Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli.

Results are presented which demonstrate that the 2-electron-reduced lipoamide dehydrogenase (EC 1.6.4.3) from Escherichia coli is a mixture of species. In catalysis, this enzyme cycles between the oxidized and the 2-electron-reduced forms. Three spectrally distinct species are indicated in the pH range 5.8 to 8.0 from measurements of the fluorescence and visible spectra during dithionite titration. These have the following properties. 1) A fluorescent form where the FAD is oxidized and the active center disulfide is reduced. This species is unable to charge transfer and predominates at low pH. 2) A form in which there is a facile charge transfer between thiolate and FAD (epsilon530 - 3300 M-1 cm-1). This species, which predominates at high pH, is very similar to the 2-electron-reduced pig heart enzyme at high pH. 3) A form where the flavin is reduced and the disulfide is oxidized. The spectra of these three species have been determined. Anaerobic reduction of the enzyme with stoichiometric dihydrolipoamide leads to the formation of the charge transfer species in less than 1 s. Subsequently, in a process requiring about 12 s, the charge transfer complex relaxes to a mixture of species observed in dithionite titrations. The pH dependence of the oxidation-reduction potential, the fluorescence, the charge transfer absorbance (530 nm), and the 455 nm absorbance indicates the presence of a base which is able to stabilize the thiolate anion generated upon reduction of the active center disulfide. The pH dependence of the oxidation-reduction potential indicates that the reduction of the enzyme by dihydrolipoamide involves 2 protons as well as 2 electrons. These potentials are somewhat more positive than those determined for the pig heart enzyme and thus explain the ready further reduction of the E. coli enzyme to the 4-electron-reduced enzyme. The pH-independent formation constant (Kf) for the disproportionation of 2-electron-reduced enzyme (2EH2 in equilibrium E + EH4) is about 55 as calculated from dithionite titrations. Therefore at equilibrium there is about 80% 2-electron-reduced enzyme, 1-% oxidized enzyme, and 10% 4-electron-reduced enzyme. The spectrum of fully formed 2-electron-reduced enzyme has been calculated at several pH values from these data. The results confirm the previous conclusion that lipoamide dehydrogenase from E. coli is qualitatively similar to the pig heart enzyme, differing only in certain quantitative features such as the distribution between the various forms at the 2-electron-reduced level.