Influences of cysteine residues and hydrogen bond networks on enzyme activity of the persulfide dioxygenase from Acidithiobacillus caldus. Persulfide dioxygenase from A. caldus

Persulfide dioxygenases (PDO) are abundant in Bacteria but are also crucial for H2S detoxification in mitochondria. The pdo-gene of the acidophilic bacterium Acidithiobacillus caldus was expressed in Escherichia coli. The protein (AcPDO) had 0.77±0.1 Fe/subunit and an average specific sulfite formation activity of 111.5 U/mg protein (Vmax) at 40 ˚C and pH 7.5 with sulfur and GSH following a Michaelis-Menten kinetic. Gel permeation chromatography and non-denaturing gels showed mostly tetramers. The temperature optimum was 40-45 ˚C, the melting point 63±1.3 ˚C in thermal unfolding experiments, whereas some activity was measurable up to 95 ˚C.

过硫化物双加氧酶（Persulfide dioxygenases，PDO）广泛存在于细菌中，同时在线粒体硫化氢（H₂S）的解毒过程中发挥关键作用。嗜酸硫杆菌（Acidithiobacillus caldus）的pdo基因在大肠杆菌（Escherichia coli）中实现了异源表达。所得重组蛋白（AcPDO）每亚基结合0.77±0.1个铁原子；在40℃、pH 7.5条件下，以硫与谷胱甘肽（GSH）为底物时，其平均亚硫酸根生成比活可达111.5 U/mg蛋白（Vmax），酶促动力学遵循米氏动力学（Michaelis-Menten kinetics）规律。凝胶过滤色谱与非变性凝胶电泳结果显示，该蛋白主要以四聚体形式存在。该酶的最适反应温度为40~45℃，热变性实验测得其熔点为63±1.3℃，但即便在95℃高温下仍可检测到部分酶活性。