Alternative proton binding mode in ATP synthases

ATP synthases are rotary engines which use the energy stored in a transmembrane electrochemical gradient of protons or sodium ions to catalyze the formation of ATP by ADP and inorganic phosphate. Current models predict that protonation/deprotonation of specific amino acids of the rotating c-ring, extracting protons from one side and delivering them to the other side of the membrane, are at the core of the proton translocation mechanism of these enzymes. In this minireview, an alternative proton binding mechanism is presented, considering hydronium ion coordination as proposed earlier. Biochemical data and structural considerations provide evidence for two different proton binding modes in the c-ring of H⁺-translocating ATP synthases. Recent investigations in several other proton translocating membrane proteins suggest, that hydronium ion coordination by proteins might display a general principle which was so far underestimated in ATP synthases.     

Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations

This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H⁺-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na⁺- instead of H⁺-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H⁺ transfers to ATP synthases via membrane-associated microcircuits between the H⁺ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.     

Association of α-subunits with nucleotide-modified β-subunits induces asymmetry in the catalytic sites of the F1-ATPase α3β3

The photoaffinity spin-labeled ATP analog, 2-N₃-SL-adenosine triphosphate (ATP), was used to covalently modify isolated β-subunits from F₁-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated α-subunits to the modified β-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2∶1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated β-subunits, binding of α-subunits induces asymmetry in the catalytic sites even in the absence of the γ-subunit. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to PDV.     

Catalysis by isolated β-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of Pyrophosphate

Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F₁ of mitochondrial H⁺-ATPase can bind inorganic pyrophosphate (PP_i) and synthesize PP_i from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PP_i. The difference spectra in the near UV CD of β-subunits with and without PP_i show that PP_i binds to the subunits. Other studies show that β-subunits hydrolyze [³²P] PP_i through a Mg²⁺-dependent process with an optimal pH of 8.3. Free Mg²⁺ is required for maximal hydrolytic rates. The Km for PP_i is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP_i. Thus, isolated β-subunits are catalytically competent with PP_i as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.     

Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli

Abstract The genes oadGAB encoding the oxaloacetate decarboxylase γ, α and β-subunits from Klebsiella pneumoniae were expressed in Escherichia coli . Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed α-subunit or a combination of a and β-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a γα-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the γα-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the a-subunit was not completely complexed to the membrane-bound γ or βγ-subunits.     

Bacterial Na(+)-ATP synthase has an undecameric rotor

Synthesis of adenosine triphosphate (ATP) by the F₁F₀ ATP synthase involves a membrane-embedded rotary engine, the F₀ domain, which drives the extra-membranous catalytic F₁ domain. The F₀ domain consists of subunits a₁b₂ and a cylindrical rotor assembled from 9–14 α-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits.     

Role of gamma-subunit N- and C-termini in assembly of the mitochondrial ATP synthase in yeast

The γ-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the γ-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. γ-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated γ-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.     

Regulatory mechanisms of β-glucan synthases in bacteria, fungi, and plants

The evidence accumulated to date indicates that 1,3-β-glucan synthase (EC 2.3.1.12) and 1,4-β-glucan synthase (EC 2.4.1.12) are regulated by different effectors. Further that the same synthase has different effectors, depending upon its presence in green plants, fungi, and bacteria. Synthases from plants require divalent cations and β-linked glucosides whereas fungal enzymes require neither cations nor β-glucosides, but most require nucleoside triphosphates for activation. Two endogenous effectors have been characterized and shown to produce activation in vitro. One is 3',5'-cyclic diguanylic acid that is the activator of cellulose synthase in bacteria. The other is a β-linked glucosyl dioleoyl diglyceride from mung bean, capable of activating synthases that produce both β-(1–3) and β-(1–4) products. The results of product analysis of the β-linked glucoside activated reaction suggest that the synthesis of (1–3) and (1–4) glucosyl linkages may share a common enzyme in plants. All synthases utilize uridine 5'-diphosphoglucose (UDPG) and are associated with the plasma membrane. Efforts to solubilize the synthases from cellular fractions enriched in plasma membranes have been generally successful. The purification of the soluble enzymes, however, remains a major obstacle to the full understanding of their regulation.     

Electrical power fuels rotary ATP synthase

ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical gradient of protons or sodium ions. The electric component of the ion motive force is crucial for ATP synthesis. Here, we incorporate recent results on structure and function of the F(0) domain and present a mechanism for torque generation with the fundamental nature of the membrane potential as driving force in the core.     

Cloning and sequencing of genes encoding the beta subunits of the ATP-synthases from Enterobacter aerogenes and Flavobacterium ferrugineum

Abstract The genes encoding the β-subunit of the ATPase from Enterobacter aerogenes and Flavobacterium ferrugineum were cloned and their sequences determined. The predicted amino acid sequences were compared with the corresponding proteins from other eubacteria. Homology values of 58–98% confirmed the highly conserved character of the ATPase β-subunit. The enterobacterial ( Escherichia coli, E. aerogenes ) β-subunits represent the shortest sequences, whereas the corresponding F. ferrugineum protein exhibits an additional 33 amino acid residues as insertions at three different locations.     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.     

Operation of the F(0) motor of the ATP synthase

ATP, the universal carrier of cell energy is manufactured from ADP and phosphate by the enzyme ATP synthase using the energy stored in a transmembrane ion gradient. The two components of the ion gradient (DeltapH or DeltapNa(+)) and the electrical potential difference Deltapsi are thermodynamically but not kinetically equivalent. In contrast to accepted wisdom, the electrical component is kinetically indispensable not only for bacterial ATP synthases but also for that from chloroplasts. Recent biochemical studies with the Na(+)-translocating ATP synthase of Propionigenium modestum have given a good idea of the ion translocation pathway in the F(0) motor. Taken together with biophysical data, the operating principles of the motor have been delineated.     

Isolation and characterization of a new phycoerythrin from the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)₆ subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.     

Acetogenesis and ATP synthesis in Acetobacterium itwoodii are coupled via a transmembrane primary sodium ion gradient

Abstract In cell suspensions of Acetobacterium woodii the acetyl-CoA pathway is coupled to net ATP formation. Acetate formation as well as ATP synthesis and the generation of a transmembrane sodium ion gradient are not inhibited by protonophores but by sodium ionophores. Acetogenesis from CO or formaldehyde + CO as catalyzed by inverted vesicles is coupled to sodium ion uptake. Both processes are not inhibited by protonophores but by sodium ionophores. These experiments are in accordance with the presence of a primary sodium ion pump connected to the acetyl-CoA pathway which enables the cells to synthesize net ATP by means of a Δμ _Na+ in concert with a Na⁺-translocating ATPase.     

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes

 The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were 49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication from a γ-subunit-like precursor. Received: 11 March 1997     

ATP synthases from archaea: The beauty of a molecular motor

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A₁A_O ATP synthases, a class of ATP synthases distinct from the F₁F_O ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V₁V_O ATPases of eukaryotes. A₁A_O ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A₁A_O ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A₁A_O ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H⁺, Na⁺ or even H⁺ and Na⁺ using enzymes. The evolution of the H⁺ binding site to a Na⁺ binding site and its implications for the energy metabolism and physiology of the cell are discussed.     

Energy conservation in the decarboxylation of dicarboxylic acids by fermenting bacteria

Decarboxylation of dicarboxylic acids (oxalate, malonate, succinate, glutarate, and malate) can serve as the sole energy source for the growth of fermenting bacteria. Since the free energy change of a decarboxylation reaction is small (around –20 kJ per mol) and equivalent to only approximately one-third of the energy required for ATP synthesis from ADP and phosphate under physiological conditions, the decarboxylation energy cannot be conserved by substrate-level phosphorylation. It is either converted (in malonate, succinate, and glutarate fermentation) by membrane-bound primary decarboxylase sodium ion pumps into an electrochemical gradient of sodium ions across the membrane; or, alternatively, an electrochemical proton gradient can be established by the combined action of a soluble decarboxylase with a dicarboxylate/monocarboxylate antiporter (in oxalate and malate fermentation). The thus generated electrochemical Na⁺ or H⁺ gradients are then exploited for ATP synthesis by Na⁺- or H⁺-coupled F₁F₀ ATP synthases. This new type of energy conservation has been termed decarboxylation phosphorylation and is responsible entirely for ATP synthesis in several anaerobic bacteria. Received: 5 December 1997 / Accepted: 16 March 1998     

Na+ Transport by the A1AO-ATP Synthase Purified from Thermococcus onnurineus and Reconstituted into Liposomes

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A₁A_O-ATP synthases use Na⁺ as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A₁A_O-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A₁A_O-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na⁺ transport. This is the first proof of Na⁺ transport by an A₁A_O-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na⁺ in the bioenergetics of archaea.