Great Britain - Electron - Paramagnetic - Resonance Studies on Cobalt ( ll ) Carbonic Anhydrase - Sulphonamide Complexes 703

Sulphonamide adducts of three Co(II) carbonic anhydrases were investigated by e.p.r. (electron paramagnetic resonance) at helium temperatures. The highly anisotropic9GHz spectra exhibited only three distinct features, with g values between 6.3 and 1.5. Such spectra arise from an electronic state with effective spin S' = i, indicating that the highspin (S= 2) ground level is split into two spin doublets differing in energy by an amount large compared with the microwave quantum, but small in relation to thermal energies at ambient temperature. This situation would occur in a tetrahedral system suffering a large rhombic distortion. Calculations based on this model accounted for apparent discrepancies in integrated spectral intensities, and yielded magnetic moments in good agreement with independent measurements, especially in the case of certain small Co(II) complexes resembling the enzyme adducts in their e.p.r. signals. Precise sets ofg values, reflecting a particular co-ordination geometry, were found to be representative of each enzyme variant and the type of sulphonamide inhibitor, whether benzocyclic or heterocyclic.A series ofsubstituted benzenesulphonamides bound to thesameenzyme gave rise to closely similar spectra despite a wide range ofpK1 values. Thus benzocyclic and heterocyclic sulphonamides were evidently held in the active-site cleft in characteristic orientations irrespective of side chains that might considerably influence the total binding strength. Visible absorption spectra of various sulphonamide adducts at room temperature showed a similar pattern ofinhibitor dependence to the e.p.r. spectra, suggesting a correspondence between the co-ordination structures in liquid and frozen solution. E.p.r. spectra of the sulphonamide complexes were remarkable not only for their range ofg values, but also for their variations in line-width and spin-lattice relaxation behaviour. Addition of glycerol to the medium produced marked enhancement in resolution, owing to the creation of a more homogeneous frozen matrix. The non-uniform spin relaxation was probably a consequence of the large anisotropy in effective g tensor.