Abstract
Oxidant pulse experiments were performed on Paracoccus and Pseudomonas denitrificans grown anaerobically on nitrate to determine the stoichiometry of respira- tion-dependent proton translocation during denitrification. In the presence of valinomycin, the ^H+/2e~ ratio for oxygen was maximally 7-8 (external acidification), in agreement with the reports of others with aerobically grown Paracoccus de- nitrificans. The ratios observed upon the reduction of nitrate, nitrite, and nitrous oxide to nitrogen gas were maximally 4.3, 3.7, and 4.5, respectively. That for the reduction of nitrite to nitrous oxide in the presence of acetylene was about 3.3. These results are qualitatively consistent with previously reported growth-yield experiments on Pseudomonas aeruginosa and Paracoccus denitrificans. When valinomycin was omitted, respiration was partially inhibited (controlled), and the ‘H+/2e” ratio diminished to about 0.2 after several additions of oxygen or nitrous oxide. Under these conditions, the initial event observed upon the addition of nitrite or nitrate was a transient alkalinization with ~~H+/anion = -1 maximally. These alkaline transients were prevented by certain inhibitors of respiration and by dicyclohexylcarbodiimide but not weak acid-type uncouplers of oxidative phosphorylation. Under appropriate conditions the transient exhibited by nitrate showed a substructure in which an acid transient appeared between two alkaline transients. These results show that the uptake of nitrite and nitrate by denitrifiers is coupled with the uptake of one proton (or exit of one hydroxide ion) per anion, and that the site of their reduction is inside the cell or on the inner aspect of the membrane. The proton (or hydroxide) transporter involved is probably the membrane ATPase (Maloney, P. C. (1977) J. Bacteriol. 132, 564), inasmuch as it was blocked by dicyclohexylcarbodiimide and exhibited a threshold for function which seems to be related to the value of the membrane potential. The entry of nitrate and nitrite would appear to be by facilitated diffusion down concentration gradients created by their reductive destruction within the cell. Concerted uptake of a proton with each nitrate (nitrite) anion renders the uptake electrically neutral and also almost exactly compensates for the net alkalinization associated with deni- trification of nitrate and nitrite.