ATP synthases are the primary source of ATP in all living cells. To catalyze ATP synthesis, these membrane-associated complexes use a rotary mechanism powered by the transmembrane diffusion of ions down a concentration gradient. ATP synthases are assumed to be driven either by H + or Na + , reflecting distinct structural motifs in their membrane domains, and distinct metabolisms of the host organisms. Here, we study the methanogenic archaeon Methanosarcina acetivorans using assays of ATP hydrolysis and ion transport in inverted membrane vesicles, and experimentally demonstrate that the rotary mechanism of its ATP synthase is coupled to the concurrent translocation of both H + and Na + across the membrane under physiological conditions. Using free-energy molecular simulations, we explain this unprecedented observation in terms of the ion selectivity of the binding sites in the membrane rotor, which appears to have been tuned via amino acid substitutions so that ATP synthesis in M. acetivorans can be driven by the H + and Na + gradients resulting from methanogenesis. We propose that this promiscuity is a molecular mechanism of adaptation to life at the thermodynamic limit.F rom archaea to man, the common enzyme in cellular bioenergetics is the ATP synthase. This membrane-bound macromolecular complex produces ATP, the primary energy source in the cell, at the expense of transmembrane ion gradients established by diverse enzymes, which vary across organisms and organelles. ATP synthases and related ATPases arose from a common ancestor and evolved into three distinct classes: the V 1 V 0 ATPase present in eukarya; the F 1 F 0 ATP synthase found in bacteria, mitochondria, and chloroplasts; and the A 1 A 0 ATP synthase present in archaea (1, 2). All of these synthases are rotary machines that comprise two coupled units, one residing in the cytoplasm (F 1 /A 1 /V 1 ), and another (F o /A o /V o ) embedded in the membrane. The former catalyzes ATP synthesis (or hydrolysis), and the latter is coupled to the transmembrane electrochemical potential (3)(4)(5)(6)(7)(8).The ion specificity of ATP synthases/ATPases is encoded by the membrane-embedded domain, and in particular in the rotor ring. This ring is an oligomeric assembly of multiple copies of subunit c, each of which carries at least one ion binding site. The architecture of the membrane domain enables the rotation of the c ring around its central axis in a manner that is tightly coupled to ion flow across the membrane (9-12). The rotation of the c ring is also mechanically transmitted to the cytoplasmic domain, where it sustains a conformational cycle conducive to ATP synthesis (13,14).Most F 1 F 0 ATP synthases and V 1 V 0 ATPases use protons as the coupling ion, but some use sodium ions instead (15-17). However, Na + -driven F 1 F 0 ATP synthases have so far been found only in anaerobic bacteria, whose metabolism leads to the generation of a primary Na + gradient, rather than a H + gradient (16,17). Na + is clearly preferred over H + in these ATP synthases (18,19). ...

The anaerobic methanogenic archaeon Methanosarcina acetivorans lives under extreme energy limitation. Methanogenesis from acetate as carried out by M. acetivorans involves an anaerobic electron transport chain with ferredoxin as electron donor and heterodisulfide as electron acceptor, and so far only the heterodisulfide reductase has been shown to translocate H + . Here, we describe a second Na + -translocating coupling site in this electron transport chain. Inside-out membrane vesicles of M. acetivorans catalyzed Na + transport coupled to an electron transport catalyzed by the ferredoxin:heterodisulfide oxidoreductase activity. Ionophore studies revealed that Na + transport was primary and electrogenic. A Δrnf mutant was unable to grow on acetate and the ferredoxin:heterodisulfide oxidoreductase-coupled Na + transport was abolished. These data are consistent with the hypothesis that the Rnf complex of M. acetivorans is an Na + -translocating coupling site and the entry point of electrons derived from reduced ferredoxin into the electron transport chain leading to the heterodisulfide.

Enzymatic hydrolysis of lupin protein isolates (LPI; Lupinus angustifolius L.) was performed with nine different protease preparations to investigate their effect on technofunctionality, sensory properties, and the integrity of the proteins to estimate the reduction of the immunoreactivity. Alcalase 2.4 L, papain, and pepsin were most effective in the degradation of the α‐ and β‐conglutin examined by SDS–PAGE analysis, although the degree of hydrolysis only slightly increased. The technofunctional properties of LPI—solubility, emulsifying, and foaming activity—were improved by most of the proteolytic enzymes with the most impressive increase from 980% foam activity for LPI up to 3,614% foam activity for pepsin hydrolysate. The formation of bitterness, most likely linked to generation of bitter peptides, was pronounced in the Alcalase hydrolysate, while the other hydrolysates did not show an extensive increase in bitterness compared to the LPI. Other sensory attributes of the hydrolysates—with the exception of Alcalase treatment—were also very similar to the LPI. The results of this study show the potential of enzymatic degradation of LPI to modify the IgE‐reacting polypeptides and to improve the technofunctionality of the isolates and therefore their use as food ingredients.

Lupin protein isolate was fermented with eight different microorganisms to evaluate the influence on sensory profile, techno-functional properties and protein integrity. All investigated microorganisms were able to grow in lupin protein isolate. The results showed that the foaming activity in the range of 1646–1703% and the emulsifying capacity in the range of 347–595 mL of the fermented lupin protein isolates were similar to those of the unfermented ones. Protein solubility at pH 4 showed no significant changes compared to unfermented lupin protein isolate, whereas the solubility at pH 7 decreased significantly from 63.59% for lupin protein isolate to solubilities lower than 42.35% for fermented lupin protein isolate. Fermentation with all microorganisms showed the tendency to decrease bitterness from 2.3 for lupin protein isolate (LPI) to 1.0–2.0 for the fermented ones. The most promising microorganisms for the improvement of the sensory properties of lupin protein isolates were Lactobacillus brevis as it reduced the intensity of characteristic aroma impression (pea-like, green bell pepper-like) from 4.5 to 1.0. The SDS-PAGE results showed the fermentation treatment appeared not to be sufficiently effective to destruct the protein integrity and thus, deplete the allergen potential of lupin proteins. Fermentation allows the development of food ingredients with good functional properties in foam formation and emulsifying capacity, with a well-balanced aroma and taste profile.