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, alpha-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 (EH(2)), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH(4)), 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 EH(2) and EH(4) 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 degrees C. EH(2) reduced d,l-lipoylpentanoate >/=100 times faster than EH(4) did. Conversely, EH(4) reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH(2), respectively. Comparison of the rates of reduction of the above substrates by EH(2) and EH(4) with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH(2) while reduction of diaphorase substrates occurs with EH(4).