Spectroscopic and Mechanistic Studies of Type-1 and Type-2 Copper Sites in Pseudomonas aeruginosa Azurin As Obtained by Addition of External Ligands to Mutant His46Gly

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• 作者：Gertie van Pouderoyen ; Colin R. Andrew ; Thomas M. Loehr ; Joann Sanders-Loehr ; Shyamalava Mazumdar ; H. Allen O. Hill ; and Gerard W. Canters
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：February 6, 1996
• 年：1996
• 卷：35
• 期：5
• 页码：1397 - 1407
• 全文大小：783K
• 年卷期：v.35,no.5(February 6, 1996)
• ISSN：1520-4995

文摘

The spectroscopic and mechanistic properties of the Cu-containingactive site of azurin fromPseudomonas aeruginosa were investigated by the constructionof a mutant in which one of the ligandsof the metal, His46, was replaced by a glycine. Although themutation creates a hole in the interior ofthe protein, the 3D structure of the protein does not change to anyappreciable extent. However, thespectroscopic (optical, resonance Raman, EPR) properties of the mutantprotein are strongly affected bythe mutation. In the presence of external ligands, the propertiesof the original wild-type protein arerestored to a smaller or larger extent, depending on the ligand.It is concluded that the hole created bythe mutation, even though it is completely buried inside the protein,can be filled by external ligands,often resulting in the creation of a mixture of so-called type-1 andtype-2 copper sites. Also, the redoxproperties (midpoint potential, kinetics of reduction/oxidation)appeared to be strongly affected by themutation and the presence of external ligands. The results arecompared with previous results obtainedon the mutant His117Gly.