formation of (AcMP-11)Fe(N(O)SR) occurs in a reaction of RS with (AcMP-11)Fe(NO) and excluded the second mechanism, indicating that the formation of (AcMP-11)Fe(RS) is a dead-end equilibrium. Theoretical calculations revealed that -coordination of RSNO to iron, forming (AcMP-11)Fe(N(O)SR), shortens the S-N bond and increases the complex stability compared to -coordination. Our work unravels the molecular mechanism of heme-iron-assisted interconversion of NO and low-molecular-weight thiols to -nitrosothiols and recognizes the reversible NO binding in the form of a heme-Fe(N(O)SR) motif as an important biological strategy of NO storage.
NO 向稳定的 - 亚硝基硫醇的转化被认为是 NO 储存和信号转导机制的重要生物学策略。过渡金属离子和金属蛋白是有能力的电子受体，可能促进 NO 形成 - 亚硝基硫醇。我们选择 - 乙酰基微过氧化物酶（AcMP-11），一种蛋白质血红素中心的模型，来研究三种生物相关的巯基（谷胱甘肽、半胱氨酸和 - 乙酰半胱氨酸）与 NO 的结合。在厌氧条件下，通过荧光光谱法和电化学分析证实了 - 亚硝基硫醇的高效形成。AcMP-11 辅助的 NO 与硫醇的结合通过一个中间体进行，该中间体被表征为 - 配位的 - 亚硝基硫醇（（AcMP-11）Fe（N（O）SR），在过量 NO 的存在下，该中间体有效地转化为（AcMP-11）Fe（NO）。在血红素铁上形成 - 亚硝基硫醇有两种可能的机制：一个硫醇盐对（AcMP-11）Fe（NO）的亲核攻击和（AcMP-11）Fe（RS）与 NO 的反应。在厌氧条件下进行的动力学研究表明，RS 与（AcMP-11）Fe（NO）的反应可逆地形成（AcMP-11）Fe（N（O）SR），排除了第二种机制，表明（AcMP-11）Fe（RS）的形成是一个死端平衡。理论计算表明，RSNO 与铁的 - 配位，形成（AcMP-11）Fe（N（O）SR），缩短了 S-N 键并增加了与 - 配位相比的配合物稳定性。我们的工作揭示了血红素铁辅助 NO 和低分子量硫醇相互转化为 - 亚硝基硫醇的分子机制，并将血红素-Fe（N（O）SR）基序的可逆 NO 结合视为 NO 储存的重要生物学策略。
