Structure of the active site of carbonic anhydrase as determined by electron spin resonance.

Abstract

High resolution electron spin resonance spectra of the stepwise formation of CN- complexes of Co(II) and Cu(II) carbonic anhydrase show that both metal enzymes form successive 1:1 and 2:1 addition products with CN- at 112 K. The 1:1 complex with the Cu(II) enzyme has a rhombic ESR spectrum similar to the spectra of the 1:1 complexes of the Cu(II) enzyme with CH3COO-, OCN-, N3-, and SH-. The 1:1 complex with the CO(II) enzyme shows a broad resonance at 10 K indicating the presence of high spin Co(II). Previous optical, ESR, and magnetic susceptibility data suggest that the 1:1 complexes are 4-coordinate. At high concentrations of 13CN- the Cu(II) enzyme forms a 2:1 CN- complex with a shift to an axial ESR signal showing ligand nuclear superhyperfine structure from two magnetically equivalent equatorial nitrogen nuclei of the protein and two magnetically equivalent equatorial carbon ligands from two 13CN- anions. Under the same conditions a structurally analogous dicyanide complex of the co(II) enzyme forms with the appearance of and axial ESR signal typical of low spin Co(II). Ligand nuclear superhyperfine structure shows the presence of an axial protein nitrogen as ligand and two magnetically equivalent equatorial carbon ligands from two 13CN- anions. The dicyanide complexes of the Co(II) and Cu(II) enzymes form completely only in frozen solutions and analysis of the ESR spectra show them to have a 5-coordinate square pyrimidal geometry. Comparison of the ligand superhyperfine structure on the ESR signals of both dicyanide complexes shows that there are three nitrogen nuclei of the protein present as ligands at the metal binding site; one axial and two equatorial in the dicyanide complexes. A transient 5-coordinate intermediate might play a role in the mechanism of action of carbonic anhydrase by facilitating ligand exchange reactions within the inner coordination sphere of the Zn(II) ion at the active center.