Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD⁺- and NAD⁺/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.

蛋白S-亚硝基化在细胞信号传导中起着基本作用，亚硝基谷胱甘肽（GSNO）被认为是主要的亚硝基化信号分子。因此，控制GSNO稳态的酶系统对于间接控制蛋白S-亚硝基硫醇的形成至关重要。GSNO还原酶（GSNOR）是在NADH存在下通过催化其降解来控制GSNO水平的关键酶。在这里，我们发现从微藻蛋白质提取物的莱茵衣藻分解代谢GSNO通过两种酶系统具有特定的NADPH或NADH活性和不同的生化特征。根据衣藻衣原体基因组的已知植物GSNOR直系同源物评分，我们发现了两个编码推定且几乎相同的GSNOR同工酶的基因。异源表达和纯化了两个，其中一个称为CrGSNOR1。确定了它的动力学性质，并确定了载脂蛋白，NAD ⁺和NAD ⁺的三维结构/ GSNO形式已解决。这些分析表明，CrGSNOR1对GSNO和NADH具有严格的特异性，并且相对于其他植物GSNOR具有保守的折叠性。然而，催化锌离子显示出配位环境的意外变化。此外，我们评估了CrGSNOR1对热变性，硫醇修饰剂和氧化修饰的催化响应以及可及的半胱氨酸的反应性和位置。尽管是富含半胱氨酸的蛋白质，CrGSNOR1仅包含两个溶剂暴露/反应性半胱氨酸。氧化和亚硝基化处理对CrGSNOR1的活性和折叠没有或仅有有限的影响，突出了藻类酶对氧化还原修饰的一定抵抗力。