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

Abstract

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pHdependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of LADH entrapped in reverse micelles reveals dimer and tetramer at both pH 5.8 and 7.5, but monomer was observed only at pH 5.8. Enzyme activity measurements in reverse Aerosol OT micelles in octane indicate that LADH dimer and tetramer possess lipoamide dehydrogenase and diaphorase activities at pH 7.5. Upon acidification to pH 5.8 only the LADH monomer is active and only the diaphorase activity is observed. These results indicate a correlation between pH-dependent changes in the LADH reaction specificity and its oligomeric state. The acidification of mitochondrial matrix that occurs during ischemia-reperfusion injury is sufficient to alter the structure and enzymatic specificity of LADH, thereby reducing mitochondrial defenses, increasing oxidative stress, and slowing the recovery of energy metabolism. Matrix acidification may also disrupt the quaternary structure of other mitochondrial protein complexes critical for cellular homeostasis and survival.

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

“…As detailed in the Introduction, LADH is greatly susceptible to the ambient conditions, and as many studies suggest, changes in LADH function are based on specific structural (conformational) changes [3,8,10,11,27,29,58]. It is important to note that several pathogenic mutations [30] or the drop in pH alone [3] can facilitate the dissociation of LADH from multienzyme complexes.…”

“…In both directions in the absence of added electron acceptors, O 2 is reduced by LADH to superoxide ("oxidase reaction" of LADH), the latter being a reactive oxygen species (ROS), which is then partly dismutated to H 2 O 2 and O 2 [3][4][5][6]. Analogous mechanism to ROS generation by LADH is the diaphorase activity of the enzyme [7], where LADH reduces via NADH various ions or organic molecules as artificial substrates [8][9][10]. The pH optimum of the diaphorase/ROS-generating activity is below 6 (4.8-5.7) [3,8,10,11], while that of the reverse reaction is 6.5-7.3 [8,10].…”

“…Analogous mechanism to ROS generation by LADH is the diaphorase activity of the enzyme [7], where LADH reduces via NADH various ions or organic molecules as artificial substrates [8][9][10]. The pH optimum of the diaphorase/ROS-generating activity is below 6 (4.8-5.7) [3,8,10,11], while that of the reverse reaction is 6.5-7.3 [8,10]. pH optimum of the forward physiological reaction is 7.9-8.3 [10,12].…”

“…FAD binds non-covalently and is thought to play a crucial role in ROS-generation [28]. Monomerization of LADH was proposed to occur due to mild acidification [8] or specific pathogenic mutations [27,29], which could potentially account for the transformation of LADH to a diaphorase [8] and/or a Ser-protease [29], respectively. Later studies partly also by our laboratory, disproved monomer formation under the above conditions and proposed that instead, specific conformational changes would be responsible for the observed phenomena [3,11,30].…”

“…Hence, there is hitherto no structural information available on the diaphorase/ROS-generating conformation. Nevertheless, pH dependence of the diaphorase/ROS-generating activity of LADH [3,8,10,11] strongly suggests the involvement of a key residue with a pK a in the range of the pH optimum of the diaphorase/ROS-generating activity.…”