Mitochondrial respiratory chain Complex II is an inner membrane-bound enzyme that directly connects the TCA cycle with oxidative phosphorylation and the production of ATP. Complex II has been suggested to play a crucial role in regulation of cellular energy metabolism in scenarios such as cancer, diabetes, and inflammation due to its bidirectional conversion of succinate to fumarate at catalytic subunit A (also referred as Complex II-F site) and concomitantly reduces ubiquinone (CoQ10) into ubiquinol (CoQ10H2) at the transmembrane quinone reaction site formed by SDHC and SDHD. It was previously reported that mouse tissues exhibited net reversal of Complex II activity and used fumarate as terminal electron acceptor to protect the ubiquinone/ubiquinol pool redox state during Complex III or IV dysfunction. The data presented here built on this by revealing a cell type-specific mechanism triggered by alterations in the ubiquinone/ubiquinol redox state where the binding of fumarate to the Complex II F-site was blocked by excess succinate dehydrogenase assembly factor 2. The measurements of polar metabolites in HEK293T cells with impaired Complex III revealed that succinate accumulated in the knockout cell lines compared to the wild-type whereas lower levels of the most TCA metabolites were observed. Using 13C6-glucose tracer, a detailed analysis of the isotopologue distribution in key TCA metabolites revealed a decrease in isotopologues associated with operation of a canonical cyclic TCA in which carbon backbones cycle multiple times with a concomitant increase in the unlabelled isotopologue of all TCA intermediates. We also followed up a 13C5-glutamine tracer experiment which showed that elevated levels of succinate dehydrogenase assembly factor 2 inhibited both forward and reverse activity of Complex II.