Technical Abstract: Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved free NADH (t = 0.4 ns) and bound NADH (t = 6.0 ns). We used this technique to investigate the effects of Mg2+ on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD for NADH with ALDH2 ranged from 334 µM to 10 µM for Mg2+ ion concentrations of 2 µM to 6000 µM, respectively The dissociation rate of NADH ranged from 0.4 s-1 (6000 µM Mg2+) to 8.3 s-1 (0 µM Mg2+) by by displacing NADH from ALDH2 with the addition of excess NAD+ and resolving the real-time NADH fluorescence signal. The calculated NADH association/re-association rates were approximately 0.04 s-1 over the entire Mg2+ ion concentration range and demonstrate that Mg 2+ ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed the biphasic behavior of the enzyme activity as a function of Mg2+ concentration. Importantly, we observed no pre-steady state burst of NADH formation in the absence of Mg2+, and our results indicate that previously reported burst activity in the absence of metal may be due to trace levels of metal present in these assays. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (t = 11.2 ns).