S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP⁺ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP⁺ and NAD⁺ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.

S-亚硝基化是一种氧化还原翻译后修饰，被广泛认为在细胞信号传导中发挥重要作用，因为它可以调节蛋白质功能和构象。在生理水平上，亚硝基谷胱甘肽 (GSNO) 被认为是主要的生理 NO 释放化合物，因为它能够将 NO 部分转移到蛋白质硫醇上，但调节其氧化还原特异性的结构决定因素尚未完全阐明。在这项研究中，我们利用来自莱茵衣藻(CrGAPA) 的光合甘油醛-3-磷酸脱氢酶 (CrGAPA) 来研究 GSNO 依赖性硫醇氧化的分子机制。我们首先观察到 GSNO 通过诱导S-亚硝基化引起可逆的酶抑制。虽然辅因子 NADP ⁺部分保护酶免受 GSNO 介导的S-亚硝基化作用，但在底物 1,3-二磷酸甘油酸酯存在的情况下未观察到蛋白质抑制，表明催化 Cys149 的S-亚硝基化是 CrGAPA 失活的原因。 CrGAPA 与 NADP ⁺和 NAD ⁺复合物的晶体结构揭示了与其他光合 GAPDH 的总体结构相似性。我们从3D结构开始，进行了分子动力学模拟，以确定参与GSNO结合的蛋白质残基。通过量子力学/分子力学计算研究了 GSNO 与 CrGAPA Cys149 的反应机理，这允许揭示蛋白质残基在调节反式亚硝基化反应的活化势垒中的相对贡献。 基于我们的发现，我们提供了 CrGAPA 对 GSNO 依赖性调节的反应的功能和结构见解，可能将机制特征扩展到易被 GSNO 氧化修饰的其他蛋白质半胱氨酸。