Crystallographic Studies of the Binding of Protonated and Unprotonated Inhibitors To Carbonic-anhydrase Using Hydrogen-sulfide and Nitrate Anions

The structures of human carbonic-anhydrase-II complexes with the anionic inhibitors hydrogen sulphide (HS-) and nitrate (NO3-) have been determined by X-ray diffraction at 0.19-nm resolution from crystals soaked at pH 7.8 and 6.0, respectively. The modes of binding of these two anions differ markedly from each other. The strong inhibitor HS- replaces the native zinc-bound water/hydroxide (Wat263) leaving the tetrahedral metal geometry unaltered and acts as a hydrogen-bonding donor towards Thr199gamma. The weak NO3- inhibitor does not displace Wat263 from the metal coordination but occupies a fifth binding site changing the zinc coordination polyhedron into a slightly distorted trigonal bipyramid. The interaction of NO3- with the metal is weak; the nearest of its oxygen atoms being at a distance of 0.28 nm from the zinc ion. The binding of nitrate to the enzyme is completed by a hydrogen bond to the metal coordinated Wat263 and a second one to a water molecule of the active-site cavity. The structures of the two complexes help to rationalize the binding of anionic inhibitors to carbonic anhydrase and the binding mode displayed by NO3- may be relevant to the catalytic mechanism.