X-ray absorption spectra are reported for native (Zn) and Coreconstituted bovine carbonic anhydrase (CA), prepared at several pH values across the acid-alkaline transition and in the presence of inhibitors (acetazolamide, imidazole, acetate, HCO3−, NO3−, Cl−, CN−, OCN−, SCN−). The extended fine structure (EXAFS) was analyzed by filtering the first-shell contribution and was compared with a series of model compounds of known structure having various coordination numbers. Co-CA showed variable coordination, as revealed by the calculated number of scatterers and by the average bond distance; four-coordination for the alkaline form and for complexes with CN- and acetazolamide, and five-coordination for the acid form and for complexes with acetate and bicarbonate. For Zn-CA, however, the average coordination number is independent of pH, or of inhibitor binding, and is judged to be four from the relatively short ~2.01-Å average first-shell distance, although the EXAFS-determined average coordination numbers were slightly higher and are less reliable than the distance as monitors of the Zn coordination. Two-atom fits to the filtered first-shell EXAFS data improved the agreement for Zn-CA, with pairs of distances suggesting a ~0.1-Å spread in the Zn-ligand bond lengths, but no improvement was obtained for Co-CA. All of the EXAFS Fourier transforms showed the outer-shell peaks attributable to imidazole ring atoms, at constant intervals from the first-shell peak, implying no alterations in the angle of the imidazole rings relative to the metal-imidazole bonds. The K-edge spectra showed prominent Is → 3d absorption bands for four-coordinate Co-CA complexes, reflecting 4p-3d mixing in near-tetrahedral geometry; the band is attenuated in the five-coordinated species. The main Co edge peak is broader for the four-coordinate species. The Zn edge peak is distinctively split in Zn-CA, as it is in some four-coordinate Zn model compounds, possibly due to 4p, 4d, and/or 5p mixing. The energy of the second component decreases slightly as the pH is raised, following the pKa for enzyme activation; the direction is that expected for ionization of a Zn-bound group, presumably H2O. The Zn- and Co-CA coordination states are discussed in relation to the enzyme mechanism.
ASJC Scopus subject areas
- Colloid and Surface Chemistry