Deoxyhypusine is a modified lysine residue. It is formed posttranslationally in the precursor of eukaryotic initiation factor 5A (eIF5A) by deoxyhypusine synthase, employing spermidine as a butylamine donor. In the initial step of this reaction, deoxyhypusine synthase catalyzes the production of NADH through dehydrogenation of spermidine. Fluorescence measurements of this reaction revealed a -22-nm blue shift in the emission peak of NADH and a approximately 15-fold increase in peak intensity, characteristics of tightly bound NADH that were not seen by simply mixing NADH and enzyme. The fluorescent properties of the bound NADH can be ascribed to a hydrophobic environment and a rigidly held, open conformation of NADH, features in accord with the known crystal structure of the enzyme. Considerable fluorescence resonance energy transfer from tryptophan 327 in the active site to the dihydronicotinamide ring of NADH was seen. Upon addition of the eIF5A precursor, utilization of the enzyme-bound NADH for reduction of the eIF5A-imine intermediate to deoxyhypusine was reflected by a rapid decrease in the NADH fluorescence, indicating a transient hydride transfer mechanism as an integral part of the reaction. The number of NADH molecules bound approached four/enzyme tetramer; not all of the bound NADH was available for reduction of the eIF5A-imine intermediate.