title: pH-dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: response to mitochondrial matrix acidification.
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.
authors.affiliation: Department of Chemical Enzymology, M. V. Lomonosov Moscow State University, 119899 Moscow, Russia.
authors.forename: Natalia L
authors.initials: NL
authors.lastname: Klyachko
authors.forename: Valentina A
authors.initials: VA
authors.lastname: Shchedrina
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Efimov
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Kazakov
authors.forename: Irina G
authors.initials: IG
authors.lastname: Gazaryan
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Kristal
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Brown
mesh_terms: D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008823:Micelles; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity / Q000502:physiology; D013552:Swine / Q000378:metabolism*
chemical_list: D008823:Micelles; D009243:NAD; D015086:2,6-Dichloroindophenol; D008058:Dihydrolipoamide Dehydrogenase