THE OXIDATION OF HEMOCYANIN - KINETICS, REACTION-MECHANISM AND CHARACTERIZATION OF MET-HEMOCYANIN PRODUCT

The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemoc yanin has been reinvestigated under several experimental conditions. V arious anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechan ism is different from that currently accepted involving a peroxide dis placement of hound dioxygen through an associative chemistry on an ope n axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. and Solomon, E. I. (1979) Biochem. Biophys. Res. Commu n. 89, 1050-1057; Solomon, E. I, in Copper proteins (Spiro, T. G., ed. ) pp. 43-108, J. Wiley, New York]. Our study suggests that the protona ted form of the anion is likely to be the species reacting with the ox ygenated form of the protein. Furthermore, it is also proposed that pr otonation of bound dioxygen generates an intermediate hydroperoxo-dico pper(II) complex to which the exogenous anion is also bound. This inte rmediate in not accumulated and precedes the release of hydrogen perox ide by reaction with water. Upon dialysis it leads to the met-hemocyan in form, The structure of this dinuclear copper(II) derivative contain s a di-mu-hydroxo bridge but there is evidence from optical and circul ar dichroism spectra for partial protonation of these bridges at low p H. As a consequence, while one azide molecule binds in the bridging mo de to met-hemocyanin with low affinity (K = 30 M(-1)) at pH 7.0, it bi nds with much higher affinity at pH 5.5 (K = 1500 M(-1)), where a seco nd azide ligand also binds in the terminal mode (K = 20 M(-1)). The co ordination mode of the azide ligands is deduced from the optical and c ircular dichroism spectra of the protein complexes.