Gogliettino Maria Angela, Tanfani Fabio, Sciré Ancrea, Ursby Thomas, Adinolfi Bianca Stella, Cacciamani Tiziana, De Vendittis Emmanuele
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini, 5, I-80131 Naples, Italy.
Biochemistry. 2004 Mar 2;43(8):2199-208. doi: 10.1021/bi035661y.
We have examined and compared the effects of mutating Y41 and H155 in the iron superoxide dismutase (SOD) from the archaeon Sulfolobus solfataricus (Ss). These two neighboring residues in the active site are known to have crucial functions in structurally related SODs from different sources. The metal analysis indicates a slightly lower iron content after either Y41F or H155Q replacement, without any significant substitution of iron for manganese. The specific activity of SsSOD referred to the iron content is 17-fold reduced in the Y41F mutant, whereas it is less than 2-fold reduced by the H155Q mutation. The noticeable pH dependence of the activity of SsSOD and H155Q-SsSOD, due to the ionization of Y41 (pK 8.4), is lost in Y41F-SsSOD. After H155Q and even more after the Y41F substitution, the archaeal enzyme acquires a moderate sensitivity to sodium azide inhibition. The hydrogen peroxide inactivation of SsSOD is significantly increased after H155Q replacement; however, both mutants are insensitive to the modification of residue 41 by phenylmethanesulfonyl fluoride. Heat inactivation studies showed that the high stability of SsSOD is reduced by the H155Q mutation; however, upon the addition of SDS, a much faster inactivation kinetics is observed both with wild-type and mutant SsSOD forms. The detergent is also required to follow thermal denaturation of the archaeal enzyme by Fourier transform infrared spectroscopy; these studies gave information about the effect of mutations and modification on flexibility and compactness of the protein structure. The crystal structure of Y41F mutant revealed an uninterrupted hydrogen bond network including three solvent molecules connecting the iron-ligating hydroxide ion via H155 with F41 and H37, which is not present in structures of the corresponding mutant SODs from other sources. These data suggest that Y41 and H155 are important for the structural and functional properties of SsSOD; in particular, Y41 seems to be a powerful regulator of the activity of SsSOD, whereas H155 is apparently involved in the organization of the active site of the enzyme.
我们研究并比较了古菌嗜热栖热菌（Ss）铁超氧化物歧化酶（SOD）中Y41和H155突变的影响。已知活性位点中的这两个相邻残基在来自不同来源的结构相关SOD中具有关键功能。金属分析表明，Y41F或H155Q替换后铁含量略有降低，没有任何铁被锰显著替代的情况。以铁含量为参照，Y41F突变体中SsSOD的比活性降低了17倍，而H155Q突变使其降低不到2倍。由于Y41（pK 8.4）的电离，SsSOD和H155Q-SsSOD活性明显的pH依赖性在Y41F-SsSOD中消失。H155Q替换后，尤其是Y41F替换后，古菌酶对叠氮化钠抑制有中等敏感性。H155Q替换后，SsSOD的过氧化氢失活显著增加；然而，两个突变体对苯甲磺酰氟对41位残基的修饰均不敏感。热失活研究表明，H155Q突变降低了SsSOD的高稳定性；然而，加入SDS后，野生型和突变型SsSOD形式均观察到更快的失活动力学。去污剂也是通过傅里叶变换红外光谱跟踪古菌酶热变性所必需的；这些研究提供了有关突变和修饰对蛋白质结构灵活性和紧凑性影响的信息。Y41F突变体的晶体结构揭示了一个不间断的氢键网络，包括三个溶剂分子，它们通过H155将铁配位的氢氧根离子与F41和H37连接起来，这在其他来源的相应突变型SOD结构中不存在。这些数据表明，Y41和H155对SsSOD的结构和功能特性很重要；特别是，Y41似乎是SsSOD活性的有力调节剂，而H155显然参与了酶活性位点的组织。
