title: Glutathionylation of α-ketoglutarate dehydrogenase: the chemical nature and relative susceptibility of the cofactor lipoic acid to modification.
abstract: α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione, resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly, producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione-lipoic acid adduct. Nevertheless, 1:1 lipoic acid-glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme's native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition, providing further evidence that specific lipoic acid residues within the E2 complex are susceptible to glutathionylation. These findings offer chemical information necessary for the identification of mechanisms and physiological implications of KGDH glutathionylation.
authors.affiliation: Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: McLain
authors.affiliation: Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Cormier
authors.affiliation: Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; Department of Geriatric Medicine, Reynolds Center on Aging, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Kinter
authors.affiliation: Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA; Department of Geriatric Medicine, Reynolds Center on Aging, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA. Electronic address: Luke-Szweda@omrf.org.
authors.forename: Luke I
authors.initials: LI
authors.lastname: Szweda
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D009206:Myocardium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000737:chemistry*
chemical_list: D008063:Thioctic Acid; C013091:lipoamide; D007655:Ketoglutarate Dehydrogenase Complex; D005978:Glutathione; D002118:Calcium
keywords: Free radicals; Glutathionylation; Lipoic acid; Mitochondria; Redox regulation; α-Ketoglutarate dehydrogenase