An x-ray absorption near edge structure spectroscopy study of metal coordination in Co(II)-substituted Carcinus maenas hemocyanin

Abstract

High-resolution x-ray absorption near edge structure spectroscopy was used to characterize the metal sites in three different cobalt-substituted derivatives of Carcinus maenas hemocyanin (Hc), including a mononuclear cobalt, a dinuclear cobalt and a copper-cobalt hybrid derivative. Co(II) model complexes with structures exemplifying octahedral, trigonal bipyramidal, pseudo-tetrahedral, and square planar geometries were also studied. The results provide structural information about the metal binding site(s) in the Co-Hcs that extend earlier results from EPR and optical spectroscopy (Bubacco et al. 1992. Biochemistry. 31: 9294-9303). Experimental spectra were compared to those calculated for atomic clusters of idealized geometry, generated using a multiple scattering approach. The energy of the dipole forbidden 1s-->3d transition and of the absorption edge in the spectra for all cobalt Hc derivatives confirmed the cobaltous oxidation state which rules out the presence of an oxygenated site. Comparisons between data and simulations showed that the mononuclear and dinuclear Co(II) derivatives, as well as the hybrid derivative, contain four-coordinate Co(II) in distorted tetrahedral sites. Although the spectra for Co(II) in dinuclear metal sites more closely resemble the simulated spectrum for a tetrahedral complex than do spectra for the mononuclear derivative, the Co(II) sites in all derivatives are very similar. The Cu K-edge high resolution x-ray absorption near edge structure spectrum of the hybrid Cu-Co-Hc resembles that of deoxy-Hc demonstrating the presence of three-coordinate Cu(I).

XAFS investigations of copper(II) complexes with tetradentate Schiff base ligands

“…The feature A at the absorption edge is originated by the tetragonal distortion in the metal cluster, and its magnitude and position depend on the degree of the distortion. Non‐negligible presence of this feature has been suggested to be an indicator of a fivefold square‐pyramidal coordination . Also, the feature B reflects essentially the difference in the axial geometry of the Cu clusters.…”

The Dinuclear Copper Site Structure of Agaricus bisporus Tyrosinase in Solution Probed by X-ray Absorption Spectroscopy

“…In the case of Hc in solution, only the standard Hc structure b fits the experimental data reasonably well (F ϭ 1.23), whereas structures with long apical distances (structures c and d; F ϭ 2.77 and 2.30, respectively) are rejected. Also in structures a and d, where the tetrahedral character of the transition metal coordination increases due to the decreasing of colinearity of MS pathways, extended spectral changes are expected (Bianconi et al, 1988;Della Longa et al, 1993) and are actually observed in the calculations (F ϭ 1.51 and 1.42, respectively), so the reproduction of the experimental spectrum is worse. The fitting does not improve by using a linear combination of more than one structure (a ϩ b in Table III, F ϭ 1.25, with a negligible percentage of structure a); hence, the only conclusion is that Hc has equivalent pyramidal copper sites with a short apical distance (as probed by EXAFS) and the dioxygen parallel to the pyramid base.…”

Similar enzyme activation and catalysis in hemocyanins and tyrosinases

“…This change results in the blue color developed by type 3 copper proteins upon oxygenation. The active sites of phenoloxidase and hemocyanins with oxygen bound to the copper atoms exhibit similar spectroscopic properties with respect to UV-resonance Raman, X-ray absorption and UV/VIS spectroscopy (Eickman et al, 1978;Himmelwright et al, 1980;Pate et al, 1989;Della Longa et al, 1993;Karlin, 1993;Solomon et al, 1994Solomon et al, , 1996Spritz et al, 1997;Itoh et al, 2001;Metz and Solomon, 2001):…”