The mechanism of proton translocation driven by the respiratory nitrate reductase complex of Escherichia coli

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Abstract
Low concentrations (1-50 .mu.M) of ubiquinol were rapidly oxidized by spheroplasts of E. coli derepressed for synthesis of nitrate reductase using either nitrate or O2 as electron acceptor. Oxidation of ubiquinol drove an outward translocation of protons with a corrected .fwdarw. H+/2e- stoichiometry of 1.49 when nitrate was the acceptor and 2.28 when O2 was the acceptor. Proton translocation driven by the oxidation of added ubiquinol was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA- menA-), whereas a heme-deficient mutant, strain A1004a, did not oxidize ubiquinol. Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ+) to the oxidized species (DQ2+) with nitrate as acceptor, nitrate was reduced to nitrite and the nitrite was further reduced. Nitrite reductase activity was inhibited by CO, leaving nitrate reductase activity unaffected. Benzyl Viologen radical (BV+) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV++. In the presence of CO, 2 protons are consumed. The consumption of these protons could not be detected by a pH electrode in the extracellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. The protons are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulfate and its sulfonated analog N-methylphenazonium-3-sulfonate (PMSH) and ubiquinol1 are all oxidized by nitrate reductase via a heme-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate .fwdarw. H+/2e- stoichiometries were 0 with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulfate or its sulfonated analog, both hydride donors, and 2.0 with ubiquinol1 (QH2), an H donor. The protons appearing in the medium evidently are derived from the reductant and the observed .fwdarw. H+/2e- stoichiometries are accounted for by a set of reactions occurring at a periplasmic aspect of the cytoplasmic membrane. The observed proton translocation in this segment of the respiratory chain arises not from the translocation of protons across the membrane, but from the translocation of electrons associated with consumption of protons at the cytoplasmic aspect of the membrane and proton liberation at the periplasmic aspect.

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