SPECTROSCOPIC STUDIES OF THE ACTIVE-SITE OF GALACTOSE-OXIDASE

X-ray absorption and EPR spectroscopy have been used to probe the copp er site structure in galactose oxidase at pH 4.5 and 7.0. The results suggest that there are no major differences in the structure of the te tragonal Cu(II) site at these pH values. Analysis of the extended X-ra y absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 Angstrom; these are presumably the equ atorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore result s obtained on the one-electron reduced enzyme, containing Cu(II) but n ot the tyrosine radical, probably also apply to the catalytically acti ve Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor bi nding, and substrate binding have been probed via nuclear magnetic rel axation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a sing le, equatorial water ligand with a Cu(II)-O separation of about 2.4 An gstrom. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effe cts on the NMRD profile of galactose oxidase, indicating that substrat es also bind to the active site Cu(II) in an equatorial position.