Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropodan hemocyanins

The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins. The first shell analysis of the EXAFS modulation is consistent with the presence of n = 3N∈2 imidazole nitrogens at an average distance of 1.92±0.03 Å from copper in all the deoxy-hemocyanins investigated. Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n = 5 with average distances of 1.93 Å. Alternatively, by separating the contribution of N∈2 imidazole nitrogens and of peroxide O-atoms, n = 3 ligands at 1.98 ± 0.03 Å and n = 2 ligands at 1.87 ± 0.03 Å are found.