Promiscuous archaeal ATP synthase concurrently coupled to Na ⁺ and H ⁺ translocation

Abstract

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). ...

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

“…All c-rings known to be physiologically coupled to transmembrane Na + gradients can also bind H + , although functional coupling to H + (i.e., catalytic activity) has been observed typically only in in vitro conditions where the concentration of Na + is sufficiently low, e.g., less than 50 μM at pH 7 (39). [Thus, far, only the ATP synthase from Methanosarcina acetivorans has been observed to mediate Na + and H + permeation concurrently in physiological conditions (17).] Nevertheless, DCCD-labeling assays of Na + -coupled c-rings have clearly demonstrated that a competition between H + and Na + exists in a broader concentration range (40,41).…”

“…In previous theoretical studies, we have addressed this question for the family of ion-motive ATPases (17,29,30), which comprises eukaryotic and prokaryotic ATP synthases, as well as vacuolar ion pumps. In these multicomponent enzymes, a membrane-embedded substructure known as the rotor ring can revolve around its axis, relative to the rest of the protein's membrane domain.…”

“…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). Moreover, specific membrane transporters and enzymes are sometimes coupled to both Na + and H + , either concurrently (using multiple binding sites), such as the multidrug efflux pump NorM of Vibrio cholera (16) and the Methanosarcina acetivorans ATP synthase (17), or alternately (using a single binding site), such as the Escherichia coli melibiose permease (18) and some membraneintegral pyrophosphatases (19).…”

Genomic composition and dynamics amongMethanomicrobialespredict adaptation to contrasting environments

“…Up to this point, based on studies of Methanobacteriales and Methanococcales methanogens, it was thought that all cytochrome-deficient hydrogenotrophic methanogens conserve energy using a Na + transmembrane chemiosmotic gradient, which is generated by tetrahydromethanopterin S-methyltransferase (Mtr) and consumed by a Na + -translocating ATP synthase (Schlegel and Müller, 2013). According to the new hypothesis, Methanomicrobiales members are expected to generate a H + gradient using Mtr (as supported by previous analyses of the MtrE subunit; Bräuer et al, 2015), and consume the H + gradient, thus generating ATP, using a H + /Na + promiscuous ATP synthase (Schlegel et al, 2012;Bräuer et al, 2015).…”

The role of proton and sodium ions in energy transduction by respiratory complex I

“…Very interesting was the observation that some ATP synthases are capable of simultaneously translocate Na 1 and H 1 . Such an example is the enzyme from Methanosarcina acetivorans, and its coupling mechanism was suggested to be an adaptation to overcome the weak energy gain resulting from methanogenesis (50). The involvement of the two coupling ions concurrently has also been observed in some cation/solute symporters (as described below for the symporter CitS) (51).…”