The Na +-translocating F 1F O-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Abstract

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3–3.9 M Na +, pH 50 °C 9.5 and 53 °C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F 1 portion and 3-subunit-F O portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na + (5 mM); hydrolysis activity was inhibited by higher Na + concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47 °C and the alkaline pH 50 °C of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N′-dicylohexylcarbodiimide and exhibited inhibition by both free Mg 2+ and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na +-ion gradient that was sensitive to the Na +-ionophore monensin. Analysis of the atp operon showed the presence of the Na +-binding motif in the c subunit (Q 33, E 66, T 67, T 68, Y 71), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F 1F O-ATPase of N. thermophilus is a Na +-translocating ATPase used primarily for expelling cytoplasmic Na + that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na +-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na + needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.