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

Authors :
Gao, Qing
Hägglund, Per
Gamon, Luke F.
Davies, Michael J.
Source :
Free Radical Biology & Medicine. Nov2024, Vol. 224, p723-739. 17p.
Publication Year :
2024

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 O 2 , a source of singlet oxygen (1O 2). A rapid loss of activity was detected, with this being dependent on light exposure, illumination time, and the presence of RB and O 2. Activity loss was enhanced by D 2 O (consistent with 1O 2 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 1O 2 , 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 1O 2 via the oxidation of specific residues on the protein subunits of the complex. [Display omitted] • α-Ketoglutarate dehydrogenase complex is a rate-determining enzyme in the tricarboxylic acid (Krebs) cycle. • Photooxidation and 1O 2 formation results in photolysis time- and O 2 -dependent loss of activity. • Protein side-chain modifications were detected by LC-MS and aggregation by SDS-PAGE. • Modifications detected at 45 sites including Met, Trp, His, Tyr residues and the lipoic acid active-site cofactor. • Widespread damage occurs on all of the complex subunits, consistent with reactions of free 1O 2. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
08915849
Volume :
224
Database :
Academic Search Index
Journal :
Free Radical Biology & Medicine
Publication Type :
Academic Journal
Accession number :
180885973
Full Text :
https://doi.org/10.1016/j.freeradbiomed.2024.09.024