Characterization of the active site of galactose oxidase and its active site mutational variants Y495F/H/K and W290H by circular dichroism spectroscopy

摘要

Circular dichroism spectroscopy (CD) has been used to investigate the generation of the tyrosine radical in wild-type galactose oxidase and the active site variants Y495F/H/K and W290H. Oxidation was observed in all the variants except Y495K and the radical was noted to have a greater stability at pH 4.6 compared to pH 7.0, especially in Y495H and W290H. In the axial tyrosine variants active site oxidation to generate the radical species was confirmed by the presence of characteristic CD bands, particularly a negative band, in the 350 to 450 nm region. The band at 810 nm in the optical absorption spectrum of WT-GO is absent in oxidized Y495 variants consistent with the Y495 --> Y272 via Cu(II) d(xz) assignment (M.L. McGlashen, D.D. Eads, T.G. Spiro and J.W. Whittaker, J. Phys. Chem., 99 (1995) 4918-922 [1]). CD spectra of either oxidized or semi-reduced proteins are pH-dependent between pH 4.6 and 7.0 with differing intensities and dispersions. The presence of a positive CD band between 309 and 321 nm (N(pi) --> Cu(II)) confirmed the coordination of histidine to the copper ion in the variants studied here. The slight wavelength and intensity shifts seen in this transition is ascribed to perturbation of coupling of the dissymmetric environment to the electronic transitions of the copper site. (C) 1998 Elsevier Science S.A. All rights reserved. [References: 21]