Mapping of oxidative modifications on the alpha-keto glutarate dehydrogenase complex induced by singlet oxygen: Effects on structure and activity

Abstract

The large multi-subunit mitochondrial alpha-keto glutarate dehydrogenase (KGDH) complex plays a key, rate-determining, role in the tricarboxylic acid (Krebs) cycle, catalyzing the conversion of alpha-keto glutarate to succinyl-CoA. This complex is both a source and target of oxidants, but the sites of modification and association with structural changes and activity loss are poorly understood. We report here oxidative modifications induced by Rose Bengal (RB) in the presence of O₂, a source of singlet oxygen (¹O₂). A rapid loss of activity was detected, with this being dependent on light exposure, illumination time, and the presence of RB and O₂. Activity loss was enhanced by D₂O (consistent with ¹O₂ involvement), but diminished by both pre- and (to a lesser extent) post-illumination addition of lipoic acid and lipoamide. Aggregates containing all three KGDH subunits were detected on photooxidation. LC-MS experiments provided evidence for oxidation at 45 sites, including specific Met, His, Trp, Tyr residues and the lipoyllysine active-site cofactor. Products include mono- and di-oxygenated species, and kynurenine from Trp. Mapping of the modifications to the 3-D structure showed that these are localized to both the inner channel and the external surface, consistent with reactions of free ¹O₂, however the sites and extent of modification do not correlate with their solvent accessibility. These products are generated concurrently with loss of activity, indicative of strong links between these events. These data provide evidence for the impairment of KGDH activity by ¹O₂ via the oxidation of specific residues on the protein subunits of the complex.