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 … Show more
“…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.…”
Section: Kinetic Equivalence Of Pmf Components May Depend On Experimementioning
confidence: 99%
“…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.…”
Section: Kinetic Equivalence Of Pmf Components May Depend On Experimementioning
confidence: 99%
“…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.…”
The ATP synthase (F-ATPase) is a highly complex rotary machine that synthesizes ATP, powered by a proton electrochemical gradient. Why did evolution select such an elaborate mechanism over arguably simpler alternating-access processes that can be reversed to perform ATP synthesis? We studied a systematic enumeration of alternative mechanisms, using numerical and theoretical means. When the alternative models are optimized subject to fundamental thermodynamic constraints, they fail to match the kinetic ability of the rotary mechanism over a wide range of conditions, particularly under low-energy conditions. We used a physically interpretable, closed-form solution for the steady-state rate for an arbitrary chemical cycle, which clarifies kinetic effects of complex free-energy landscapes. Our analysis also yields insights into the debated "kinetic equivalence" of ATP synthesis driven by transmembrane pH and potential difference. Overall, our study suggests that the complexity of the F-ATPase may have resulted from positive selection for its kinetic advantage.ATP synthase | kinetic mechanism | free-energy landscape | nonequilibrium steady state | evolution T he F-ATPase performs molecular-level free-energy (FE) transduction to phosphorylate ADP and yield ATP, the primary energy carrier that drives a vast range of cellular processes. It spurred Mitchell's chemiosmotic hypothesis (1) and Boyer's now-validated proposal for the binding-change mechanism (2) in which a proton electrochemical gradient is transduced to rotationbased mechanical energy and then back to chemical FE as ADP is phosphorylated. The F-ATPase's two-domain uniaxial rotary structure is conserved across all three domains of life (3-6), and details of its function have been the subject of a multitude of studies (e.g., refs. 7-30).Here, we address a relatively narrow question with potentially significant evolutionary implications: Why is ATP synthesized by a rotary mechanism instead of a potentially much simpler alternating-access mechanism (Fig. 1)? Using thermodynamic and kinetic constraints, we address the question of whether evolution tends to arrive at optimal molecular processes (31), building on established concepts of optimality-derived evolutionary convergence (32, 33). Presumably, performance advantages in the central task of ATP synthesis would be under significant evolutionary pressure. Previous modeling studies of the F-ATPase have addressed structural and mechanistic questions about the rotary mechanism (e.g., refs. 11-20), but not the metaissue of the mechanism itself compared with alternatives.To assess whether the rotary mechanism possesses any intrinsic performance advantage, we constructed a series of kinetic models abstracted from known mechanisms (Fig. 1). Beyond the rotarybased model, we considered a series of alternating-access analogs (Fig. 2), building on the demonstrated capacity for ATP-hydrolyzing transporters to be driven in reverse to synthesize ATP (34, 35). The discrete-state models do not include structural details, bu...
“…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.…”
Section: Kinetic Equivalence Of Pmf Components May Depend On Experimementioning
confidence: 99%
“…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.…”
Section: Kinetic Equivalence Of Pmf Components May Depend On Experimementioning
confidence: 99%
“…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.…”
The ATP synthase (F-ATPase) is a highly complex rotary machine that synthesizes ATP, powered by a proton electrochemical gradient. Why did evolution select such an elaborate mechanism over arguably simpler alternating-access processes that can be reversed to perform ATP synthesis? We studied a systematic enumeration of alternative mechanisms, using numerical and theoretical means. When the alternative models are optimized subject to fundamental thermodynamic constraints, they fail to match the kinetic ability of the rotary mechanism over a wide range of conditions, particularly under low-energy conditions. We used a physically interpretable, closed-form solution for the steady-state rate for an arbitrary chemical cycle, which clarifies kinetic effects of complex free-energy landscapes. Our analysis also yields insights into the debated "kinetic equivalence" of ATP synthesis driven by transmembrane pH and potential difference. Overall, our study suggests that the complexity of the F-ATPase may have resulted from positive selection for its kinetic advantage.ATP synthase | kinetic mechanism | free-energy landscape | nonequilibrium steady state | evolution T he F-ATPase performs molecular-level free-energy (FE) transduction to phosphorylate ADP and yield ATP, the primary energy carrier that drives a vast range of cellular processes. It spurred Mitchell's chemiosmotic hypothesis (1) and Boyer's now-validated proposal for the binding-change mechanism (2) in which a proton electrochemical gradient is transduced to rotationbased mechanical energy and then back to chemical FE as ADP is phosphorylated. The F-ATPase's two-domain uniaxial rotary structure is conserved across all three domains of life (3-6), and details of its function have been the subject of a multitude of studies (e.g., refs. 7-30).Here, we address a relatively narrow question with potentially significant evolutionary implications: Why is ATP synthesized by a rotary mechanism instead of a potentially much simpler alternating-access mechanism (Fig. 1)? Using thermodynamic and kinetic constraints, we address the question of whether evolution tends to arrive at optimal molecular processes (31), building on established concepts of optimality-derived evolutionary convergence (32, 33). Presumably, performance advantages in the central task of ATP synthesis would be under significant evolutionary pressure. Previous modeling studies of the F-ATPase have addressed structural and mechanistic questions about the rotary mechanism (e.g., refs. 11-20), but not the metaissue of the mechanism itself compared with alternatives.To assess whether the rotary mechanism possesses any intrinsic performance advantage, we constructed a series of kinetic models abstracted from known mechanisms (Fig. 1). Beyond the rotarybased model, we considered a series of alternating-access analogs (Fig. 2), building on the demonstrated capacity for ATP-hydrolyzing transporters to be driven in reverse to synthesize ATP (34, 35). The discrete-state models do not include structural details, bu...
“…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.…”
Section: Subunit C Of the Central Stalk And Its Interaction With The mentioning
confidence: 99%
“…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).…”
The eubacterium Thermus thermophilus uses a macromolecular assembly closely related to eukaryotic V-ATPase to produce its supply of ATP. This simplified V-ATPase offers several advantages over eukaryotic V-ATPases for structural analysis and investigation of the mechanism of the enzyme. Here we report the structure of the complex at ∼16 Å resolution as determined by single particle electron cryomicroscopy (cryo-EM). The resolution of the map and our use of cryo-EM, rather than negative stain EM, reveals detailed information about the internal organization of the assembly. We could separate the map into segments corresponding to subunits A and B, the threefold pseudosymmetric C-subunit, a central rotor consisting of subunits D and F, the L-ring, the stator subcomplex consisting of subunits I, E, and G, and a micelle of bound detergent. The architecture of the V O region shows a remarkably small area of contact between the I-subunit and the ring of L-subunits and is consistent with a two half-channel model for proton translocation. The arrangement of structural elements in V O gives insight into the mechanism of torque generation from proton translocation. membrane protein | single particle analysis V acuolar-type ATPases (V-ATPases) in eukaryotes function as ATP-driven proton pumps that acidify intracellular compartments including lysosomes, endosomes, and secretory vesicles. This acidification, in turn, affects diverse processes including protein sorting and degradation, overall ion homeostasis, and protection of cells from oxidative stress (1). Extracellular acidification by V-ATPases is linked to tumor invasion and metastasis and osteoporosis (2). F-type ATP synthases and V-type ATPases are evolutionarily related but differ in the details of subunit composition and arrangement. Both F-and V-type ATPases use a rotary catalytic mechanism where proton translocation through the membranebound F O or V O region, respectively, generates a torque on a rotor subcomplex that drives ATP synthesis in the F 1 or V 1 region. The enzymes can also run in the opposite direction with ATP hydrolysis in the F 1 or V 1 region resulting in proton pumping through F O or V O . This mechanism has been the subject of a large body of research for F-type ATP synthases (e.g., 3-5), but there has also been direct demonstration of rotary catalysis for V-ATPases (6). Some archaea and eubacteria use a complex more closely related to VATPase than F-type ATP synthase, sometimes called an A-ATPase, to generate their supply of ATP (7).The V-ATPase from the eubacterium Thermus thermophilus is composed of nine different subunits with a stoichiometry of A 3 B 3 CDE 2 FG 2 IL 12 (Fig. S1). Subunit nomenclature for this family of enzymes differs between F-and V-type complexes and from organism to organism. Where the eukaryotic ATP synthase F 1 catalytic region consists of α 3 β 3 γδε, the V-ATPase V 1 catalytic region consists of B 3 A 3 DF with no equivalent of the ε-subunit. The catalytic A-subunit of V-ATPase is homologous to the F-type ATP synthas...
Antibiotic‐associated infections with Clostridioides difficile are a severe and often lethal risk for hospitalized patients, and can also affect populations without these classical risk factors. For a rational design of therapeutical concepts, a better knowledge of the metabolism of the pathogen is crucial. Metabolic modeling can provide a simulation of quantitative growth and usage of metabolic pathways, leading to a deeper understanding of the organism. Here, we present an elaborate genome‐scale metabolic model of C. difficile 630Δerm. The model iHD992 includes experimentally determined product and substrate uptake rates and is able to simulate the energy metabolism and quantitative growth of C. difficile. Dynamic flux balance analysis was used for time‐resolved simulations of the quantitative growth in two different media. The model predicts oxidative Stickland reactions and glucose degradation as main sources of energy, while the resulting reduction potential is mostly used for acetogenesis via the Wood–Ljungdahl pathway. Initial modeling experiments did not reproduce the observed growth behavior before the production of large quantities of a previously unknown polysaccharide was detected. Combined genome analysis and laboratory experiments indicated that the polysaccharide is an acetylated glucose polymer. Time‐resolved simulations showed that polysaccharide secretion was coupled to growth even during unstable glucose uptake in minimal medium. This is accomplished by metabolic shifts between active glycolysis and gluconeogenesis which were also observed in laboratory experiments.
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