The Oxidation of Hemocyanin

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 bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. 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(I1) 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(I1) derivative contains a di-k-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-I) at pH 7.0, it binds with much higher affinity at pH 5.5 ( K = 1500 M-'), where a second azide ligand also binds in the terminal mode ( K = 20 M-'). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.Keywords: hemocyanin ; copper active site; oxidation; azide binding ; circular dichroism.Hemocyanins are copper proteins found in the hemolymph of several invertebrate species belonging to the phyla of Mollusca and Arthropoda, where they have an oxygen transport and/ or storage function. The biological role of this family of proteins is based on the reversible binding of dioxygen to a dinuclear copper active site. The protein exists in two functional forms, deoxy-hemocyanin and oxy-hemocyanin, their relative abundance depending on oxygen concentration.The deoxy-hemocyanin form contains a dinuclear Cu(1) site. X-ray crystallography data on two deoxy forms, of the lobster Panulirus interruptus hemocyanin (Volbeda and Hol, 1989) and of the horseshoe crab Limulus polyphemus subunit I1 hemocyanin(Hazes et al., 1993; Magnus et al., 1994) are now available.In both proteins each copper ion is bound to three histidine residues. The active-site structure of Panulirus deoxy-hemocyanin consists of a coplanar system of four imidazole nitrogens together with the two metal ions (Cu,, Cu,); the Cu-N and the Dioxygen is bound as a bridge between the two copper ions in a p-q2:q2 mode (Magnus et al., , 1994. This coordination mode is substantiated by a study on a dinuclear copper model complex where pyrazolyl groups replace the imidazole donors, and peroxide is bound as it is proposed for oxy-hemocyanin (Kitajima et al., 1992, Kitajima and Moro-oka, 1994); the absorption properties of this model ...

Oxidized Derivatives of Octopus vulgaris and Carcinus aestuarii Hemocyanins at pH 7.5 and Related Models by X-ray Absorption Spectroscopy

“…The proposed active site model for the met-Hc form assumes a Cu(II) binuclear structure with a di-hydroxo bridge. The pH dependence of the CD features of the met-Hcs and the pH dependence of the azide interaction is suggesting a partial protonation of these bridges at low pH (Beltramini et al, 1995;Alzuet et al, 1997). An extended x-ray absorption fine structure (EXAFS) study (Woolery et al, 1984) shows that there are no differences in the coordination number between the oxy-Hc and metaquo-Hc forms, however the fundamental question of the origin of the diamagnetism in the met-Hc form is still unresolved.…”

“…Assuming the same bis-hydroxo adducts of the binuclear site for both met-Hcs, different reaction models for the binding of azide to O. vulgaris and C. aestuarii met-Hcs have been proposed (Beltramini et al, 1995;Alzuet et al, 1997). The substitution of the hypothetical exogenous bridging ligands with azide allows for disturbing, in a controlled manner, the structural properties of the binuclear site on the met-Hc forms and for evaluating the effect of different coordination modes of the ligand on the Hcs from the two phyla.…”

“…Native Hcs were isolated from the hemolymph of O. vulgaris and C. aestuarii and were prepared as described, respectively, in Beltramini et al (1995) and Bubacco et al (1992). The protein solutions were stored at Ϫ20°C in the presence of 20% (w/w) sucrose as cryoprotectant.…”

“…The starting spectroscopic hypothesis for the azide-binding mode is an end-on geometry for C. aestuarii derivative (Alzuet et al, 1997) and a -bridge geometry for O. vulgaris, with a preferred -1,3 bridging mode (Beltramini et al, 1995). For the O. vulgaris met-azido-Hc, the best fits were obtained by considering a structure with a coordinated -1,3 azido bridge as the starting model for the calculations of the theoretical signal (Fig.…”

The Binding of Azide to Copper‐Containing and Cobalt‐Containing Forms of Hemocyanin from the Mediterranean Crab Carcinus Aestuarii

“…The stoichiometry of binding of this ligand between the two proteins differs at pH 5.5, because a second azide binds to Octopus met-Hc with CD features similar to those observed here for the azide bound to Carcirzus met-Hc. Therefore, with respect to azide coordination, Carcinus and Octopus met-Hc differ from each other, since in the latter case the CD spectral features are indicative of a bridging binding mode for the first azide and a terminal binding mode for the second azide (at pH 5.5; Beltramini et al, 1995).…”