Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration

Abstract

Aerobic respiration by cells of P. denitrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. denitrificans respiring aerobically in 100 mM Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid]/NaOH or 100 mM-NaH2PO4/NaOH. This potential is decreased by > 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM K+. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150 mV in described suspension media. The presence of these species under aerobic conditions has a negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. None of these molecules exert a significant uncoupling effect on the proton motive force. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore is not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified do not posses K+ transport systems of either the Kdp or TrkA types that have been described in Escherichia coli.