Adducts of Co(II) and Cu(II) Bovine carbonic anhydrase with bidentate ligands

Abstract

The Zn(II) ion of bovine carbonic anhydrase can be substituted with Co(II), a useful spectroscopic probe, without loss of enzymic activity. The Co(II)-enzyme binds bidentate anionic ligands such as oxalate [1], 2-pyridinecarboxylate [2] and N,N-diethyldithiocarbamate [3], giving pentacoordinate metal derivatives. The ligand displaces the water molecule bound to the metal in the unreacted enzyme, causing a decrease in the 1H NMR relaxation rate to about the same value as in the native diamagnetic Zn(II)-enzyme. The Cu(II)-substituted bovine carbonic anhydrase has no catalytic activity, but is able to bind the same type of ligands as the Co(II)-enzyme. The 1H NMR relaxation rate of the Cu(II)-enzyme, which is almost unaffected by binding of monodentate anions [4], is decreased by binding of the bidentate ones [1, 3, 4]. The substantial residual relaxivity, about 20% that of the unreacted enzyme, may indicate that water coordination is retained in these adducts as it is in the case of monodentate anions, the decrease being related to a different geometry, involving a longer CuO distance. EPR spectra of the adducts with bidentate ligands are compatible with a hexacoordinate tetragonal geometry. The spectroscopic properties of the bicarbonato-derivatives of both Co(II) [1] and Cu(II) [5] enzymes suggest that bicarbonate, the real enzyme substrate, does behave as a bidentate ligand.