The Role of Tyr41 and His155 in the Functional Properties of Superoxide Dismutase from the Archaeon Sulfolobus solfataricus

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• 作者：Maria Angela Gogliettino ; Fabio Tanfani ; Ancrea Sciré ; Thomas Ursby ; Bianca Stella Adinolfi ; Tiziana Cacciamani ; and Emmanuele De Vendittis
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：March 2, 2004
• 年：2004
• 卷：43
• 期：8
• 页码：2199 - 2208
• 全文大小：210K
• 年卷期：v.43,no.8(March 2, 2004)
• ISSN：1520-4995

文摘

We have examined and compared the effects of mutating Y41 and H155 in the iron superoxidedismutase (SOD) from the archaeon Sulfolobus solfataricus (Ss). These two neighboring residues in theactive site are known to have crucial functions in structurally related SODs from different sources. Themetal analysis indicates a slightly lower iron content after either Y41F or H155Q replacement, withoutany significant substitution of iron for manganese. The specific activity of SsSOD referred to the ironcontent is 17-fold reduced in the Y41F mutant, whereas it is less than 2-fold reduced by the H155Qmutation. The noticeable pH dependence of the activity of SsSOD and H155Q-SsSOD, due to the ionizationof Y41 (pK 8.4), is lost in Y41F-SsSOD. After H155Q and even more after the Y41F substitution, thearchaeal enzyme acquires a moderate sensitivity to sodium azide inhibition. The hydrogen peroxideinactivation of SsSOD is significantly increased after H155Q replacement; however, both mutants areinsensitive to the modification of residue 41 by phenylmethanesulfonyl fluoride. Heat inactivation studiesshowed that the high stability of SsSOD is reduced by the H155Q mutation; hovewer, upon the additionof 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 transforminfrared spectroscopy; these studies gave information about the effect of mutations and modification onflexibility and compactness of the protein structure. The crystal structure of Y41F mutant revealed anuninterrupted hydrogen bond network including three solvent molecules connecting the iron-ligatinghydroxide ion via H155 with F41 and H37, which is not present in structures of the corresponding mutantSODs from other sources. These data suggest that Y41 and H155 are important for the structural andfunctional properties of SsSOD; in particular, Y41 seems to be a powerful regulator of the activity ofSsSOD, whereas H155 is apparently involved in the organization of the active site of the enzyme.