Catalysis of O-18 exchange between CO2 and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of (H2O)-O-18 from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound O-18-labeled hydroxide, and was observed in a site-specific mutant of carbonic anhydrase II in which a prominent proton shuttle residue His64 was replaced by alanine, which does not support proton transport. Upon increasing the concentration of bicarbonate, the rate of release of (H2O)-O-18 increased in a saturable manner to a maximum of 4 x 10(5) s(-1), consistent with proton transfer from bicarbonate to the Co(II)-bound hydroxide. The same mutant of carbonic anhydrase containing Zn(II) had the rate of release of (H2O)-O-18 Smaller by 10-fold, but rate of interconversion of CO2 and HCO3- about the same as the Co(II)-containing enzyme. These data as well as solvent hydrogen isotope effects suggest that the bicarbonate transferring the proton is bound to the cobalt in the enzyme. The enhancement of O-18 exchange caused by increasing bicarbonate concentration during catalysis by the Zn(II)containing carbonic anhydrase from the archaeon Methanosarcina thermophila suggests that a very similar mechanism for proton donation by bicarbonate occurs with this wild-type enzyme.