New Type 2 Copper−Cysteinate Proteins. Copper Site Histidine-to-Cysteine Mutants of Yeast Copper−Zinc Superoxide Dismutase

Abstract

Preparation and characterization of two new site-directed mutant copper-zinc superoxide dismutase proteins from Saccharomyces cerevisiae, i.e., His46Cys (H46C) and His120Cys (H120C), in which individual histidyl ligands in the copper-binding site were replaced by cysteine, are reported here. These two mutant CuZnSOD proteins may be described as type 2 (or normal) rather than type 1 (or blue) copper-cysteinate proteins and are characterized by their yellow rather than blue color, resulting from intense copper-to-sulfur charge transfer bands around 400 nm, their type 2 EPR spectra, with large rather than small nuclear hyperfine interactions, and their characteristic type 2 d-d electronic absorption spectra. An interesting difference between these two copper site His-to-Cys mutations is that the imidazolate bridge between the two metal sites that is characteristic of the wild-type protein remains intact in the case of the H46C mutant but is not present in the case of the H120C mutant.

A New Type 2 Copper Cysteinate Azurin

“…1A) and is characteristic of a type 2 copper site. It can be assigned as a RS Ϫ Ϫ Cu(II) 3 * band (15)(16)(17)(18). These features are strikingly different from the spectroscopic characteristics of Cu-H117G azurin mentioned above (Fig.…”

“…The first step is very fast: under pseudo-first order conditions an intermediate is formed within 10 ms with an absorption maximum at 385 nm, indicative of a RS Ϫ Ϫ Cu(II) 3 * band in a Cu(II)-thiolate center with a tetragonal geometry (15)(16)(17). The formation of such an intermediate has been observed during the incorporation of copper in a Cu A azurin (45).…”

Cloning and sequencing the genomic encoding region of copper–zinc superoxide dismutase enzyme from several marine strains of the genus Debaryomyces (Lodder & Kreger‐van Rij)

“…As was mentioned above, this is not the first report of an incomplete 'histidine package' in a CuZnSOD sequence; in the SODC2 of D. hansenii, the three absent His amino acids are apparently replaced by Tyr. Although evidence (amino acid composition of purified proteins, cloned sequences from DNA, and specific activity observed on native PAGE) indicates that the linking function of His could be replaced by other amino acids, it is necessary to do sitedirected mutation and other analyses (including biological activity, internal peptide sequencing, amino acid content determination from purified proteins, and sequencing of cloned coding sequences) to confirm the replaced and functional roles of unusual amino acids in the active site conformation, although successful replacements have been reported, especially for the corresponding His 46 , strongly related with Cu 2+ binding (Lu et al, 1996). Finally, Cys 57 and Cys 146 , related to the tertiary spatial conformation, remain in the correct positions in all sequences analysed.…”

“…Other researchers prepared two site-directed mutant CuZnSOD proteins from S. cerevisiae, in which individual histidyl ligands in the copper-binding site were replaced by cysteine (H46C and H120C). These two mutant SODC proteins were characterized by their yellow colour (Lu et al, 1996). An interesting difference between these two copper site His-to-Cys mutations is that the imidazolate bridge between the two metal sites (which is characteristic of the wild-type protein) remains intact in the case of the H46C mutant but is not present in the case of the H120C mutant.…”

Electrophoretic Studies of Binary and Ternary Complexes of Some Metal Ions with Alizarin

“…It is well known that the ease of formation of a complex with dye, thus stability of the complex decides its applicatory importance. Zn(II),Cu(II),Ni(II) and Cr(III) are well known for their biomedical applications and play essential role in different physiological processes of the body [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] In recent years a number of papers have described paper electrophoresis for the study of complexation reactions in solution [30][31][32][33][34][35][36] .…”