The binding 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. 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.
Alzuet
G
G
Department of General Chemistry, University of Pavia, Italy.
Bubacco
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L
Casella
L
L
Rocco
G P
GP
Salvato
B
B
Beltramini
M
M
D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D003386:Brachyura; D002942:Circular Dichroism; D003035:Cobalt / Q000032:analysis / Q000378:metabolism*; D003300:Copper / Q000032:analysis / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006433:Hemocyanins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation
D001059:Apoproteins; D001386:Azides; D003035:Cobalt; D003300:Copper; D006433:Hemocyanins