The sensitivity of lipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase E3) from Azotobacter vinelandii to inhibition by NADH requires measurement of the activity in the initial phase of the reaction. Stopped-flow turnover experiments show that kcat is 830 s-1 compared with 420 s-1 found in standard steady-state experiments. Mutations at the si-side of the flavin prosthetic group that cause severe inhibition by NADH were studied. Tyr16 was replaced by phenylalanine and serine, which causes the loss of two intersubunit H-bonds. [F16]E3 shows only 5.7% of wild-type activity in the standard assay procedure, but analyzed by stopped-flow the activity is 70% of the wild-type enzyme. The NADH-->Cl2Ind (dichloroindophenol) activity was normal or slightly increased. The inhibition by NADH is competitive with respect to NAD+, Ki = 50 microM. Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin. It is concluded that subunit interaction plays an important role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin. The interaction of wild-type or mutant enzymes with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent. In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme. The stabilizing effect of E2 on [F16]E3 was used as an assay to analyze the stoichiometry of interaction of E3 with E2p as well as E2o. 1 mol E2p monomer was sufficient to saturate 1 mol E3 dimer with a Kd of about 1 nM. Similarly, 1 mol E2o saturated the E3 dimer with a Kd of 30 nM. From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit interface of E3 near the dyad axis, thus preventing sterically the interaction with a second molecule of the binding domain. This mode of interaction, which causes asymmetry in the complex, explains the stabilization against over-reduction by tightening the subunit interaction. Subgene cloning of the E2p component of the pyruvate dehydrogenase complex is described in order to obtain a complex between the lipoamide dehydrogenase component (E3) and the binding domain of E2p. A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Bal31 was used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from beta-galactosidase. The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and E1p. The shortest fusion protein containing a functional binding domain was expressed and purified. [F16]E3 was combined with this fusion protein in a stoichiometric ratio and the resulting complex was subjected to limited proteolysis to remove the catalytic domain. The resulting [F16]E3-binding domain preparation was purified to homogeneity.