Hemocyanins are the oxygen transport/storage proteins, present in the hemolymph of molluscs and arthropods. Their physiological role is based on the reversible binding of molecular oxygen to a binuclear copper containing active site. In the deoxygenated form, three Ne-imidazole nitrogens coordinate each Cu(I) ion. Binding of oxygen occurs via a reversible two-electrons transfer and the resulting complex is described as Cu(II)O22-Cu(II) complex, with peroxide bound as bridging ligand. Arthropod hemocyanins are giant oligomers made by aggregation of hexameric building blocks (Mr ~450 kDa) that represent the allosteric unit that can adopt two different conformational state corresponding respectively to high oxygen affinity (R) and low oxygen affinity state (T).

In this work the functional and structural properties of the conformational states of Carcinus aestuarii hemocyanin were investigated embedding the protein into a sol-gel. This silica host matrix allows to trap functional hemocyanin either in the oxygenated or deoxygenated forms blocking it in a single conformational R or T state. The Hill-plot of the oxygen binding demonstrates that the hemocyanin can be blocked in either the T and R states. To characterize the properties of the T and R states, fluorescence, circular dichroism, U.V. and X-ray absorption spectroscopy (XAS) were used. Analysis of XAS spectra allows to correlate the coordination geometry of the copper ions to R and T state of both oxy and deoxy-hemocyanin.