X-ray Analysis and Spectroscopic Characterization of M121Q Azurin

Romero, Antonio; Hoitink, Carla W. G.; Nar, Herbert; Huber, Robert; Messerschmidt, Albrecht; Canters, Gerard W.

doi:10.1006/jmbi.1993.1101

Cited by 178 publications

(235 citation statements)

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“…Sequence alignment [ 121 and molecular modeling studies [13] led Freeman and co-workers to propose that Gin-97 may be the elusive axial ligand. This is consistent with a recent study on the M121Q mutant of azurin, which exhibits spectroscopic features similar to those of stellacyanin [14]. Two other models for St have been proposed, both suggesting that a sulfur atom from a disulfide bridge may be the fourth ligand [l&16].…”

Section: Introductionsupporting

confidence: 90%

“…Blue copper proteins (BCPs) have attracted the attention of researchers from different fields due to their puzzling spectroscopic and functional properties [14]. One of the main questions in the field of BCPs is how the protein framework is able to tune the redox potential of the Cu site, which ranges from 184 mV in stellacyanin to 680 mV in rusticyanin.…”

Section: Introductionmentioning

confidence: 99%

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A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Vila

1994

FEBS Letters

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The 'H NMR spectrum of Co(H) stellacyanin is reported, in which four signals not previously observed have been detected. NOE experiments were performed to assign the hyperfine shifted signals corresponding to a Cys and two His residues. Both His residues are solventaccessible and are shown to bind the metal ion through their NJ1 atoms. The j?-CH, Cys proton shifts indicate the presence of a strong axial ligand.

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Section: Introductionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Vila

1994

FEBS Letters

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“…Stellacyanin contains no methionines. Modelling studies, based in part on amino acid sequence comparisons [20], and site-directed mutagenesis experiments [21] have made it highly likely, that the position normally occupied by a methionine, in stellacyanin is taken up by a glutamine which coordinates with its side chain 0" to the Cu (see Fig. 1).…”

Section: Type 1 Copper Sitesmentioning

confidence: 99%

Engineering type 1 copper sites in proteins

1993

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The use of site-directed mutagenesis methods has revolutionalized the study of the so-called type 1 and type 2 copper sites in proteins. In particular our understanding of the relation between the structure, and the mechanistic and spectroscopic features of these sites is benefitting from the application of these techniques. Recent progress in the field is reviewed with emphasis on the study of type 1 sites. Topics covered comprise the characteristics of the natural type 1 and type 2 sites, the genetics of blue copper proteins, the modification of Cu sites, the spectroscopy of natural and engineered type 1 and type 2 sites, the effect of mutations on midpoint potentials and the mechanism of electron transfer as carried out by the blue copper proteins.

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“…There are some studies on the replacement of the Cu ligands to shed light on the copper site of stellacyanin (16)(17)(18)(19). In the case of azurin, axial ligand substitution (Met121→Gln) causes significant change of the spectroscopic and electrochemical characters (16).…”

mentioning

confidence: 99%

“…In the case of azurin, axial ligand substitution (Met121→Gln) causes significant change of the spectroscopic and electrochemical characters (16). In this study, to elucidate the copper center of mavicyanin, we have synthesized and expressed a gene coding for zucchini mavicyanin, and have prepared the mutant that the putative ligand (Gln95) is replaced with Met by site-directed mutagenesis.…”

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confidence: 99%

Gene Synthesis, Expression, and Mutagenesis of Zucchini Mavicyanin: The Fourth Ligand of Blue Copper Center Is Gln

Kataoka

Nakai

Yamaguchi

et al. 1998

Biochemical and Biophysical Research Communications

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The gene coding for the 109 amino acid, non-glycosylated form of mavicyanin was synthesized and expressed in Escherichia coli. The recombinant protein refolded from E. coli inclusion bodies was purified and characterized. Its spectroscopic properties are fully identical to those of mavicyanin isolated from zucchini, even in the absence of its carbohydrate moiety. The blue cooper center of mavicyanin strongly binds three ligands (2His and Cys) as well as many blue copper proteins. To disclose the fourth ligand of mavicyanin, Met was substituted for Gln95 by site-directed mutagenesis. The replacement changes from a rhombic EPR signal to an axial one and exhibits the quite similar absorption and CD spectra to those of plastocyanin. The midpoint potential of Gln95→Met mavicyanin shows the positive shift of 187 mV compared with the recombinant protein, being close to the values of plastocyanins. The differences of the spectroscopic and electrochemical properties between mavicyanin and its mutant demonstrate that the fourth ligand of mavicyanin is Gln95.

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X-ray Analysis and Spectroscopic Characterization of M121Q Azurin

Cited by 178 publications

References 0 publications

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Engineering type 1 copper sites in proteins

Gene Synthesis, Expression, and Mutagenesis of Zucchini Mavicyanin: The Fourth Ligand of Blue Copper Center Is Gln

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X-ray Analysis and Spectroscopic Characterization of M121Q Azurin

Cited by 178 publications

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A 1H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

A 1H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Engineering type 1 copper sites in proteins

Gene Synthesis, Expression, and Mutagenesis of Zucchini Mavicyanin: The Fourth Ligand of Blue Copper Center Is Gln

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Product

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A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site