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SUMMARYAntibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure and also demonstrate that MsrD twin-ATPase sites work asymmetrically to mediate the dynamic of MsrD interaction with the ribosome. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into an undocumented conserved crevice of the ribosomal exit tunnel concomitantly with 23S rRNA rearrangements that prevent peptide bond formation and preclude accommodation of release factors. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

Streptococcus mutans, a bacterium mainly inhabiting the tooth surface, is a major pathogen of dental caries. The bacterium metabolizes sugars to produce acids, resulting in an acidic microenvironment in the dental plaque. Hence, S. mutans should possess a mechanism for surviving under acidic conditions. In the current study, we report the effects of inhibitors of Escherichia coli proton-pumping F-type ATPase (F-ATPase) on the activity of S. mutans enzyme, and the growth and survival of S. mutans under acidic conditions. Piceatannol, curcumin, and demethoxycurcumin strongly reduced the ATPase activity of S. mutans F-ATPase. Interestingly, these compounds inhibited the growth of S. mutans at pH 5.3 but not at pH 7.3. They also significantly reduced the colony-forming ability of S. mutans after incubation at pH 4.3, while showing essentially no effect at pH 7.3. These observations indicate that S. mutans is highly sensitive to F-ATPase inhibitors under acidic conditions and that F-ATPase plays an important role in acid tolerance of this bacterium.

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used sitedirected mutagenesis to investigate the role of amino acid residues β139, β158, β189, and β319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The βN158L substitution produced no effect on the enzyme activity, while substitutions βF139Y, βF189L, and βV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations βF189L and βV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues β139, β189, and β319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.

Hydrolysis of ATP by the mitochondrial F-ATPase is inhibited by a protein called IF1 . In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1 , is expressed exclusively in the procyclic stage, where the F-ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F-ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1 -ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C-terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1 , the inhibitory capacity of the C-terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1 . This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C-terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1 -ATPase, and this difference in the mechanism of regulation of the F-ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases.

The Rv2477c protein of Mycobacterium tuberculosis (Mtb) belongs to the ATP-binding cassette (ABC) subfamily F that contains proteins with tandem nucleotide-binding domains but lacking transmembrane domains. ABC-F subfamily proteins have been implicated in diverse cellular processes such as translation, antibiotic resistance, cell growth and nutrient sensing. In order to investigate the biochemical characteristics of Rv2477c, we expressed it in Escherichia coli, purified it and characterized its enzymatic functions. We show that Rv2477c displays strong ATPase activity (Vmax = 45.5 nmol/mg/min; Km = 90.5 μM) that is sensitive to orthovanadate. The ATPase activity was maximal in the presence of Mn2+ at pH 5.2. The Rv2477c protein was also able to hydrolyze GTP, TTP and CTP but at lower rates. Glutamate to glutamine substitutions at amino acid residues 185 and 468 in the two Walker B motifs of Rv2477c severely inhibited its ATPase activity. The antibiotics tetracycline and erythromycin, which target protein translation, were able to inhibit the ATPase activity of Rv2477c. We postulate that Rv2477c could be involved in mycobacterial protein translation and in resistance to tetracyclines and macrolides. This is the first report of the biochemical characterization of an ABC-F subfamily protein in Mtb.

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity – similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.HighlightsThylakoid membranes exposed to low p2H studied by small-angle neutron scatteringAcidification causes reversible shrinkage and diminished lamellar orderSANS changes induced by low pH resemble those due to light-induced lumenal acidificationAbbreviationsNPQnon-photochemical quenchingqEthe energy-dependent component of NPQΔμH+transmembrane electrochemical potential gradientPSIphotosystem IPSIIphotosystem IILETlinear electron transportCDcircular dichroismSANSsmall-angle neutron scatteringqscattering vector or momentumtransferIintensityq*center position of the Bragg peakRDrepeat distanceφazimuthal angleI(φ)angular dependency of the scattering intensityFWHMfull width at half maximum

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

F1-ATPase (F1) is a multisubunit water-soluble domain of FoF1- ATP synthase and is a rotary enzyme by itself. Earlier genetic studies using yeast suggested that two factors, Atp11p and Atp12p, contribute to F1 assembly. Here, we show that their mammalian counterparts, AF1 and AF2, are essential and sufficient for efficient production of recombinant bovine mitochondrial F1 in Escherichia coli cells. Intactness of the function and conformation of the E. coli-expressed bovine F1 was verified by rotation analysis and crystallization. This expression system opens a way for the previously unattempted mutation study of mammalian mitochondrial F1.

F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells.

Among the many types of bioenergy-transducing machineries, F- and V-ATPases are unique bio- and nano-molecular rotary motors. The rotational catalysis of F1-ATPase has been investigated in detail, and molecular mechanisms have been proposed based on the crystal structures of the complex and on extensive single-molecule rotational observations. Recently, we obtained crystal structures of bacterial V1-ATPase (A3B3 and A3B3DF complexes) in the presence and absence of nucleotides. Based on these new structures, we present a novel model for the rotational catalysis mechanism of V1-ATPase, which is different from that of F1-ATPases.

The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases., The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F1–ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.

The aim of our study was to investigate the role of protons in regulating energy distribution between the two photosystems in the thylakoid membranes. Low pH-induced changes were monitored in the presence of a proton blocker, N,N'-dicyclohexylcarbodiimide (DCCD). When thylakoid membranes were suspended in a low-pH reaction mixture and incubated with DCCD, then a decrease in the fluorescence intensity of photosystem II (PSII) was observed, while no change in the intensity of photosystem I (PSI) fluorescence occurred according to the measured fluorescence emission spectra at 77 K. Since low pH induced distribution of energy from PSII to PSI was inhibited in the presence of DCCD, we concluded that pH/proton concentration of the thylakoid membranes plays an important role in regulating the distribution of the absorbed excitation energy between both photosystems.

Curcumin, a dietary phytopolyphenol isolated from a perennial herb (Curcuma longa), is a well-known compound effective for bacterial infections and tumors, and also as an antioxidant. In this study, we report the inhibitory effects of curcumin and its analogs on the Escherichia coli ATP synthase F1 sector. A structure-activity relationship study indicated the importance of 4'-hydroxy groups and a β-diketone moiety for the inhibition. The 3'-demethoxy analog (DMC) inhibited F1 more strongly than curcumin did. Furthermore, these compounds inhibited E. coli growth through oxidative phosphorylation, consistent with their effects on ATPase activity. These results suggest that the two compounds affected bacterial growth through inhibition of ATP synthase. Derivatives including bis(arylmethylidene)acetones (C5 curcuminoids) exhibited only weak activity toward ATPase and bacterial growth.

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.

F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid ‘plug’ in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase., Rotary ATPases are membrane-embedded motors that produce or consume ATP and control pH within cells. Schmidt et al. use mass spectrometry to characterize the intact chloroplast ATPase from spinach and, using comparative cross-linking, show that phosphorylation affects stability and nucleotide occupancy.

Trabajo presentado en la 67th Annual Meeting of the International Society of Electrochemistry, celebrada en La Haya del 21 al 26 de agosto de 2016., ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0-ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products., This research was funded by the Spanish MINECO (project CTQ2012-32448) and by the Fundacão para a Ciência e a Tecnologia (project PTDC/BBB-EP/0934/2012 and UID/Multi/04551/2013). O.G.-S. thanks MINECO for an FPI grant. ILM acknowledges the European Research Council (ERC), grant no. ERC-StG-2013-338133 titled “mitochon” and the “Ramón y Cajal” program (RyC-2013-12609) form the Spanish Ministry of Economy.

Everything you do needs energy to power it, whether it is walking up a flight of stairs, eating dinner, fighting off an infection, or even just making new proteins for hair growth. The energy form used by all cells, from bacteria to man is adenosine triphosphate (ATP). Every process within a species, including DNA and protein synthesis, muscle contraction, active transport of nutrients, nerve activity, maintaining osmosis, or carbon fixation, requires a source of ATP.

金沢大学ナノ生命科学研究所 / 金沢大学理工研究域生命理工学系, Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo. © 2012 The Author.

Adenosine triphosphate (ATP) is one of the most important energy sources in living cells, which can drive serial key biochemical processes. However, generation of a proton gradient for ATP production in an artificial way poses a great challenge. In nature, photophosphorylation occurring in chloroplasts is an ideal prototype of ATP production. In this paper we imitate the light-to-ATP conversion process occurring in the thylakoid membrane by construction of FoF1-ATPase proteoliposome-coated PSII-based microspheres with well-defined core@shell structures using molecular assembly. Under light illumination, PSII can split water into protons, oxygen, and electrons and can generate a proton gradient for ATPase to produce ATP. Thus, an artificially designed chloroplast for PSII-driven ATP synthesis is realized. This biomimetic system will help to understand the photophosphorylation process and may facilitate the development of ATP-driven devices by remote light control.

Two proton pumps, the F-ATPase (ATP synthase, FoF1) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F1 or V1) and a membrane intrinsic (Fo or Vo) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F1 sector was found to rotate at surprisingly high speeds (>400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the beta subunits, or between the beta and gamma subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F1-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.

The plasma membrane Ca 2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca 2+ at a rate near 1.5% of that attained at saturating concentrations of Ca 2+ . Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca 2+ -independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca 2+ -independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca 2+ and is catalyzed by E 2 -like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.

The effect of high salt concentration on photosystem II (PS II) electron transport rates and chlorophyll a fluorescence induction kinetics was investigated in coupled and uncoupled spinach thylakoid membranes. With increase in salt concentration, the rates of electron transport mediated by PS II and the F(v)/F(m) ratio were affected more in uncoupled thylakoids as compared to coupled thylakoid membranes. The uncoupled thylakoid membranes seemed to behave like coupled thylakoid membranes at high NaCl concentration (approximately 1 M). On increasing the salt concentration, the uncoupler was found to be less effective and Na+ probably worked as a coupling enhancer or uncoupling suppressor. We suggest that positive charge of Na+ mimics the function of positive charge of H+ in the thylakoid lumen in causing coupled state. The function of NaCl (monovalent cation) could be carried out by even lower concentration of Ca2+ (divalent cation) or Al3+ (trivalent cation). We conclude that this function of NaCl as coupling enhancer is not specific, and in general a positive charge is required for causing coupling in uncoupled thylakoid membranes.

Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3 degrees C to 42.6 degrees C and from 47.5 degrees C to 44.3 degrees C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5 degrees C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the LHCII trimers at 25 degrees C. These data strongly suggest that phosphorylation of the membranes considerably facilitates the heat- and light-inducible reorganizations in the thylakoid membranes and thus enhances the structural flexibility of the membrane architecture.

F(1)-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F(0) to synthesize ATP in vivo. Therefore elucidation of the behavior of F(1) under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F(1) rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 microM), 120 degrees stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.

The abundance of E. coli F1-ATPase molecules observed to rotate using gold nanorods attached to the gamma-subunit was quantitated. Individual F1 molecules were determined to be rotating based upon time dependent fluctuations of red and green light scattered from the nanorods when viewed through a polarizing filter. The average number of F1 molecules observed to rotate in the presence of GTP, ATP, and without nucleotide was approximately 50, approximately 25, and approximately 4% respectively. In some experiments, the fraction of molecules observed to rotate in the presence of GTP was as high as 65%. These data indicate that rotational measurements made using gold nanorods provide information of the F1-ATPase mechanism that is representative of the characteristics of the enzyme population as a whole.

ATP synthesis assisted by the F1F0-ATP synthase enzyme is considered in the context of chemical thermodynamics. It is shown that the proton gradient itself has no energy. For this reason, ATP synthesis with the participation of protons’ gradient has no privileges in comparison with direct ATP synthesis.

ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a e subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli e (eCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.

This chapter deals with the primary active transport systems that use metabolic energy directly to pump ions out of cells or organelles. The source of metabolic energy in the different primary active transporters is, in the P-type ATPases, the splitting of adenosine triphosphate (ATP); in the light-driven pumps, the absorption of the energy of a photon; and in the rotary ATPases, the interconversion of the energy present in ATP with that in an electrochemical gradient of protons, which can itself be produced by the flow of electrons in oxidation–reduction reactions. We discuss the kinetics of two of the P-type ATPases (the calcium ATPase of the sarcoplasmic reticulum and the ubiquitous sodium/potassium ATPase) and how this relates to the molecular structural details of these pumps as this has been determined during the last decade and a half. We touch on the evolution of these pumps. The new structural information on the rotary ATPases (especially the F0F1 ATPase) and bacteriorhodopsin is presented and discussed in terms of the proton movements in these systems. The discussion extends to the ABC transporters, including the multidrug resistance protein, P-glycoprotein, that move a great range of substrates across cell membranes, harnessing the energy in ATP to do so.

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex., his work was funded by the intramural programme of the Medical Research Council via MRC programme U105663150 to J.E.W., and by support from the Biotechnology and Biological Sciences Research Council to M.J.O.W.

The Na + -translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two dimensions. From electron microscopy of these c-ring crystals, a projection map was calculated to 7 Å resolution. In the projection map, each c ring consists of two concentric, slightly staggered, packed rings, each composed of 11 densities representing the α-helices. On the basis of these results, it was determined that the F-ATPase from C. paradoxum contains an undecameric c ring.

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F1 or V1) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase.

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.

A mathematical model for describing electron and proton transport in chloroplasts of higher plants with nonuniform distribution of photosystem 1 (PS1) and photosystem 2 (PS2) between granal and stromal thylakoids was proposed. Along with the noncyclic (linear) electron transport from PS2 to terminal acceptor PS1, we considered cyclic electron transport around PS1, ATP synthesis coupled to transmembrane proton transfer through ATP synthase, and ATP consumption in the Calvin cycle. Numerical simulations of the kinetics of the photoinduced redox transformations of the primary PS1 donor (P700) and the behavior of pH in the intrathylakoid space (pHi) and interthylakoid space (pH0), as well as the time evolution of the ATP concentration for various conditions of the functioning of the chloroplast were performed.

Oxidized ATP synthase of spinach thylakoid membranes catalyzes high rates of ATP synthesis in the light, but very low rates of ATP hydrolysis in the dark. Reduction of the disulfide bond in the gamma subunit of the ATP synthase in the light enhances the rate of Mg2+-ATP hydrolysis in the dark. The light plus thiol-activated state decays in a few minutes in the dark after illumination in Tris buffer, but not when Tricine was used in place of Tris. In this paper, it is shown that Tris in the assay mixture is an inhibitor of the light plus thiol-activated ATPase activity of thylakoids, but only after the activated membranes had incubated in the dark. Aminopropanediols and diethanolamine, also selectively inhibited ATPase activity of activated membranes after storage in the dark, whereas NH4Cl and imidazole inhibit the ATPase activity of activated thylakoids almost equally whether they are added directly after the illumination or several minutes later. The fluorescence of 9-amino-6-chloro-2-methoxyacridine (ACMA) is quenched by the establishment of proton gradients by ATP-dependent proton uptake. Addition of ATP to activated membranes results in rapid quenching of ACMA fluorescence. If the activated membranes were incubated in the dark prior to ATP addition, a lag in the ATP-dependent ACMA fluorescence quenching as well as a similar lag in the rate ATP hydrolysis were seen. It is concluded that ADP rebinds to CF1 in the dark following illumination and inhibits the activity of the ATP synthase. Reactivation of the ATP synthase in the dark can occur by the slow generation of proton gradients by ATP hydrolysis in the dark. This reactivation takes place in Tricine buffer, but not in Tris because of its uncoupling action. Whether ADP binding plays a role in the regulation of the activity of the ATP synthase in situ remains to be established.

A modified 'cold chase' technique was used to study tight [(14)C]ADP and [(14)C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF(0)F(1)). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF(0)F(1 )or CF(1) reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k (d) of about 5 min(-1). The exposure of thylakoid membranes to 0.7-24.8 muM nucleotides leads to filling of up to two noncatalytic sites of CF(0)F(1). The sites differ in their specificity: one preferentially binds ADP, whereas the other - ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF(1) dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the alpha subunits induced by its interaction with the delta subunit and/or subunit I-II when CF(1) becomes bound to a thylakoid membrane.

During ATP hydrolysis, the gammaepsilon c10 complex (gamma and epsilon subunits and a c subunit ring formed from 10 monomers) of F0F1 ATPase (ATP synthase) rotates relative to the alpha3beta3delta ab2 complex, leading to proton transport through the interface between the a subunit and the c subunit ring. In this study, we replaced the two pertinent residues for proton transport, cAsp-61 and aArg-210 of the c and a subunits, respectively. The mutant enzymes exhibited lower ATPase activities than that of the wild type but exhibited ATP-dependent rotation in planar membranes, in which their original assemblies are maintained. The mutant enzymes were defective in proton transport, as shown previously. These results suggest that proton transport can be separated from rotation in ATP hydrolysis, although rotation ensures continuous proton transport by bringing the cAsp-61 and aArg-210 residues into the correct interacting positions.

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans . Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis . Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans ; however, it is not induced to the same extent in the nonpathogenic S. sanguis . Expression studies with an atpD deletion strain of E. coli showed that S. sanguis - E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.

The rotor stoichiometry of F-ATPases has been revealed by the combined approaches of X-ray diffraction (XRD), electron crystallography, and atomic force microscopy (AFM). XRD showed the rotor from the yeast mitochondrial F-ATPase to contain 10 subunits. AFM was used to visualize the tetradecameric chloroplast rotors, and electron crystallography and AFM together revealed the rotors from Ilyobacter tartaricus to be composed of 11 subunits. While biochemical methods had determined an approximate stoichiometric value, precise measurements and new insights into a species-dependent rotor stoichiometry became available by applying the three structural tools together. The structures of AQP1, a water channel, and GlpF, a glycerol channel, were determined by electron crystallography and XRD. The combination of both of these structural tools with molecular dynamics simulations gave a differentiated description of the mechanisms determining the selectivity of water and glycerol channels. This illustrates that the combination of different methods in structural biology reveals more than each method alone.Key words: AQP1, GlpF, F-ATPase, XRD, electron crystallography, AFM.

Structural Biology Synthesis of adenosine triphosphate (ATP) in mitochondria is accomplished by a large molecular machine, the F1FO ATP synthase. Proton translocation across the FO region that spans the mitochondrial inner membrane drives ATP synthesis in the F1 region through a rotational mechanism

ATP is synthesized using ATP synthase by utilizing energy either from the oxidation of organic compounds, or from light, via redox reactions (oxidative- or photo phosphorylation), in energy-transforming membranes of mitochondria, chloroplasts, and bacteria. ATP synthase undergoes several changes during its functioning. The generally accepted model for ATP synthesis is the well-known rotatory model (see e.g., Junge et al., Nature 459:364-370, 2009; Junge and Müller, Science 333:704-705, 2011). Here, we present an alternative modified model for the coupling of electron and proton transfer to ATP synthesis, which was initially developed by Albert Lester Lehninger (1917-1986). Details of the molecular mechanism of ATP synthesis are described here that involves cyclic low-amplitude shrinkage and swelling of mitochondria. A comparison of the well-known current model and the mechano-chemiosmotic model is also presented. Based on structural, and other data, we suggest that ATP synthase is a Ca(2+)/H(+)-K(+) Cl(-)-pump-pore-enzyme complex, in which γ-subunit rotates 360° in steps of 30°, and 90° due to the binding of phosphate ions to positively charged amino acid residues in the N-terminal γ-subunit, while in the electric field. The coiled coil b 2-subunits are suggested to act as ropes that are shortened by binding of phosphate ions to positively charged lysines or arginines; this process is suggested to pull the α 3 β 3-hexamer to the membrane during the energization process. ATP is then synthesized during the reverse rotation of the γ-subunit by destabilizing the phosphated N-terminal γ-subunit and b 2-subunits under the influence of Ca(2+) ions, which are pumped over from storage-intermembrane space into the matrix, during swelling of intermembrane space. In the process of ATP synthesis, energy is first, predominantly, used in the delivery of phosphate ions and protons to the α 3 β 3-hexamer against the energy barrier with the help of C-terminal alpha-helix of γ-subunit that acts as a lift; then, in the formation of phosphoryl group; and lastly, in the release of ATP molecules from the active center of the enzyme and the loading of ADP. We are aware that our model is not an accepted model for ATP synthesis, but it is presented here for further examination and test.

In ATP synthase (F(O)F(1)-ATPase) ion flow through the membrane-intrinsic portion, F(O), drives the central "rotor", subunits c(10)epsilongamma, relative to the "stator" ab(2)delta(alphabeta)(3). This converts ADP and P(i) into ATP. Vice versa, ATP hydrolysis drives the rotation backwards. Covalent cross-links between rotor and stator subunits have been shown to inhibit these activities. Aiming at the rotary compliance of subunit gamma we introduced disulfide bridges between gamma (rotor) and alpha or beta (stator). We engineered cysteine residues into positions located roughly at the "top," "center," and "bottom" parts of the coiled-coil portion of gamma and suitable residues on alpha or beta. This part of gamma is located at the center of the (alphabeta)(3) domain with its C-terminal part at the top of F(1) and the bottom part close to the F(O) complex. Disulfide bridge formation under oxidizing conditions was quantitative as shown by SDS-polyacrylamide gel electrophoresis and immunoblotting. As expected both the ATPase activities and the yield of rotating subunits gamma dropped to zero when the cross-link was formed at the center (gammaL262C alphaA334C) and bottom (gammaCys(87) betaD380C) positions. But much to our surprise disulfide bridging impaired neither ATP hydrolysis activity nor the full rotation of gamma and the enzyme-generated torque of oxidized F(1), which had been engineered at the top position (gammaA285C alphaP280C). Apparently the high torque of this rotary engine uncoiled the alpha-helix and forced amino acids at the C-terminal portion of gamma into full rotation around their dihedral (Ramachandran) angles. This conclusion was supported by molecular dynamics simulations: If gammaCys(285)-Val(286) are attached covalently to (alphabeta)(3) and gammaAla(1)-Ser(281) is forced to rotate, gammaGly(282)-Ala(284) can serve as cardan shaft.

Five widely documented mechanisms of chloride transport across plasma membranes are: anion-coupled antiport; sodium and hydrogen-coupled symport; Cl − channels; and an electrochemical coupling process. No genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl − -stimulated ATPases co-existing, in the same tissue, with uphill chloride transport that could not be accounted for by the five common chloride transport processes. Cl − -stimulated ATPase activity is a common property of practically all biological cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl − -stimulated ATPase activity. Recent studies of Cl − -stimulated ATPase activity and active chloride transport in the same membrane system, including liposomes, suggest a mediation by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl − -stimulated ATPases.

This review focuses on ion-transporting ATPases which play an essential role in the generation and maintenance of ionic gradients between cell compartments and environment. There are three types of...

Photochemical characteristics and lipid composition of thylakoid membranes from 12 day-old pea leaves that were exposed to slow clino-rotation were examined and compared with a vertical control. Proton permeability of thylakoid membranes was estimated from light-induced proton uptake (ΔH + ) and post-illumination proton efflux in chloroplast suspensions. The ΔpH magnitude was calculated from the level of light-induced quenching of 9-aminoacridine fluorescence. Proton permeability of thylakoid membranes increased during exposure to clino-rotation. When subsequently transferred to darkness, proton efflux increased almost 2-fold in clinorotated leaves. The results were compared with data on pigment and polar lipid composition of photosynthetic membranes in clino-rotated and control plants. It was concluded that both the increase of proton permeability and the decrease of polar lipid content in chloroplasts were induced by clino-rotation.

Coupling with electrochemical proton gradient, ATP synthase (F(0)F(1)) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the beta subunit of F(1) sector and roles of the gamma subunit in energy coupling. The gamma-subunit rotation during catalysis is also discussed.

The Mg 2+ cofactor of the F 1 F 0 ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (V IV O) 2+ is a functional surrogate for Mg 2+ in the F 1 -ATPase. The 51 V-hyperfine parameters derived from EPR spectra of VO 2+ bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the 51 V-hyperfine parameters of the bound VO 2+ , thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to β E in mitochondrial F 1 , activation of the chloroplast F 1 -ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the γ subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the β DP site of mitochondrial F 1 , the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from βE197 in Chlamydomonas CF 1 . Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.

A single molecule of F 1 –ATPase is by itself a rotary motor in which a central γ–subunit rotates against a surrounding cylinder made of α 3 β 3 –subunits. Driven by the three βs that sequentially hydrolyse ATP, the motor rotates in discrete 120° steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central γ–subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F motor produces a constant torque of ca . 40 pN nm. The work done in a 120° step, or the work per ATP molecule, is thus ca . 80 pN nm. In cells, the free energy of ATP hydrolysis is ca . 90 pN nm per ATP molecule, suggesting that the F 1 motor can work at near 100% efficiency. We confirmed in vitro that F 1 indeed does ca . 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F 1 motor: the ATP synthase, of which F 1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F motor. Possible mechanisms of F 1 rotation are discussed.

The Mg2+ dependent asymmetry of the F(1)-ATPase catalytic sites leads to the differences in affinity for nucleotides and is an essential component of the binding-change mechanism. Changes in metal ligands during the catalytic cycle responsible for this asymmetry were characterized by vanadyl (V(IV) + O)2+, a functional surrogate for Mg2+. The (51)V-hyperfine parameters derived from EPR spectra of VO2+ bound to specific sites on F(1) provide a direct probe of the metal ligands. Site-directed mutations of metal ligand residues cause measurable changes in the (51)V-hyperfine parameters of the bound VO2+, thereby providing a means to identification. Initial binding of the metal-nucleotide to the low-affinity catalytic site conformation results in metal coordination by hydroxyl groups from the P-loop threonine and catch-loop threonine. Upon conversion to the high-affinity conformation, carboxyl groups from the Walker homology B aspartate and MF(1)betaE197 become ligands in lieu of the hydroxyl groups.