NEW TYPE-2 COPPER-CYSTEINATE PROTEINS - COPPER SITE HISTIDINE-TO-CYSTEINE MUTANTS OF YEAST COPPER-ZINC SUPEROXIDE-DISMUTASE

Preparation and characterization of two new site-directed mutant coppe r-zinc superoxide dismutase proteins from Saccharomyces cerevisiae, i. e., His46Cys (H46C) and His120Cys (H120C), in which individual histidy l ligands in the copper-binding site were replaced by cysteine, are re ported here. These two mutant CuZnSOD proteins may be described as typ e 2 (or normal) rather than type 1 (or blue) copper-cysteinate protein s and are characterized by their yellow rather than blue color, result ing from intense copper-to-sulfur charge transfer bands around 400 Mn, their type 2 EPR spectra, with large rather than small nuclear hyperf ine interactions, and their characteristic type 2 d-d electronic absor ption spectra. An interesting difference between these two copper site His-to-Cys mutations is that the imidazolate bridge between the two m etal sites that is characteristic of the wild-type protein remains int act in the case of the H46C mutant but is not present in the case of t he H120C mutant.