Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO₃^-, NO₂^-, N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO₂^-to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO₃^-, N₂O or O₂ was added to cells in the dark or with these compounds and NO₂^-in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO₃^-, NO₂^-, N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO₃^-0.5N₂, 4.82; NO₂^-0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO₃^-, NO₂^-or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO₃^-NO₂^-,-1.95; NO₂^-0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide