ATP synthesis in cell envelope vesicles of Halobacterium halobium driven by membrane potential and/or base-acid transition.

Abstract

Cell envelope vesicles active in ATP synthesis were prepared from Halobacterium halobium cells, which genetically lack bacteriorhodopsin, by sonication in the presence of substrates. ATP was synthesized when vesicles were illuminated to build up membrane potential through the action of halorhodopsin. The threshold value of membrane potential for ATP synthesis was about -100 mV relative to the external medium, i.e., inside-negative. ATP synthesis also occurred in the dark upon acidification of the external medium of a suspension of cell envelope vesicles. This base-acid transition ATP synthesis took place when the pH difference was greater than 1.6 units. The threshold pH difference was lowered when the base-acid transition was carried out under dim light which induced a membrane potential of about -100 mV. Regardless of the sort of driving force, ATP synthesis was optimum at the intravesicular pH of around 6.5 and almost nil at 8, where ATP syntheses by F0F1 type ATPases in other organisms are most active. The synthesis could be inhibited by N,N'-dicyclohexylcarbodiimide (DCCD) with a half-maximum inhibition at around 25 microM/2 mg protein/ml. These results strongly suggest that in halobacteria a DCCD-sensitive H+-translocating ATP synthase is in operation which is driven by membrane potential and/or pH gradient, and obeys chemiosmotic energetics. The results also suggest that the ATP synthase may not be identical to F0F1 type H+-translocating ATPases found in mitochondria, chloroplasts and eubacteria.