Lipoamide dehydrogenase catalyzes the reversible NAD+-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, α-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 (O2). 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 O2, in a stopped-flow spectrophotometer at pH 7.5 and 4 °C. EH2 reduced D,L-lipoylpentanoate ≥100 times faster than EH4 did. Conversely, EH4 reduced 2,6-dimethyl-1,4-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.
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