PhD Scientific Days 2021

Budapest, 7-8 July 2021

PO_I_P: Pathology and Oncology I. Posters

Proline Oxidation in Mitochondria with Inhibited Complex I Maintains Forward Mode of F0-F1 ATP Synthase Due to High Electron Flow and Ubiquinone Reduction

Gergely Pallag1, Sara Nazarian1, Dora Ravasz1, David Bui1, Jeonghyoun Lee1, Seung Won Jeong1, Judit Doczi1, Christos Chinopoulos1

1 Department of Biochemistry and Molecular Biology, Semmelweis University, 1094 Budapest, Hungary

Text of the abstract

The malfunction or inhibition of the mitochondrial electron transport chain (ETC) complexes can lead to the depletion of cytosolic ATP, which terminates in cell death. It can give rise to several diseases. However, at membrane potentials more negative than the reversal potential of ATP synthase and adenine nucleotide transporter (ANT), these enzymes are still functioning in forward mode. Consequently, they produce ATP and transport it out of the mitochondrial matrix, respectively. Hence, substrates bypassing the blocked components of the mitochondrial ETC and therefore maintaining the sufficient mitochondrial membrane potential may have therapeutic utilization.

Our aim was to investigate the effect of the proline catabolism on the mitochondrial membrane potential and its effect on the ANT and ATP synthase directionality in isolated mouse liver mitochondria and permeabilized cells.

The directionality of the ANT and ATP synthase enzymes was examined fluorimetrically with safranine O dye, recorded in a Hitachi F-7000 spectrofluorimeter.
The oxygen consumption was measured polarographically using Oxygraphy-2k fluorimeter.
The proline dehydrogenase activity was measured spectrophotometrically.
For measurement of the oxidation state of coenzyme Q (CoQ) pool, we used a NextGen-O2k machine.

Proline catabolism in isolated liver mitochondria and permeabilized cells contribute to the maintenance of the forward operation of ANT and ATP synthase under complex I inhibition, but not under the inhibition of complex III and complex IV. This effect of proline catabolism is blocked by the inhibition of the proline dehydrogenase enzyme. Proline catabolism also leads to the reduction of mitochondrial CoQ pool.

During the proline catabolism, electrons are transferred from proline to CoQ by proline dehydrogenase. Reduced CoQ is oxidized by the complex III, which subsequently pumps out protons from the mitochondrial matrix to the intermembrane space. This process helps for ANT and ATP synthase to process in the forward mode, preventing the mitochondria to become a cytosolic ATP consumer when the complex I is blocked.

University and Doctoral School

Semmelweis University, János Szentágothai Doctoral School of Neurosciences