Functional production of an archaeal ATP synthase with a V-type c subunit in Escherichia coli.

ATP synthases are the key elements of cellular bioenergetics and present in any life form and the overall structure and function of this rotary energy converter is conserved in all domains of life. However, ancestral microbes, the archaea, have a unique and huge diversity in the size and number of ion-binding sites in their membrane-embedded rotor subunit c. Due to the harsh conditions for ATP synthesis in these life forms it has never been possible to address the consequences of these unusual c subunits for ATP synthesis. Recently, we have found a Na ⁺ -dependent archaeal ATP synthase with a V-type c subunit in a mesophilic bacterium and here, we have cloned and expressed the genes in the ATP synthase-negative strain Escherichia coli DK8. The enzyme was present in membranes of E. coli DK8 and catalyzed ATP hydrolysis with a rate of 35 nmol·min ^-1 ·mg protein ^-1 . Inverted membrane vesicles of this strain were then checked for their ability to synthesize ATP. Indeed, ATP was synthesized driven by NADH oxidation despite the V-type c subunit. ATP synthesis was dependent on Na ⁺ and inhibited by ionophores. Most importantly, ATPase activity was inhibited by DCCD and this inhibition was relieved by addition of Na ⁺ , indicating a functional coupling of the F ₁ and F _O domains, a prerequisite for studies on structure-function relationship. A first step in this direction was the exchange of a conserved arginine (Arg ⁵³⁰ ) in the F _O motor subunit a which led to loss of ATP synthesis whereas ATP hydrolysis was retained.
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