A sodium ion‐dependent A₁A_O ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus

Abstract

Membrane-bound, multisubunit, ion-translocating ATP synthases ⁄ ATPases are present in every domain of life. They arose from a common ancestor, but evolved into three distinct classes of ATP synthases ⁄ ATPases: the F 1 F O ATP synthase present in bacteria, mitochondria and chloroplasts, the A 1 A O ATP synthase present in archae, and the V 1 V O ATPase associated with cytoplasmic organelles such as vacuoles in eukaryotes [1][2][3][4]. A common feature of ATP synthases ⁄ ATPases is their organization into two domains, a hydrophilic and a membrane-bound domain, that are connected by (at least) two stalks, one central and one to two peripheral [5][6][7][8][9][10][11][12]. The hydrophilic domain catalyzes ATP hydrolysis [13][14][15][16], and the membranebound domain translocates ions from one side of the membrane to the other against their electrochemical gradient [17][18][19][20].ATP synthases ⁄ ATPases are rotary machines that work as a pair of coupled motors: a chemically driven motor (F 1 ⁄ A 1 ⁄ V 1 ) that is attached to the membrane, and an ion gradient-driven membrane-embedded motor (The membrane-embedded motor is composed of a stator and a rotor. The rotor is composed of multiple copies of subunit c that form an oligomeric ring of noncovalently linked subunits, and rotation of the c ring is obligatorily coupled to ion flow across the membrane [28][29][30][31] The rotor subunit c of the A 1 A O ATP synthase of the hyperthermophilic archaeon Pyrococcus furiosus contains a conserved Na + -binding motif, indicating that Na + is a coupling ion. To experimentally address the nature of the coupling ion, we isolated the enzyme by detergent solubilization from native membranes followed by chromatographic separation techniques. The entire membrane-embedded motor domain was present in the preparation. The rotor subunit c was found to form an SDS-resistant oligomer. Under the conditions tested, the enzyme had maximal activity at 100°C, had a rather broad pH optimum between pH 5.5 and 8.0, and was inhibited by diethystilbestrol and derivatives thereof. ATP hydrolysis was strictly dependent on Na + , with a K m of 0.6 mm. Li + , but not K + , could substitute for Na + . The Na + dependence was less pronounced at higher proton concentrations, indicating competition between Na + and H + for a common binding site. Moreover, inhibition of the ATPase by N¢,N¢-dicyclohexylcarbodiimide could be relieved by Na + . Taken together, these data demonstrate the use of Na + as coupling ion for the A 1 A O ATP synthase of Pyrococcus furiosus, the first Na + A 1 A O ATP synthase described.Abbreviation DES, diethylstilbestrol.

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…The A 1 A o -type ATPase (A 1 A o -ATPase) of Pyr. furious uses a sodium rather than a proton gradient (457), and there is some evidence that the proton gradient generated by the Mbh hydrogenase module (comprising the subunits MbhH to MbhN) is converted to a sodium gradient by the Mrp system (Mrp-type Na ϩ /H ϩ antiporter homologs, comprising the subunits MbhA to MbhH) of Mbh (456). This has been discussed as a "simple" form of respiration, which enables the organism to improve the limited energy yield during fermentative growth (see below).…”

The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

“…Determination of Na ϩ Concentration-The Na ϩ concentration in the buffer was determined with an Orion 84-11 ROSS sodium electrode (Thermo Electron Corp., Witchford, United Kingdom) as described (15).…”

“…In the assay that was used previously (6,11), these were provided by Ti 3ϩ (E 0 Ј ϳ Ϫ480 mV) (15). However, Ti 3ϩ is a very strong and unspecific reducing agent.…”

A Na+-translocating Pyrophosphatase in the Acetogenic Bacterium Acetobacterium woodii

“…ϩ Concentration-The Na ϩ concentration in the buffer was determined with an Orion 84-11 ROSS sodium electrode (Thermo Electron Corp., Witchford, UK) as described (18).…”