Dodecamer rotor ring defines H ⁺ /ATP ratio for ATP synthesis of prokaryotic V-ATPase from Thermus thermophilus

Abstract

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (⌬pH) and the electrical potential differences (⌬⌿) across membranes. The optimum rate of synthesis reached 85 s ؊1 , and the H ؉ /ATP ratio of 4.0 ؎ 0.1 was obtained. ATP was synthesized at a considerable rate solely by ⌬pH, indicating ⌬⌿ was not absolutely required for synthesis. Consistent with the H ؉ /ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 Vo-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases. ATP synthase ͉ rotary motor ͉ membrane protein ͉ bioenergetics ͉ two-dimensional crystal M embers of the F o F 1 and V-ATPase superfamily (rotary ATPase/synthase) use a rotary catalytic mechanism to perform their specific function (1, 2). The F o F 1 mainly catalyzes ATP synthesis in mitochondria, chloroplasts, and aerobic bacteria (3, 4). In contrast, V-ATPases exist in the endomembranes of all eukaryotic cells and in the plasma membrane of some specific eukaryotic cells functioning as a proton pump with a variety of cellular functions (2). The homologues of eukaryotic V-ATPases are also found in the plasma membrane of some bacteria (5, 6). Like the F o F 1 , V-ATPase from the thermophilic eubacterium Thermus thermophilus catalyzes ATP synthesis (7,8). In addition, it has the simplest known subunit structure (Fig. 1a) and is thus an excellent model for studying the mechanism of action of these important molecules. Subunits A and B of V-ATPase are the counterparts of subunits ␤ and ␣ of F o F 1 ATPase. Three copies of each subunit are arranged around the central rotor, which is made of single copies of subunits D and F. The A 3 B 3 DF moiety, termed V 1 , is responsible for the ATP hydrolysis or ATP synthesis reaction. The remaining subunits, V o -a (sometimes referred to as subunit I), V o -c (sometimes referred to as subunit L), and V o -d, E, and G form the V o domain of T. thermophilus V-ATPase (9). The V o -c subunits, which are folded into two transmembrane helices, constitute a membraneembedded oligomeric ring structure (10). The V o -c rotor ring and subunit V o -a form a proton channel, as seen in the c rotor ring of the F o -a subunit, despite low sequence similarity between the proteins.The basic mechanism of ATP synthesis for F o F 1 is well understood, as described below. Briefly, the ring of the F o -c subunit oligomer and ␥-subunits of F 1 comprise the central rotor, and together these rotate as a single body (11). The transmembrane electrochemical potential gradient of proton [⌬ H ϩ ϭ PMF ϫ F (PMF, proton motive force; F, Faraday constant)] drives rotation of the roto...

Biophysical comparison of ATP synthesis mechanisms shows a kinetic advantage for the rotary process

“…Our analysis suggests that variant experimental conditions likely underlie conflicting evidence regarding kinetic equivalence of pmf components Δ pH and Δψ. Some in vitro studies have found that the effect of the two components of pmf are equivalent (39)(40)(41)(42), whereas others have concluded that they are not (43)(44)(45). There is no inherent reason to expect kinetic equivalence (53) because Δ pH and Δψ affect kinetics through different mechanisms: Δ pH through H + concentration, and Δψ most likely through binding affinity dictated by the thermodynamic cycle constraint, as described in SI Appendix.…”

“…Green arrows represent range of productive operating condition for each of the ATPases shown. Red circles represent examples of reported stoichiometry and physiological conditions (42,48,60,61,(63)(64)(65)(66)(67). The diagonal represents equilibrium behavior where the efficiency, as defined in the text, is maximum but the rate of synthesis or pumping would vanish.…”

“…Additional analysis sheds light on contradictory reports on the "kinetic equivalence" of different components of the driving protonmotive force (pmf) (39)(40)(41)(42)(43)(44)(45)(46): should the synthesis rate be sensitive to whether the pmf is generated by a pH difference or transmembrane potential difference? We show that kinetic behavior is approximately equivalent under most physiological conditions, but that variant conditions can clearly exhibit nonequivalence.…”

Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound V O motor

“…3B) further supports the hypothesis that the map represents an average sampled from images of particles that have been trapped with the rotor in a single position. The L-subunit is known to form a dodecameric ring (14). Automatic docking of a comparative model of the L 12 -ring positioned the ring with the connecting loops of the transmembrane helices of the L-subunits facing towards the V 1 region (Fig.…”

“…Instead of a single peripheral stalk, as found in the F-type ATP synthase (11,12), V-ATPases have more than one peripheral stalk, each consisting of an EG heterodimer in T. thermophilus (13). The c-ring in F O is homologous to the c-ring of eukaryotic V-ATPase and the L 12 -ring of the T. thermophilus enzyme (14), whereas the membrane-bound a-subunit contains an additional N-terminal domain in V-ATPase and is called subunit I in the T. thermophilus enzyme. The C-subunit of T. thermophilus forms a part of the central stalk unique to the V-ATPase family, coupling V 1 to the proteolipid-ring of L-subunits (15).…”