Specific labelling of the (Ca2+ + Mg2+)-ATPase of Escherichia coli with 8-azido-ATP and 4-chloro-7-nitrobenzofurazan

1. 8-Azido-ATP is a substrate for Escherichia coli (Ca²⁺ + Mg²⁺)-ATPase (E. coli F₁).2. Illumination of E. coli F₁ in the presence of 8-azido-ATP causes inhibition of ATPase activity. The presence of ATP during illumination prevents inhibition.3. 8-Azido-ATP and 4-chloro-7-nitrobenzofurazan (NbfCl) bind predominantly to the α subunit of the enzyme, but also significantly to the β subunit.4. The α subunit of E. coli F₁ seems to have some properties that in other F₁-ATPases are associated with the β subunit.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

Thymoquinone Inhibits Escherichia coli ATP Synthase and Cell Growth

We examined the thymoquinone induced inhibition of purified F₁ or membrane bound F₁F_O E. coli ATP synthase. Both purified F₁ and membrane bound F₁F_O were completely inhibited by thymoquinone with no residual ATPase activity. The process of inhibition was fully reversible and identical in both membrane bound F₁F_o and purified F₁ preparations. Moreover, thymoquinone induced inhibition of ATP synthase expressing wild-type E. coli cell growth and non-inhibition of ATPase gene deleted null control cells demonstrates that ATP synthase is a molecular target for thymoquinone. This also links the beneficial dietary based antimicrobial and anticancer effects of thymoquinone to its inhibitory action on ATP synthase.     

Effects of Ellman’s reagent and other thiol compounds on ion transport and ATPase activity in anaerobically grown Escherichia coli cells

It was found that modification of thiol (SH-) groups of membrane proteins by Ellman’s reagent (5,5′-dithiol-bis-(2-nitrobenzoic) acid) results in inhibition of proton efflux and K⁺ influx in anaerobically grown (pH 7.5) wild-type strains of Escherichia coli and causes disturbances in K⁺-dependent, N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity and molecular hydrogen production. No such effects were observed after substitution of the cysteine residue in the b-subunit of F₀ of proton F₀F₁-ATPase for alanine. Moreover, the redox potential (RP) decreased as a result of H₂ release during glucose fermentation and formate utilization was partly restored in the presence of Ellman’s reagent. Similar changes were established when another specific SH-reagent, succinimidyl-6(β-maleimidopropionamido)hexanoate, was used. Another thiol reagent, N-ethylmaleimide, did not exert such effects despite its inhibitory action on ion transport and ATPase activity. The data obtained provide conclusive evidence in favor of essential role of thiol groups and the cysteine residue in the b-subunit of F₀ of F₀F₁-ATPase in proton-potassium exchange and H₂ production in E. coli cells. The results also point to a possible involvement of SH-groups in the TrkA system of K⁺ uptake and an involvement of hydrogenases 3 or 4 in the interactions of these integral proteins with each other.     

Residue 249 in subunit beta regulates ADP inhibition and its phosphate modulation in Escherichia coli ATP synthase

ATPase activity of proton-translocating F_OF₁-ATP synthase (F-type ATPase or F-ATPase) is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conservative of these mechanisms found in all enzymes studied so far is allosteric inhibition of ATP hydrolysis by MgADP (ADP-inhibition). When MgADP is bound without phosphate in the catalytic site, the enzyme lapses into an inactive state with MgADP trapped.In chloroplasts and mitochondria, as well as in most bacteria, phosphate prevents MgADP inhibition. However, in Escherichia coli ATP synthase ADP-inhibition is relatively weak and phosphate does not prevent it but seems to enhance it.We found that a single amino acid residue in subunit β is responsible for these features of E. coli enzyme. Mutation βL249Q significantly enhanced ADP-inhibition in E. coli ATP synthase, increased the extent of ATP hydrolysis stimulation by sulfite, and rendered the ADP-inhibition sensitive to phosphate in the same manner as observed in F_OF₁ from mitochondria, chloroplasts, and most aerobic\photosynthetic bacteria.     

F0F1 ATP synthase of Streptomycetes: Modulation of activity and oligomycin resistance by protein Ser/Thr kinases

Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F₀F₁ ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F₀F₁ complex is Mg²⁺-dependent and moderately stimulated by high concentrations of Ca²⁺ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F₀F₁ ATPase inhibitors that react with the membrane-bound F₀ complex. The assay of biochemical properties of the F₀F₁ ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F₀F₁ complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F₀F₁ ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis.     

Complementation of the Fo c Subunit of Escherichia coli with That of Streptococcus mutans and Properties of the Hybrid FoF1 ATP Synthase

The c subunit of Streptococcus mutans ATP synthase (F_oF₁) is functionally exchangeable with that of Escherichia coli, since E. coli with a hybrid F_oF₁ is able to grow on minimum succinate medium through oxidative phosphorylation. E. coli F₁ bound to the hybrid F_o with the S. mutans c subunit showed N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity similar to that of E. coli F_oF₁. Thus, the S. mutans c subunit assembled into a functional F_o together with the E. coli a and b subunits, forming a normal F₁ binding site. Although the H⁺ pathway should be functional, as was suggested by the growth on minimum succinate medium, ATP-driven H⁺ transport could not be detected with inverted membrane vesicles in vitro. This observation is partly explained by the presence of an acidic residue (Glu-20) in the first transmembrane helix of the S. mutans c subunit, since the site-directed mutant carrying Gln-20 partly recovered the ATP-driven H⁺ transport. Since S. mutans is recognized to be a primary etiological agent of human dental caries and is one cause of bacterial endocarditis, our system that expresses hybrid F_o with the S. mutans c subunit would be helpful to find antibiotics and chemicals specifically directed to S. mutans.     

Reconstitution of ATPase from the isolated subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3

ATPase was reconstituted from mixtures of isolated subunits of coupling factor, F₁ ATPase of E. coli (EF₁) and thermophilic bacterium PS3 (TF₁); ability to hydrolyze ATP was attained from the combination of α and β subunits from EF₁ and γ subunit from TF₁, α and β from TF₁ and γ from EF₁, and α and γ from EF₁ and β from TF₁. The β subunit of TF₁ also could complement the EF₁ from an E. coli mutant defective in this subunit. This is the first demonstration of interspecies in vitro recombination of ATPase activity from isolated subunits.     

The DCCD-binding polypeptide is close to the F1 ATPase-binding site on the cytoplasmic surface of the cell membrane of Escherichia coli

Removal of the F₁ ATPase from membrane vesicles of $\text{Escherichia}\text{coli}$ resulted in leakage of protons across the membrane through the F_O portion of the ATPase complex. The leakage of protons was prevented by antiserum to the N,N′-dicyclohexylcarbodiimide (DCCD)-binding polypeptide in everted but not in “right-side out” membrane vesicles. The antiserum prevented the rebinding of F₁ ATPase to F₁-stripped everted membrane vesicles. It is concluded that in F₁-depleted vesicles the DCCD-binding polypeptide is exposed on the cytoplasmic surface of the cell membrane at or close to the binding site of the F₁ ATPase.     

ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin

The coupling factor ATPase complex extracted by Triton X-100 from the photosynthetic bacterium Rhodospirillum rubrum could be incorporated into phospholipid vesicles after removal of the Triton. Vesicles reconstituted with this F₀ · F₁-type ATPase together with bacteriorhodopsin were found to catalyze, in the light, net ATP synthesis which was inhibited by the energy transfer inhibitors oligomycin and N,N-dicyclohexylcarbodiimide as well as by uncouplers. In vesicles reconstituted with the crude ATPase up to 50% of the observed rate of phosphorylation was independent on light and bacteriorhodopsin and insensitive to the above-listed inhibitors. This dark activity was, however, completely blocked by the adenylate kinase inhibitor, p¹,p⁵-di(adenosine-5′)pentaphosphate, which did not affect at all the net light-dependent phosphorylation nor the ATP-³²P_i exchange reaction. Vesicles reconstituted with the purified ATPase catalyzed only the light- and bacteriorhodopsin-dependent diadenosine pentaphosphate-insensitive phosphorylation. The rate of this photophosphorylation was found to be proportional to the amount of ATPase and bacteriorhodopsin, and linear for at least 20 min of illumination. These results indicate that the purified ATPase contains the complete assembly of subunits required to transduce electrochemical gradient energy into chemical energy.     

F-ATP-ase of Escherichia coli membranes: The ubiquitous MgADP-inhibited state and the inhibited state induced by the ε–subunit's C-terminal domain are mutually exclusive

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F₁ complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for F_OF₁ from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF₁, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F₁ with the membrane-embedded F_O complex modulate inhibition by the εCTD. Here, we study membranes containing EcF_OF₁ with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).     

Inhibition of ATPase activity of Escherichia coli ATP synthase by polyphenols

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-β-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the γ-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the γ-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-β-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (～0 residual activity), inhibition by other compounds was partial with ～20% residual activity by quercetin, ～50% residual activity by quercetin-3-β-d glucoside, and ～60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC₅₀ ～14 μM) followed by quercetin (IC₅₀ ～33 μM), quercetin-3-β-d glucoside (IC₅₀ ～71 μM), resveratrol (IC₅₀ ～94 μM), quercitrin (IC₅₀ ～120 μM). Inhibition was identical in both F₁F_o membrane preparations as well as in isolated purified F₁. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-β-d glucoside inhibited only ATPase activity and not ATP synthesis.     

Determination of Fatty Acid in Surfactin and Elucidation of the Total Structure of Surfactin

Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N,N’- dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃ Vo₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.     

The Effects of Low Doses of Gamma-Radiation on Growth and Membrane Activity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> GRP3 and <Emphasis Type="Italic">Escherichia coli</Emphasis> M17

Microorganisms are part of the natural environments and reflect the effects of different physical factors of surrounding environment, such as gamma (γ) radiation. This work was devoted to the study of the influence of low doses of γ radiation with the intensity of 2.56?μW (m²?s)^?1 (absorbed doses were 3.8 mGy for the radiation of 15?min and 7.2 mGy—for 30?min) on Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild type cells. The changes of bacterial, growth, survival, morphology, and membrane activity had been studied after γ irradiation. Verified microbiological (specific growth rate, lag phase duration, colony-forming units (CFU) number, and light microscopy digital image analysis), biochemical (ATPase activity of bacterial membrane vesicles), and biophysical (H⁺ fluxes throughout cytoplasmic membrane of bacteria) methods were used for assessment of radiation implications on bacteria. It was shown that growth specific rate, lag phase duration and CFU number of these bacteria were lowered after irradiation, and average cell surface area was decreased too. Moreover ion fluxes of bacteria were changed: for P. aeruginosa they were decreased and for E. coli—increased. The N,N′-dicyclohexylcarbodiimide (DCCD) sensitive fluxes were also changed which were indicative for the membrane-associated F₀F₁-ATPase enzyme. ATPase activity of irradiated membrane vesicles was decreased for P. aeruginosa and stimulated for E. coli. Furthermore, DCCD sensitive ATPase activity was also changed. The results obtained suggest that these bacteria especially, P. aeruginosa are sensitive to γ radiation and might be used for developing new monitoring methods for estimating environmental changes after γ irradiation.     

Mechanistic basis for differential inhibition of the F1Fo‐ATPase by aurovertin

The mitochondrial F₁F_o‐ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F₁F_o‐ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F₁ domain and inhibits F₁F_o‐ATPase‐catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F₁F_o‐ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K_i values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F₁F_o‐ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single‐molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F₁F_o‐ATPase catalyzes ATP synthesis and hydrolysis. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 830–840, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1.

Incubation of F₁ in the presence of Mg²⁺ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg²⁺ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F₁ treated to remove tightly bound nucleotides was preincubated in the presence of Mg²⁺. Mg²⁺-induced inhibition of ATPase activity of nucleotide-depleted F₁ can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg²⁺-induced inhibition of the ATPase activity of F₁ is due to the formation of an inactive F₁. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, $\text{86}$, 434/ is directed to F₁ component of the complete mitochondrial ATPase system.     

Proton-transporting urinary epithelia. Reactivity with N,N′-dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD), a potent inhibitor of the F₀F₁-type H⁺-translocating ATPase, was employed to determine the possible involvement of such an ATPase in urinary acidification. Two methods were used in this approach: (1) the reaction of [¹⁴C]DCCD with tissues involved in urinary acidification and (2) the inhibition of ATPase activity by DCCD. Membrane components from epithelial cells of toad and turtle urinary bladder and brush borders of rabbit kidney were reacted with [¹⁴C]DCCD and analyzed by polyacrylamide gel electrophoresis both before and after extraction with organic solvents. Although a DCCD-binding component was extracted from toad and turtle bladder membranes by chloroform/methanol (2:1, $\text{v}\text{v}$), the binding was not saturable. Analysis of this DCCD-binding component by thin-layer chromatography indicated that there was no ninhydrin reactivity associated with the [¹⁴C]DCCD binding. Moreover, all attempts to precipitate a DCCD-binding protein were unsuccessful. This and other evidence suggested that the observed DCCD binding was to phospholipid. In the second type of experiments, the ATPase activity present in brush borders from rabbit kidney was partially inhibited by DCCD, but at a concentration that is over two orders of magnitude greater than that required for typical DCCD-sensitive ATPase. We conclude from our failure to find positive evidence of a DCCD-reactive protein and from the relative insensitivity of the ATPase to DCCD that either urinary acidification is not accomplished by a typical F₀F₁-type translocating ATPase, or the F₀ has been modified so that the sensitivity to DCCD has been altered or lost.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F₁ were compared. Both compounds induced a maximal inhibition of 60–80% in various preparations of F₁ studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F₁ bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F₁ is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F₁ to Sepharose-hexylammonium is prevented by high concentrations of Na⁺ or K⁺.