Previous studies found the lipid peroxidation product 4-hydroxynon-2-enal (4HNE) to be both a substrate and an inhibitor of mitochondrial aldehyde dehydrogenase (ALDH2). Inhibition of the enzyme by 4HNE was demonstrated kinetically to be reversible at low micromolar aldehyde but may involve covalent modification at higher concentrations. Structurally analogous to 4HNE is the lipid peroxidation product 4-oxonon-2-enal (4ONE), which is more reactive than 4HNE toward protein nucleophiles. The goal of this work was to determine whether 4ONE is a substrate or inhibitor of human ALDH2 (hALDH2) and elucidate the mechanism of enzyme inhibition by 4HNE and 4ONE. Both 4ONE and its glutathione conjugate were found to be substrates for the enzyme in the presence of NAD. At low concentrations of 4ONE (< or = 10 microM), hALDH2 catalyzed the oxidation of 4ONE to 4-oxonon-2-enoic acid (4ONEA) with a maximal yield of 5.2 mol 4ONEA produced per mol of enzyme (monomer). However, subsequent analysis of hALDH2 activity toward propionaldehyde revealed that both 4ONE and the oxidation product, 4ONEA, were potent, irreversible inhibitors of the enzyme. In contrast, inhibition of hALDH2 by a high concentration of 4HNE (i.e., 50 microM) was primarily reversible. The reactivity of 4ONEA toward glutathione was measured and found to be comparable to that of 4HNE, indicating that the 4ONE-oxidation product is a reactive electrophile. hALDH2/NAD was incubated with 4HNE, 4ONE, and 4ONEA, and mass spectral analysis of tryptic peptides revealed covalent modification of an hALDH2 active site peptide by both 4ONE and 4ONEA. These data demonstrate that hALDH2 catalyzes the oxidation of 4ONE to 4ONEA; however, the product 4ONEA is a reactive electrophile. Furthermore, both 4ONE and 4ONEA are potent, irreversible inhibitors of the enzyme.