The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii

Abstract

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotohucter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, t, = 80°C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyine or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass > 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu4.59 and/ or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.Dihydrolipoamide dehydrogenase is a member of the class of flavoprotein disulfide oxidoreductases [l]. The dimeric enzyme catalyzes the NAD+-dependent oxidation of dihydrolipoyl groups, which in vivo are covalently attached to the lipoate acyltransferase component of different multienzyme complexes [2]. The active site of the enzyme is composed of both subunits, each containing FAD and a redox-active disulfide group [3]. Mechanistically and kinetically, lipoamide dehydrogenase from pig heart and Escherichiu coli have been most extensively characterized [4, 51. These enzymes differ strongly with rcspect to the stabilization of different twoelectron reduced enzyme species [6-91. Inhibition of the E . coli enzyme by the product NADH was shown to be mainly due to over-reduction of the enzyme to the inactive fourelectron reduced state [lo].Recent

Denaturation and Reactivation of Dimeric Human Glutathione Reductase

“…In addition, the data collected at 450 nm showed the presence of free FAD; they could be fitted under the assumption that proteinbound FAD was exclusively associated with dimeric human GR. Similar results from fluorescence studies were reported for the Gdn/HCl-induced unfolding of the related flavoenzyme lipoamide dehydrogenase (van Berkel et al, 1991).…”

pH-dependent Substrate Preference of Pig Heart Lipoamide Dehydrogenase Varies with Oligomeric State

“…Chemical modification allowed the preparation of monomer, which was reported to retain diaphorase activity (21). In addition, conformational instability of the fully reduced LADH from Azotobacter vinelandii has been reported (22). We have observed inactivation of LADH under reducing conditions, which was favored under more dilute conditions.…”

“…It is important to note that DLS and sedimentation experiments were performed with the native, oxidized form of LADH (i.e. without substrate), whereas diaphorase activity measurements were done under reducing conditions, which is known to stimulate enzyme dissociation (22). The results of sedimentation analysis suggest that the equilibrium between LADH oligomeric forms is pH-sensitive, i.e.…”

Deflavination and reconstitution of flavoproteins

“…A disadvantage of this method is that one needs to find conditions in which the partially unfolded apoprotein is capable of refolding. Circular dichroism spectroscopy [46,118,125–127] and fluorescence spectroscopy [127–129] are useful here to probe the folding behaviour of the protein of interest.…”

“…Then, dimerization occurs as reflected by the lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum [151]. For lipoamide dehydrogenase from A. vinelandii , the conformational stability of the monomeric apoprotein was compared with that of the dimeric holoenzyme [128]. Unfolding of the apoenzyme in guanidinium hydrochloride follows a simple two‐state mechanism and is fully reversible.…”