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.