Morphinone reductase (MR) catalyzes the NADH-dependent reduction of alpha/beta unsaturated carbonyl compounds in a reaction similar to that catalyzed by Old Yellow Enzyme (OYE1). The two enzymes are related at the sequence and structural levels, but key differences in active site architecture exist which have major implications for the reaction mechanism. We report detailed kinetic and solution NMR data for wild-type MR and two mutant forms in which residues His-186 and Asn-189 have been exchanged for alanine residues. We show that both residues are involved in the binding of the reducing nicotinamide coenzyme NADH and also the binding of the oxidizing substrates 2-cyclohexen-1-one and 1-nitrocyclohexene. Reduction of 2-cyclohexen-1-one by FMNH(2) is concerted with proton transfer from an unknown proton donor in the active site. NMR spectroscopy and flavin reoxidation studies with 2-cyclohexen-1-one are consistent with His-186 being unprotonated in oxidized, reduced, and ligand-bound MR, suggesting that His-186 is not the key proton donor required for the reduction of 2-cyclohexen-1-one. Hydride transfer is decoupled from proton transfer with 1-nitrocyclohexene as oxidizing substrate, and unlike with OYE1 the intermediate nitronate species produced after hydride transfer from FMNH(2) is not converted to 1-nitrocyclohexane. The work highlights key mechanistic differences in the reactions catalyzed by MR and OYE1 and emphasizes the need for caution in inferring mechanistic similarities in structurally related proteins.