The kinetics of oxygen-induced proton efflux and membrane energization in Escherichia coli

The kinetics of respiration-dependent proton efflux and membrane energization have been studied in intact cells of logarithmic phase Escherichia coli. Proton efflux following a small O2 pulse is slow (t1/2 approximately equal to 10 sec) and inefficient (H+/O approximately equal to 0.5), taking 5-10 times longer than expected from the time required for the cells to reduce the O2 added in the pulse. A much closer agreement is found in cells treated to enhance counter ion fluxes and eliminate the transmembrane electric potential (deltapsi). In cells treated with SCN-, or with colicin E1 (which enhances K+ permeability), the rates of proton efflux are much faster (t1/2 less than or equal to 1 sec) than in untreated cells. The kinetics of formation and dissipation of deltapsi were estimated from changes in the fluorescence properties of the cell envelope bound probe N-phenyl-l-naphthylamine. In untreated cells, a small O2 pulse induces a rapid (t1/2 less than or equal to 0.5 sec) decrease in fluorescence intensity followed by a slower (t1/2 approximately equal to 40 sec) return of the fluorescence to the original level. The extent of the initial fluorescence decrease is proportional to the amount of O2 added, although the half-time for the relaxation is independent of the amount of O2 added. Colicin E1 (plus K+) and the uncoupler FCCP greatly decrease the half-time of the relaxation, while only slightly affecting the extent of the initial decrease, indicating that the initial fluorescence decrease is reporting the energization of the membrane while its relaxation is reporting the subsequent deenergization of the membrane resulting from counterion redistributions. The fact that the efflux of H+ into the medium after an O2 pulse is small and much slower (t1/2 approximately equal to 10 sec) than the actual energization of the membrane (t1/2 less than or equal to 0.5 sec) suggests that the current of respiratory H+ involved in membrane energization is confined within the bacterial cell envelope.