X-RAY-ABSORPTION STUDIES AND HOMOLOGY MODELING DEFINE THE STRUCTURAL FEATURES THAT SPECIFY THE NATURE OF THE COPPER SITE IN RUSTICYANIN

Rusticyanin, a blue copper protein, possessing the highest redox poten tial among this class of proteins and a high stability at acidic pH re veals homology with the C-terminal end of the other single copper cont aining blue proteins and an interesting homology to parts of the blue copper domain of the multi-copper proteins such as the nitrite reducta ses. Extended X-ray absorption fine structure (EXAFS) data at pH 2.0 r eveal that Cu is ligated to two His and a Cys in the inner coordinatio n sphere, similar to other blue copper centers. Modeling studies sugge st that His85 is the ligating histidine from the N-terminal end. Its n eighboring residue is a serine rather than the asparagine found in all known blue Cu proteins. The high stability of the copper site may ari se in part due to this substitution. The Cu binding site is surrounded by aromatic residues which may provide further protection for the met al in an acidic environment. In addition, the high number of solvent-e xposed uncompensated lysine residues is likely to be of functional rel evance under low pH conditions. EXAFS data show a very small change (r elative to azurin) in the copper site upon reduction, consistent with a more constrained copper center in rusticyanin compared to azurin and a higher redox potential.