MITOCHONDRIAL SUBSTRATE-LEVEL PHOSPHORYLATION: FROM METABOLIC INTERFERENCE TO SMALL MOLECULE INTERACTIONS
Bui Dávid
János Szentágothai Neurosciences Division
Dr. Bereczki Dániel
SE Bőr-, Nemikórtani és Bőronkológiai Klinika előadóterme
2026-06-16 14:00:00
Functional neurosciences
Dr. Sperlágh Beáta
Dr. Chinpoulos Christos
Dr. Gallyas Ferenc
Dr. Kardon Tamás
Dr. Mandl József
Dr. Káldi Krisztina
Dr. Pázmándi Krisztina
We investigated 2-oxobutyrate-mediated substrate-level phosphorylation. Using isolated mitochondria, we demonstrated their ability to take up and metabolise exogenous 2-oxobutyrate. Under rotenone-induced pseudoanoxic conditions, we found that 2-oxobutyrate competed with the metabolism of 2-oxoglutarate and decreased the amount to ATP generated by substrate-level phosphorylation in a concentration-dependent way due to the ATP consumption by the propionyl-CoA carboxylase.
We investigated the ability of MDH2 to support substrate-level phosphorylation under SDH inhibition. Oxaloacetate was supplied to the mitochondrial matrix by aspartate transamination. In anoxia, under atpenin inhibition, we found that isolated mitochondria were capable of sufficient NAD regeneration to maintain substrate-level phosphorylation from 2-oxoglutarate and marginally from glutamate. Omitting the MDH2 step by replacing aspartate with malate caused ANT reversal under the same conditions.
We investigated the possibility of SDH reversal functioning as the electron acceptor pathway in anoxia when malate is used as a respiratory co-substrate with isolated mitochondria. Using the combination of stable isotope tracing with GC-MS and mathematical modelling, we found a coordinated effort by KGDHC and SDH that maintained substrate-level phosphorylation. The NAD for KGDHC is regenerated by oxidation through complex I, coupled to fumarate reduction by SDH. Fumarate is provided by the reversible dehydration of malate.
We investigated the potential for safranin to interfere with electron transfer processes in mitochondria. We demonstrated safranin’s photoinduced reduction by NADH. Under aerobic conditions, reduced safranin is rapidly oxidised by oxygen and produces hydrogen peroxide. With intact mitochondria, the light-excitation-associated reaction is very little. In the absence of light excitation, intact mitochondria were still capable of reducing safranin. Fumarate and indirectly malate facilitated the oxidation of reduced safranin through SDH. This was sensitive to malonate but not to atpenin with solubilized mitochondria, suggesting the participation of the flavin site.
To find an inhibitor for STK, we first expressed and purified the recombinant ATP-specific human enzyme. We determined the conditions for its stability and then screened a library of 10 million compounds to find the leads with the highest binding affinity.
