Residual Complex I activity supports glutamate catabolism and mtSLP via canonical Krebs cycle activity during acute anoxia without OXPHOS

Abstract

Anoxia halts oxidative phosphorylation (OXPHOS) causing hyper-reduction of mitochondrial matrix redox compounds which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone redox state in organello in real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U-13C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia and in the presence of site-specific inhibitors of the electron transfer system inferred that NAD+ arising from Complex I is reduced by the oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U-13C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.