COMPARISON OF HALF-MET AND MET APO HEMOCYANIN - LIGAND BRIDGING AT THE BINUCLEAR COPPER ACTIVE-SITE

Two series of Busycon canaliculatum hemocyanin derivatives have been prepared which have allowed a systematic study of ligand binding to the binuclear copper active site. Half-met-? hemocyanin, where L = CN-, NO2-, N3-, SCN-, OCN-, F-, Cl-, Br-, CH3CO2- and aquo contains a [Cu(II)ȦCu(I)] active site; for the met apo-L form, however, one copper has been selectively removed and the remaining copper oxidized, producing a [Cu(II)Ȧ( )] active site. A comparison of the ligand substitution chemistry of these forms has led to two general observations. First, ligands bind far more tightly to the half-met active site. This, combined with spectroscopic data and the effects of CO coordination, requires the exogenous ligand to bridge the coppers. Second, an additional coordination position is shown to be available at the Cu(II) site for only certain half-met-L forms (L = CN-, N3-, SCN-), where the ligand is expected to keep the coppers >5 Å apart. No second coordination position is observed for any met apo derivative. These observations strongly support the presence of an endogenous protein bridge between the coppers. Furthermore, spectroscopic results have shown that certain half-met-L forms (L = Cl-. Br-,I-, N3-) exhibit class II mixed valence properties (intervalence-transfer transitions and delocalized EPR) which directly correlate with the nature of the bridging ligand. Finally, half-met-L's, where L = N3-, Cl-, CH3CO2-, and aquo, are found to undergo reversible CO reactions which perturb the binding of L to the half-met active site. © 1979, American Chemical Society. All rights reserved.