The enzymatic properties of octopus vulgaris hemocyanin: o-Diphenol oxidase activity

Hemocyanin and tyrosinase are dinuclear copper proteins capable of reversibly binding dioxygen. Despite the great similarity of structure and properties of their active site, the two proteins perform different biological functions (oxygen transport/storage versus monooxygenase and oxidase activity). In this paper, we show that Octopus Vulgaris hemocyanin exhibits a tyrosinase-like activity; namely, it is capable of utilizing dioxygen for the oxidation of o-diphenol to quinone. The reaction is specific for this isomer of diphenol, the meta and para isomers being unreactive, and is strongly controlled by steric factors. Dioxygen represents a cosubstrate of the reaction, and it is involved in the catalytic turnover by binding to the dinuclear copper site of the protein to form, under steady-state conditions, oxy-Hc, which is the active species. The generation of semiquinone radicals, detected by EPR and by their reaction with N,N,N\ua2,N\ua2-tetramethyl-1,4-phenylenediamine, strongly supports a reaction mechanism in which such radicals represent the reaction products of one-electron oxidation of the substrate, quinone being generated by dismutation of semiquinones. Met-Hc is regenerated by the substrate to the deoxy form. To close the catalytic cycle, the proposed reaction mechanism also involves the participation of two transient protein forms with the total oxidation state of the active site (V and IV) intermediate between that of oxy-Hcy, [CuIIO22-CuII]VI, and deoxy-Hc, [CuICuI]II. A mathematical model has been elaborated to describe the reaction kinetics. The differences in reaction mechanisms between hemocyanin and tyrosinase are discussed in terms of accessibility to exogenous molecules of their active sites

The binnding of azide to copper-containing and cobalt-containing forms of hemocyanin from the mediterranean crab Carcinus aestuarii

To establish the competence of the active site of hemocyanin to acquire diverse coordination geometries, the binding of azide to three forms of a crab hemocyanin, the dinuclear cupric or met-hemocyanin, the mononuclear cupric or met-apo-hemocyanin. and the mononuclear Co(II)-substituted derivative has been studied by near-ultraviolet circular dichroism and EPR spectroscopies. The near-ultraviolet circular dichroism spectra of the various derivatives present qualitatively similar features, namely a negative peak around 335 nm in the case of the two copper-containing derivatives and a three-component pattern with the Co(II) derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optical activity is observed with all protein samples. The characteristic CD features, attributable to N(imidazole)-to-metal and to OH--to-metal charge transfer transitions, are strongly affected by azide binding. In particular, the intensity of the negative band exhibited by the two copper-containing protein forms decreases with the onset of a new negative feature with maximum around 400 nm diagnostic for azide-to-Cu(II) charge-transfer transitions. The visible region is affected as well, indicating that changes in the coordination sphere of copper take place. The affinity for azide of the different protein forms is higher at low pH. EPR measurements on the paramagnetic met-apo-hemocyanin derivative as a function of pH demonstrate heterogeneity in the coordination environment at low pH. Tn the presence of azide an increase of rhombic distortion of the EPR spectra is observed and on the basis of the identified sets of copper hyperfine features in the course of azide titration experiments two different azide bound forms of met-apo-hemocyanin can be detected. The CD and EPR data at the different pH values are consistent with a reaction scheme in which azide replaces a fourth ligand in the metal-coordination sphere, identified as a water or hydroxide molecule

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

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