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

• Published in: Inorganic chemistry 1996-03, Vol.35 (6), p.1692-1700
• Main Authors: Lu, Yi, Roe, James A, Bender, Christopher J, Peisach, Jack, Banci, Lucia, Bertini, Ivano, Gralla, Edith B, Valentine, Joan Selverstone
• Format: Article
• Language: English
• ISSN: 0020-1669; 1520-510X
• DOI: 10.1021/ic9513189

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.