Conformational Studies of Copper Proteins: PSEUDOMONAS BLUE PROTEIN AND POLYPORUS LACCASE

Abstract

Abstract Optical rotatory dispersion and circular dichroism spectra of the Pseudomonas blue protein and laccase B from Polyporus versicolor are presented from 190 to 700 mµ. Circular dichroism spectra of both proteins show six ellipticity bands above 300 mµ associated with the copper chromophore, indicating more transitions to be present in these copper protein complexes than the three parity-forbidden d-d transitions associated with Cu(II) in its common symmetries. A Gaussian analysis of the visible spectra of these proteins is presented. The same set of bands generates both the absorption and the circular dichroism spectra by the adjustment of the signs and the heights of the bands, while keeping the positions and the band widths constant. Below 1.8 x 104 cm-1, both proteins show three bands in the circular dichroism spectrum with signs (+, +, -) for the blue protein and (+, -, +) for laccase B. The uniqueness of the analysis has been established by using the Kronig-Kramers transform to generate the optical rotatory dispersion spectrum of the blue protein. Comparisons are made to the circular dichroism spectra of a Cu(II) complex of known donor atoms and geometry, [Cu(II)(glycyl-l-valine)]. Each protein creates a unique chemical environment for the aromatic residues. The Pseudomonas blue protein shows at least seven aromatic ellipticity bands, both positive and negative ([θ] ∼ ±6 x 103 deg cm2 per decimole), whereas laccase B shows three large positive aromatic bands ([θ] = 1 to 2.7 x 104 deg cm2 per decimole). In the region of the absorption bands of the peptide bonds, the blue protein shows a negative ellipticity band at 222 mµ ([θ] = 4.9 x 103 on a mean residue basis) and a positive band at 196 mµ ([θ] = 1.28 x 104 on a mean residue basis). A prominent negative band is not present at 206 mµ, and the circular dichroism spectrum exhibits a crossover point at 206 mµ. A preliminary analysis based on the computed contours of both the optical rotatory dispersion and circular dichroism spectra indicates a mixture of approximately 40% α-helix, 37% β structure, and 23% random coil. Laccase B shows a negative ellipticity band at 216 mµ ([θ] ∼ 4.1 x 103 on a mean residue basis) and a positive band at 196 mµ ([θ] ∼ 5.5 x 103 on a mean residue basis). Both features fit a β structure; however, the crossover occurs at 204 mµ and the computed curves indicate a mixture of β structure and random coil. The chemical interactions leading to the three-dimensional structure of the blue protein are apparently inherent in the primary chain. Acid denaturation, pH 1.5, destroys the ellipticity bands associated with the peptide bonds, the aromatic residues, and the copper chromophore. All are restored by return of the protein to neutral pH.