In metalloenzymes the side chains that ligand the metal ion are nested in a hydrogen bonding network with other protein residues. In carbonic anhydrase II (CAII) the four zinc ligands, H94, H96, H119, and water, donate hydrogen bonds to the carboxamide of Q92, the-backbone carbonyl oxygen of N244, a carboxylate oxygen of E117, and the hydroxyl of T199, respectively. To investigate the functional role of these-hydrogen bonds, we determined the effects of varying the structure of the side chain at positions 92, 117, and 199 on zinc binding and catalysis. These functional changes are further illuminated by the X-ray crystal structures of these variants (following paper by Lesburg and Christianson). The data demonstrate that zinc affinity is increased by a factor of 10 per hydrogen bond, presumably by decreasing the entropic cost of zinc binding. The observed decrease in zinc affinity, is additive for the Q92A/E117A CAII variant suggesting a maximal decrease of 10(4)-fold for removal of all four hydrogen bonds. Furthermore, the hydrogen bond between H119 and E117 determines slow zinc dissociation. Finally, this hydrogen bond network "fine-tunes" the catalytic efficiency and the zinc-water pK(a) of CAII by modulating the electrostatic environment of the zinc. The resulting negative Bronsted correlation indicates that electrostatic stabilization of both the transition state and hydroxide ion by zinc is the main determinant of catalytic efficiency in CAII. Therefore, indirect ligands are an essential feature of metalloenzymes and should be included in the structure-assisted design of metal binding sites in order to obtain high affinity and reactivity.