Biochemical and Molecular Characterization of a Na + -Translocating F 1 F o -ATPase from the Thermoalkaliphilic Bacterium Clostridium paradoxum

Abstract

Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56°C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N , N′ -dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum . The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F ₁ F _o -ATP synthase but also included the presence of a stable oligomeric c -ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q ²⁸ , E ⁶¹ , and S ⁶² ). On the basis of these properties, the F ₁ F _o -ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of Na ⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the ε subunit that has been shown to be essential for coupled ATP synthesis.