Role of the Axial Ligand in Type 1 Cu Centers Studied by Point Mutations of Met148 in Rusticyanin
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- 作者：John F. Hall ; Lalji D. Kanbi ; Richard W. Strange ; and S. Samar Hasnain
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：September 28, 1999
- 年：1999
- 卷：38
- 期：39
- 页码：12675 - 12680
- 全文大小：186K
- 年卷期：v.38,no.39(September 28, 1999)
- ISSN：1520-4995
文摘
Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize thesame trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur.In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of theseaxial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuningthe redox potential, spectroscopic properties, and rack-induced or entatic state properties of the coppersites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presenceof an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of thesesubstitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys,and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpointredox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidizedproteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH valuesbetween 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR propertiesof this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra inthis pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture oftype 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential(~800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site ofceruloplasmin where this axial ligand is also a Leu.