The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of hound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. and Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 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 protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and precedes the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met-hemocyanin form, The structure of this dinuclear copper(II) derivative contains a di-mu-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity (K = 30 M(-1)) at pH 7.0, it binds with much higher affinity at pH 5.5 (K = 1500 M(-1)), where a second azide ligand also binds in the terminal mode (K = 20 M(-1)). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.

The oxidation of hemocyanin: kinetics, reaction mechanism and characterization of the met-hemocyanin product

BELTRAMINI, MARIANO;BUBACCO, LUIGI;
1995

Abstract

The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of hound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. and Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 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 protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and precedes the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met-hemocyanin form, The structure of this dinuclear copper(II) derivative contains a di-mu-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity (K = 30 M(-1)) at pH 7.0, it binds with much higher affinity at pH 5.5 (K = 1500 M(-1)), where a second azide ligand also binds in the terminal mode (K = 20 M(-1)). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.
1995
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2458968
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