The Na+-translocating F1F0-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9 M Na+, pH(50 degrees C)9.5 and 53 degrees 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-0 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 degrees C and the alkaline pH(50 degrees C) of 93. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg2+ 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 a subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F1F0-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 a 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. (C) 2011 Elsevier B.V. All rights reserved.