AN X-RAY-ABSORPTION NEAR-EDGE STRUCTURE SPECTROSCOPY STUDY OF METAL COORDINATION IN CO(II)-SUBSTITUTED CARCINUS-MAENAS HEMOCYANIN

High-resolution x-ray absorption near edge structure spectroscopy was used to characterize the metal sites in three different cobalt-substit uted derivatives of Carcinus maenas hemocyanin (Hc), including a monon uclear cobalt, a dinuclear cobalt and a copper-cobalt hybrid derivativ e. Co(II) model complexes with structures exemplifying octahedral, tri gonal bipyramidal, pseudo-tetrahedral, and square planar geometries we re also studied. The results provide structural information about the metal binding site(s) in the Co-Hcs that extend earlier results from E PR and optical spectroscopy (Bubacco et al. 1992. Biochemistry. 31:929 4-9303). Experimental spectra were compared to those calculated for at omic clusters of idealized geometry, generated using a multiple scatte ring approach. The energy of the dipole forbidden 1s-->3d transition a nd 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 tetrah edral 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 a ll derivatives are very similar. The Cu K-edge high resolution x-ray a bsorption near edge structure spectrum of the hybrid Cu-Co-Hc resemble s that of deoxy-Hc demonstrating the presence of three-coordinate Cu(I ).