Abstract We have found that d-amino acid oxidase is rapidly and irreversibly inhibited by cyanide during oxidative turnover of the carbanion of nitroethane. The inhibited enzyme has an absorption spectrum which is characteristic of a covalent flavin-substrate adduct. The following summarizes the results and interpretation of experiments which establish the mechanism of cyanide inhibition as well as the chemical mechanism of oxidation of nitroalkanes by d-amino acid oxidase. 1. The kinetic mechanism of oxidation of the nitroethane carbanion (S-) to form acetaldehyde (P), hydrogen peroxide, and nitrite, was deduced from a combination of stopped flow and O2-monitored kinetic measurements and is the following. [see PDF for equation] Evaluation of all the rate and equilibrium constants showed that the major pathway for flavin oxidation is the oxidation of ErP by O2. 2. The rate of inhibition of the enzyme by cyanide is regulated by κ2, and O2 is not consumed during the inhibition process. Since the rate of cyanide inhibition increases as the O2 concentration is raised, cyanide does not react with ErP or with a species in rapid equilibrium with ErP. Consequently cyanide must react rapidly with an intermediate, EX, which is formed from E0S in a reaction, or reactions, controlled by κ2. This is depicted kinetically as follows: [see PDF for equation] The inhibited enzyme, EI, does not react with substrate or O2 under the conditions, and within the time scale, of routine kinetic experiments. 3. The inhibited enzyme (EI) contains, per FAD, 1 eq each of substrate and cyanide, but is lacking the nitro group. 4. Treatment of EI with hot methanol produces a free flavin-substrate adduct in good yield and with no irreversible spectral or chemical modification. At 70° and pH values greater than 8 the free flavin-substrate adduct releases cyanide and is readily converted (l10 min) under aerobic conditions to FAD, acetaldehyde, and, presumably, H2O2. Anaerobically, FADH2 is produced. These solvent-catalyzed reactions of the adduct in large part mimic the enzyme-catalyzed oxidation of the substrate. 5. The spectral and ionization properties, as well as the chemical reactivity, of the free flavin-substrate adduct closely resemble those of 5-substituted dihydroflavins in general, and those of 5-cyanomethyl-1,5-dihydroflavin in particular. For these reasons we assign the structure 5-cyanoethyl-1,5-dihydro-FAD to the free flavin-substrate adduct. The kinetic turnover mechanism, the locus of action of cyanide, and the structure of the free flavin-substrate adduct, taken together, enable us to propose a detailed chemical mechanism for the oxidation of nitroethane carbanion by d-amino acid oxidase. E0S is a noncovalent complex in which the substrate carbanion is sufficiently close to the flavin (presumably the N5 position) to perturb the electronic properties of the latter. Attack of the carbanion at N5 of the flavin is controlled by κ2 and results in the formation of 5-nitroethyl-1, 5-dihydro-FAD. Elimination of nitrite forms a highly reactive cationic imine (EX) at the N5 flavin position to which solvent adds to [see PDF for equation] form a carbinolamine. Finally, FADH2, is eliminated from the carbinolamine, leaving acetaldehyde (P) noncovalently bound to Er. Cyanide attacks the cationic imine (EX) in competition with solvent to form 5-cyanoethyl-1,5-dihydro-FAD (EI). This adduct is not reactive with O2 under conditions normally used to study the enzyme.