Transition metal binding sites in proteins are typically comprised of 3-4 protein ligands, most of which are also embedded in hydrogen bond networks. For instance, in human carbonic anhydrase II (CAII) the carboxamide side chain of Q92 accepts a hydrogen bond from H94, the carboxylate side chain of E117 accepts a hydrogen bond from H119, and the backbone carbonyl oxygen of N244 accepts a hydrogen bond from H96. In order to probe the structural importance of these hydrogen bond networks, we have determined the three-dimensional structures of Q92A, Q92N, Q92E, Q92L, and E117A CAIIs by X-ray crystallographic methods. When interpreted in light of functional measurements (catalytic activity, protein-zinc affinity) made by Kiefer and colleagues (Kiefer, L. L.; Paterno, S. A.; Fierke, C. A. J. Am. Chem. Sec., preceding paper in this issue), these high-resolution structures allow for detailed structure-function correlations which illuminate the general role of hydrogen bond networks with the second shell of residues surrounding protein-metal binding sites. Due to their structural and electrostatic contributions, these second shell residues, i.e., "indirect" metal ligands, fine-tune the pK(a) and reactivity of zinc-bound solvent; additionally, these residues may contribute a factor of up to 10(4) to protein-metal affinity in a tetracoordinate metal site. It is therefore imperative that indirect metal ligands be considered in de novo designs of avid protein-metal binding sites. Additionally, indirect ligand-direct Ligand-metal networks are important for protein-nucleic acid recognition, e.g., in the C2H2 class of zinc-finger transcription factors.