Purification and Biochemical Characterization of the F ₁ F _o -ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

Abstract

We describe here purification and biochemical characterization of the F 1 F o -ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F 1 F o -ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F 1 subunits ␣, ␤, ␥, ␦, and and F o subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F 1 F o holoenzyme or the isolated F 1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F 1 . After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (⌬). A transmembrane proton gradient or sodium ion gradient in the absence of ⌬ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na ؉ /H ؉ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na ؉ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

Single-molecule Analysis of F0F1-ATP Synthase Inhibited by N,N-Dicyclohexylcarbodiimide

“…To confirm that DCCD predominantly reacted with the c subunit in our experiments, we tested whether the bulk ATPase activities of F 0 F 1 recovered from the DCCD inhibition by adding the detergent LDAO. LDAO is known to decouple the ATPase activity of F 1 from the H ϩ -conducting activity of F 0 and thus restore the F 0 F 1 ATPase activity in the presence of LDAO (32). As expected, when 0.2% (w/v) LDAO was added after a 10-min incubation of 1 mM DCCD, more than 80% of the ATPase activity was recovered (Fig.…”

“…After the ATP hydrolysis activity reached steady state, 0.2% (w/v) lauryldimethylamine oxide (LDAO) was added to confirm the reactivation of the activity. It has been reported that LDAO activates DCCD-inhibited F 0 F 1 by inducing the uncoupling of F 0 and F 1 (32).…”

Mutants of Mycobacterium smegmatis unable to grow at acidic pH in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone

“…Proton permeability was determined in membrane vesicles by monitoring the change in external pH with the fluorescent pH probe pyranine (pK a 7?3, excitation and emission wavelengths of 450 and 508 nm respectively), as described by van de Vossenberg et al (1995). The preparation of membrane vesicles of M. smegmatis and B. subtilis was based on previously described protocols (Cook et al, 2003;Rao et al, 2001). These vesicles were prepared with a high buffering capacity on the inside (50 mM MOPS, pH 7?0, 75 mM KCl and 25 mM choline chloride), and dispersed into a solution (2 mg protein ml 21 ) of low buffering capacity (0?5 mM MOPS, pH 7?0, 75 mM KCl and 75 mM sucrose), containing 10 mM pyranine.…”

On the principle of ion selectivity in Na + /H + -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

“…Organism-specific environmental factors, such as temperature or pH, also do not provide a consistent rationale; for example, ATP synthases from thermoalkaliphilic bacteria use a H + gradient despite the scarcity of H + and the potentially greater degree of H + leakage across the membrane at high temperatures, compared with Na + (12,13). Indeed, pmf-and smf-driven systems are often found within the same organism, and sometimes with the same or similar function; for example, malate uptake in Bacillus subtilis is mediated by the H + -coupled symporter CimH (14) and by the Na + -coupled MaeN (15).…”