Passive penetration of nitrate through the plasma membrane of Paracoccus denitrificans and its potentiation by the lipophilic tetraphenylphosphonium cation

Previously, it has been shown that treatment of Paracoccus denitrificans cells with phenylglyoxal inhibits the methyl-viologen-linked nitrate reductase activity by blocking the nitrate transporter. This inhibition disappears if tetraphenylphosphonium cation (TPP+) is added to the assay medium. In the present paper, the following evidence suggests that the effect of TPP+ results from an increased transmembrane anion permeability and not from transporter reactivation or cell lysis. (1) Beside nitrate, TPP+ also mediated the utilisation of chlorate, which normally lacks access to the cytoplasm. (2) The TPP+ pathway had about hundred-times higher K. values for nitrate and chlorate than nitrate reductase in Triton X-100 permeabilised cells. (3) Although the uncoupler CCCP alone failed to overcome the PG block, it stimulated the operation of the TPP+ pathway. (4) The method of continuous variations allowed the transport stoichiometry TPP+/NO3- to be determined as 3, indicating charge compensation for nitrate movement and the subsequent transmembrane two-electron redox reaction. Anion uptake was also measured independently from passive swelling of uncoupled spheroplasts in iso-osmotic solutions of ammonium salts. The permeability to nitrate lay in the permeability sequence Cl-<NO3-<ClO4-<SCN- and was further enhanced by TPP+. (C) 2003 Elsevier Science B.V. All rights reserved.