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 pH-dependent 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.
Highlights
Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury
PH Dependence of Lipoamide and Diaphorase Activities— The pH dependence of the activity of pig heart lipoamide dehydrogenase (LADH) in NADHdependent reduction of LA (Fig. 1) displays a broad activity maximum at pH 6.5– 8, which is in agreement with earlier reports [18, 19]
The optimum DCPIP activity was centered below pH 6 (Fig. 1), which is in agreement with measurements done using benzoquinone [20], ferrycianide [20], and oxygen3 as electron acceptors in the NADH dehydrogenase reaction
Summary
Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. 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. We have demonstrated previously that Zn2ϩ inhibits the thiol-disulfide oxidoreductase activity of LADH, whereas it enhances the oxidase activity [17] In this example, the optimal conditions for dehydrogenase and oxidase reactions catalyzed by LADH are mutually exclusive. PH-dependent Oligomeric States of Lipoamide Dehydrogenase which was favored under more dilute conditions.2 This suggests that dissociation of the pig heart LADH dimer may be involved in the inactivation process PH-dependent Oligomeric States of Lipoamide Dehydrogenase which was favored under more dilute conditions. This suggests that dissociation of the pig heart LADH dimer may be involved in the inactivation process
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