Non-equilibrium thermodynamic assessment of redox-driven H+ pumps in mitochondria

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 (less than 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 labelled markers. Suitable procedures to correct for binding of the markers are described. It is found that, for a constant affinity of the electron in the suspending medium, electron flow and the flow of K+ ions, which indicates the 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. It is found that, under the present experimental condition, such constants most likely vanish thus yielding symmetric flow-force relations. It is concluded that the redox-driven H+ pumps are not tightly coupled due to molecular slipping in the pumps and that the molecular stoichiometry is 2 H+ ions/electron for coupling site I and 4 H+ ions/electron for coupling sites II and III together.