Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level

Lipoamide dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, (x-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH2 the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH4), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen O-2. To determine the mechanism of these "diaphorase" reactions, we generated the EH2 and EH4 forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with D,L-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O-2, in a stopped-flow spectrophotometer at pH 7.5 and 4 degreesC. EH2 reduced D,L-lipoylpentanoate : 100 times faster than EH4 did. Conversely, EH4 reduced 2,6-dimethyl1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH2, respectively. Comparison of the rates of reduction of the above substrates by EH2 and EH4 with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH2 while reduction of diaphorase substrates occurs with EH4.