Non‐equilibrium Thermodynamic Assessment of Redox‐Driven H+ Pumps in Mitochondria

Abstract

Isolated mitochondria suspended in an aerobic medium with 3-hydroxybutyrate or succinate serving as electron donor attain a stationary state with vanishing net flow of H+ ions (state 4). Adding valinomycin to such a suspension in the presence of various concentrations of K+ ions and a weak acid system such as acetate or phosphate creates new stationary states for the mitochondria which are characterized by a constant influx of K+ ions, while the net flow of H+ ions again vanishes due to the recycling of these ions by the weak acid system. Sufficiently low concentrations of K+ ions (< 4 mM) cause these stationary states to last long enough for a separation of the mitochondria by centrifugation. The difference in electrochemical potential for H+ ions can then be determined by means of the partitioning of radioactively labeled markers. Suitable procedures to correct for binding of the markers are described. For a constant affinity of the electron in the suspending medium, electron flow and the flow of K+ ions, which indicates a flow of pumped H+ ions, are linearly dependent on the electrochemical potential difference of H+ ions. The phenomenological coefficients obtained from these correlations are discussed with respect to the contributions of additive constants in the linear relations. Under the present experimental condition, such constants most likely vanish thus yielding symmetric flow-force relations. The redox-driven H+ pumps are not tightly coupled due to molecular slipping in the pumps and that the molecular stoichiomery is 2 H+ ions per electron for coupling site I and 4 H+ ions per electron for coupling sites II and III together.