Spectral properties of “non-blue” cupric copper in proteins. Circular dichroism and optical spectra of galactose oxidase

Galactose oxidase is a protein which contains a single cupric copper atom per molecule. Concentrated solutions of galactose oxidase are a light, murky green. Circular dichroism and optical spectra were obtained for galactose oxidase in order to compare the spectral properties of a “nonblue” Cu^2-in a protein to those reported for “blue” Cu²⁺in proteins. Unusually large copper optical activity of galactose oxidase solutions is exhibited by circular dichroism extrema at 314, 395, 500, and 610 nm with molecular ellipticities equal to + 18.9 × 10³, +3.0 × 10³, +1.5 × 10³, and-8.2 (deg cm²)/dmol, respectively. The optical spectrum contains three broad overlapping bands centered at 445, 630, and 775 nm with molar extinction coefficients equal to 1155, 1015, and 905, respectively. An absorbance transition corresponding to the 314-nm circular dichroism extremum was unmasked by recording the direct difference spectrum between the holoand apoenzymes. Consideration of the optical and circular dichroism spectra suggests that there is ligand orbital and/or charge-transfer character to one or more of the detected transitions since five rather than four maximally allowed d-d transitions are observed. As expected, the molar absorptivity near 600 nm is somewhat lower for “non-blue” Cu²⁺than for “blue” Cu²⁺in proteins. In addition, the “non-blue” Cu²⁺in proteins exhibits an absorption transition below 400 nm that has not been detected in the spectra of “blue” Cu²⁺. Analysis of the far-ultraviolet circular dichroism spectrum suggests that galactose oxidase contains a small amount of a helix, but that its peptide bonds are principally in unordered conformations. The presence of a positive peak at 292 nm in the apo-holoenzyme optical difference spectrum suggests that a tryptophan residue or residues is in a more hydrophobic environment in the apoenzyme than in the holoenzyme.