High spin and low spin forms of Co(II) carbonic anhydrase. Temperature-dependent changes in spin state and coordination geometry.

Electron spin resonance (ESR) and absorption spectral studies are described which demonstrate two types of cyanide complex of Co(I1) human carbonic anhydrase B. High spin (S = 3/2) Co(I1) is present at the active site of the cobalt enzyme in the presence of both 1 and 2 eq of CN-’ at room temperature. At liquid nitrogen temperature the 1: 1 complex remains high spin, while the enzyme in the presence of 2 eq of CNshifts to low spin (S = l/2). ESR spectra at 111°K and 10°K and absorption spectra at 299°K and 77°K are compatible with tetrahedral coordination geometry for the 1: 1 complex at all temperatures and for the enzyme in the presence of 2 eq of CNat room temperature. Determination of the temperature dependence of the ESR and absorption spectra of the enzyme in the presence of 2 eq of CNshows a low spin dicyanide complex to form when the sample freezes (-273°K). The ESR spectra at liquid nitrogen temperature of the dicyanide complex formed with 12C14N-, 12CSN-, and 13C4Nshow it to contain two ligands along the z-axis of the complex, a nitrogen atom from the protein and the carbon atom of a C-coordinated cyanide. A coordination complex consisting of a distorted trigonal bipyramid with five ligands to the Co(I1) or an octahedral complex with six ligands would account for the ESR and absorption spectra of the low spin dicyanide complex at liquid nitrogen temperature. These changes illustrate a certain flexibility of the ligands at the active site as well as a difference in molecular conformation between the liquid and the frozen state.