Proton Translocation Associated with Denitrification in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

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H^+ translocation driven by NO_3^-, NO_2^- and N_2O reductions with endogenous substrates in cells of Rhodopseudomonas sphaeroides forma sp. denitrlficans was investigated by the oxidant pulse method. Upon injection of nitrogenous oxides to anaerobic cells in darkness, an alkaline transient in the external medium was observed, followed by acidification. The alkaline transient was enhanced by carbonyl cyanide m-chloro-phenylhydrazone. When a viologen dye was used as an electron donor in the presence of 1 mM N-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide to preclude respiration-linked H^+ extrusion, addition of KNO_3, KNO_2 and N_2O caused only a rapid alkalinization. The H^+ consumption stoichiometries, H^+/2e^- ratios for NO_3^- reduction to NO_2^-, NO_2^- reduction to 1/2 N_2O and N_2O reduction to N_2 were -1.90, -3.18 and -2.04, respectively. These values agreed well with the fact that all reductions of nitrogenous oxides in denitrification occur on the periplasmic side of the cytoplasmic membrane. When corrected for H^+ consumption in the periplasm, the H^+ extrusion stoichiometries, H^+/2e^- ratios with endogenous substrates in the presence of K^+/valinomycin for NO_3^- reduction to NO_2^-, NO_2^- reduction to 1/2 N_2O and N_2O reduction to N_2 were 4.05, 4.95 and 6.01, respectively.
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