Electron Spin Resonance of Metallocarbonic Anhydrases

Electron spin resonance (ESR) spectra of Cu(II) and Co(II) derivatives of human, monkey, and bovine carbonic anhydrases and their anion complexes are reported. The ESR signals of most of the Cu(II) derivatives indicate a coordination geometry with axial symmetry. Copper carbonic anhydrase complexes appear to have one coordination site filled by a monodentate ligand from solution. This ligand causes increasing electron delocalization from the copper ion in the order CH 3 COO - < OCN - < ethoxzolamide < HCO 3 - < azosulfamide < H 2 O < N 3 - < SH - < CN - as indicated by decreasing values of g || . The presence of highly resolved nitrogen superhyperfine structure in the spectra of the cyanide complexes of Cu(II) human carbonic anhydrases B and C identifies at least 2 equivalent nitrogen atoms as ligands to the metal ion. Carboxymethylation of the human B enzyme modifies the coordination site, but appears to leave the metal ligands unchanged. A new 2:1 cyanide complex of Co(II) carbonic anhydrase is described with an intense ESR signal typical of low spin Co(II) in an axially symmetrical environment. This is in contrast to the broad, weak absorption of the cobalt enzyme and its 1:1 anion complexes which we have examined and which appear to be high spin Co(II). While the ESR spectra of the Cu(II) and Co(II) carbonic anhydrase complexes which show intense signals are best explained by a square planar or five-coordinate square pyramidal geometry, the geometry of these inactive derivatives probably deviates from that of the catalytically active complex. These studies suggest that there is some flexibility of the ligands at the active site and that certain metal ions or anions may force deviation from the active highly distorted four-coordinate geometry.