The coordination environment of the active site metal ion of horse liver alcohol dehydrogenase is investigated by EPR and steady-state kinetic methods with use of the native (ZnLADH) and the active site specific Co 2+-reconstituted enzyme (COLADH) described by Zeppezauer and coworkers [1]. The pH dependence of kinetic parameters for the oxidation of benzylalcohol reveals two ionizations (pK 1∼6.7; pK 2∼ 10.6) that govern k cat and belong to the ternary enzyme-NAD +-alcohol complex and two ionizations (pK' 1∼ 7.5; pK' 2∼ 8.9) that govern k cat/K m and belong to the binary enzyme-NAD + complex. Only pK 2 and PK' 2 are substantially influenced by metal substitution. Comparable results are observed for the oxidation of isopropanol. We attribute these ionizations to metal-bound water that occur separately at the ternary and binary complex level in the course of alcohol oxidation. In parallel studies from this laboratory [2, 3], we have demonstrated that the magnitude of the zero-field splitting (ZFS) on the high-spin Co 2+ ion falls into three ranges according to coordination number: 0–13 cm −1 for tetracoordinate; 20–60 cm −1 for penta-coordinate; and 90–310 cm −1 for hexa-coordinate environments. We have determined the ZFS energy (2 D) of the Co 2+ ion in a variety of binary and ternary complexes of CoLADH to assign the coordinates number of the active site metal ion. The results are 9.3 cm −1 (CoLADH); 3.1 cm −1 (CoLADH-CF 3CH 2OH); 13 cm −1 (CoLADHNADH.N,N-dimethylaminocinnamaldehyde); and 8.3 cm −1 (CoLADH-tetrahydroNADH), indicative of tetracoordinate sites, while the ZFS constants of the CoLADH-NADH, CoLADH-NADH-benzylalcohol, CoLADH-NADH-CF 3CH 2OH, and CoLADH-NAD +CF 3CH 2OH complexes are >20 cm −1, indicative of pentacoordinate environments. The results taken together indicate that the active site metal ion is pentacoordinate in catalytically component reaction intermediates and is ligated by a neutral water molecule in the physiologically active ternary enzymeNAD +alcohol complex. We suggest that the neutral metal-bound water molecule serves as the base catalyst for abstraction of the proton from the alcoholic hydroxyl group of the substrate. We present a mechanism for the catalytic action of LADH consistent with these observations and indicate how the metal-bound water molecule may modulate the Lewis acid reactivity of the active site metal to control the catalytic action of LADH. (Supported by NIH grant GM 21900).