PMID: 33177Feb 10, 1979

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

The Journal of Biological Chemistry
K D Wilkinson, C H Williams

Abstract

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 de...Continue Reading

Related Concepts

Sodium Dithionite
Alkalescens-Dispar Group
AS 2
Fluorescence Spectroscopy
Charge transfer complex
Fluorescent stain
Dihydrolipoamide Dehydrogenase
Spectrophotometry
DLD gene
QPCT

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