The crystal structure of F65A/Y131C murine a-carbonic anhydrase V (CAV), covalently modified at cysteine residues with 4-chloromethylimidazole, is reported at 1.88 .ANG. resoln. This modification introduces a methylimidazole (MI) group at residue C131 in the active site with important consequences. Previous studies showed that F65A/Y131C-MI CAV exhibits an up to 3-fold enhancement of catalytic activity over that of wild-type CAV. In this modified CAV variant, C131-MI acts as a proton shuttle,facilitating the deprotonation of a zinc-bound water mol. to regenerate the nucleophilic zinc-bound hydroxide ion. A network of three hydrogen-bonded water mols., across which proton transfer likely proceeds, bridges the zinc-bound water mol. and the C131-MI imidazole group. The structure of F65A/Y131C-MI CAV is compared to structures of Y64H/F65A murine CAV, wild-type human a-carbonic anhydrase II, and the g-carbonic anhydrase from Methanosarcina thermophila in an effort to outline common features of catalytic proton shuttles.