The proton pumping inorganic pyrophosphatase from Rhodospirillum rubrum

Sammanfattning: The aim of the present investigation was to study the mechanism by which light-energy is transformed to biologically useful chemical-energy in the form of inorganic pyrophosphate (PPi). The study was performed on chromatophores (subcellular membrane structures) from the photosynthetic nonsulphur bacterium Rhodospiri Hum rubrum.The enzyme catalyzing the synthesis of PPi, the H+-PPase, was solubilized from the chromatophores and subsequently purified using hydroxyapatite chromatography. The purified enzyme was very specific for PPi and required the presence of magnesium ions. The purified enzyme was activated by phospholipids. Cardiolipin was shown to have the strongest activating effect. We suggest that the H+-PPase in the chromatophores is in direct contact with some cardiolipin molecules.We have demonstrated that the H+-PPase, after reconstitution into liposomes, regains certain characteristics, which have been lost upon solubilization and which are typical for proton translocating enzymes, such as stimulation of hydrolysis by uncoupler. The results indicate that the enzyme is a proton pump.We have also shown that PPi can drive ATP synthesis in a model system, assembled by incorporation of the H+-PPase together with the H+-ATPase into pre-formed liposomes.Two methods for detecting the enzyme activity were developed. The first method, a pH change method, was used for screening the hydrolytic activity, e.g., after different purification steps. The second, very sensitive method made continuous monitoring of PPi synthesis possible for the first time. This method opens up new possibilities for obtaining a more detailed picture of the events involved in the photophosphorylation of Pi to PPi.