H+/PPi Stoichiometry of Membrane-Bound Pyrophosphatase of Rhodospirillum rubrum

Two kinetic methods have been used to measure the H+/PPi stoichiometry in the chomatophores of the photosynthetic bacteria Rhodospirillum rubrum. In the first method, the fluorescent probe acridine orange was employed to infer the proton pump activity at the steady state of the ΔpH generation. At this point the translocation of protons by the H+-PPiase in one direction is balanced exactly by the leak of protons in the opposite direction. Pyrophosphatase activity was then quickly stopped by adding EDTA, producing a relaxation of ΔpH. From the initial rate of this relaxation and the rate of PPi hydrolysis measured under the same condition, the H+/PPi stoichiometry was obtained. In the second method, a mathematical model was used to describe the time course of ΔpH formation. In the two methods an apparent H+/PPi stoichiometry of nearly 2 was obtained. The H+/ATP stoichiometry was determined also as an internal control, giving a value of nearly 3.6, which is in agreement with the value in different F-type H+-ATPases.