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

Abstract

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(I1)-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(1I) 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(I1) 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. In 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.Keywords: hemocyanin ; copper coordination ; cobalt coordination ; oxidation ; azide binding.Heinocyanin (Hc) is a copper-containing protein found in the hemolymph of organisms belonging to the mollusc and arthropod phyla, where it acts as a carrier and storage protein for dioxygen (Ellerton et al., 1983;Van Holde and Miller, 1995). This function is related to the capacity of Hc to reversibly bind molecular oxygen at an active site containing a binuclear copper center. The protein exists in two different forms: deoxy-Hc, containing a [Cu(l) Cu(l)] copper pair, and oxy-Hc. The copper complex in the latter form is currently formulated as [Cu(II) Oz--Cu(II)1 reflecting electron transfer from Cu(1) to dioxygen (Solomon, 1981 ;Solomon et al., 1992).The X-ray crystallographic studies of deoxy-Hc from Panulirus interruptus (Volbeda and Hol, 1989) and the deoxy and oxy forms of subunit I1 from Limulus polyphaemus (Hazes et al., 1993; Magnus et al., 1994a, b) provided a detailed picture of the protein active site. In the deoxy form, each Cu (1) Ahhreviurions. CT, charge transfer; ESEEM...

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

“…It is thus possible to determine the type and the number of atoms in the metal‐binding sites in metalloproteins, where the interaction with one nitrogen atom, for example, leads to three‐line multiplets and the interaction with two atoms leads to five‐line multiplets and so on. This makes EPR spectroscopy superior as analytical tool in the investigation of the structure and coordination of paramagnetic metal ions in metalloproteins such as hemoglobin and hemocyanin . In addition, the widespread popularity of EPR spectroscopy for the investigation of paramagnetic metalloproteins could be ascribed to its sensitivity and selectivity in which the metal‐binding site can be a selective monitor regardless of the protein's size, whereas protein size constraints are the main limitation of NMR spectroscopy in the study of protein structures …”

“…This makes EPR spectroscopy superior as analytical tool in the investigation of the structure and coordination of paramagnetic metal ions in metalloproteins such as hemoglobin and hemocyanin. [245][246][247][248][249][250][251] In addition, the widespread popularity of EPR spectroscopy for the investigation of paramagnetic metalloproteins could be ascribed to its sensitivity and selectivity in which the metal-binding site can be a selective monitor regardless of the protein's size, whereas protein size constraints are the main limitation of NMR spectroscopy in the study of protein structures. [252][253][254][255]…”

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.…”

“…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.…”