| Abstract
| - Lipoamide dehydrogenase catalyzes the reversible NAD+-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvatedehydrogenase, α-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It containstwo redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which canaccommodate two electrons. In the two-electron-reduced enzyme (EH2), the disulfide is reduced whilethe 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 acceptorssuch as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O2). To determinethe mechanism of these “diaphorase” reactions, we generated the EH2 and EH4 forms of Mycobacteriumtuberculosis 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. EH2reduced 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 ofthe rates of reduction of the above substrates by EH2 and EH4 with their corresponding steady-state kineticparameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs withEH2 while reduction of diaphorase substrates occurs with EH4.
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