Iron transport by proteoliposomes containing mitochondrial F1Fo ATP synthase isolated from rat heart

In this work, we present evidence of Fe²⁺ transport by rat heart mitochondrial F₁F_o ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 °C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the α, β, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg⁺², Ca⁺², Mn⁺², Zn⁺², Cu⁺², Fe⁺³, and Al⁺³ was observed. Moreover, Cu⁺², even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H⁺.     

Effects of aluminum chloride on the kinetics of rat cortex synaptosomal ATP diphosphohydrolase 9EC 3.6.1.5)

Aluminum chloride (AlCl₃), a neurotoxic compound, inhibited ATP diphosphohydrolase activity of synaptosomes obtained from cerebral cortex of adult rats. The metal ion significantly inhibited ATPase and ADPase activities of the enzyme at all concentrations tested in vitro (0.01, 0.05, 0.5, 5 and 10 mM) in the presence of 1.5 mM calcium. When tested in the absence of Ca²⁺, and with increasing amounts of Al³⁺, enzyme activity remained below basal levels, suggesting that the trivalent cation Al³⁺ is not a substitute for the divalent cation Ca²⁺ in ATP-Ca²⁺ and ADP-Ca²⁺ complexes. The Al³⁺ inhibition was competitive with respect to Ca²⁺. The enzyme inhibition was reversed by the addition of deferoxamine (DFO). NaF significantly inhibited ATP diphosphohydrolase activity, and this inhibition was reversed by the addition of Ca²⁺ to the medium. Such inhibition was not potentiated by AlF₄, which is an inhibitor of cation-transport ATPases.     

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.     

Bacterial sodium ion-coupled energetics

For many bacteria Na⁺ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na⁺ pumps in bacteria, the Na⁺-translocating oxaloacetate decarboxylase ofKlebsiella pneumoniae and the Na⁺-translocating F₁F₀ ATPase ofPropionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na⁺ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound -subunits including two conserved aspartates that may be important for Na⁺ translocation. The coupling ratio of the decarboxylase Na⁺ pumps depended on and decreased from two to zero Na⁺ uptake per decarboxylation event as increased from zero to the steady state level.InP. modestum, is generated in the course of succinate fermentation to propionate and CO₂. This is used by a unique Na⁺-translocating F₁F₀ ATPase for ATP synthesis. The enzyme is related to H⁺-translocating F₁F₀ ATPases. The F₀ part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of anEscherichia coli deletion mutant was completely functional as a Na⁺-ATP synthase conferring theE. coli strain the ability of Na⁺-dependent growth on succinate. The hybrid consisted of subunits a, c, b, and part of fromP. modestum and of the remaining subunits fromE. coli. Studies on Na⁺ translocation through the F₀ part of theP. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na⁺ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane.     

Tight nucleotide binding sites and ATPase activities of theRhodospirillum rubrum RrF1-ATPase as compared to spinach chloroplast CF1-ATPase

SolubilizedRhodospirillum rubrum RrF₁-ATPase, depleted of loosely bound nucleotides, retains 2.6 mol of tightly bound ATP and ADP/mol of enzyme. Incubation of the depleted RrF₁ with Mg²⁺-ATP or Mg²⁺-AMP-PNP, followed by passage through two successive Sephadex centrifuge columns, results in retention of a maximal number of 4 mol of tightly bound nucleotides/mol of RrF₁. They include 1.5 mol of nonexchangeable ATP, whereas all tightly bound ADP is fully exchangeable. A similar retention of only four out of the six nucleotide binding sites present on CF₁ has been observed after its passage through one or two centrifuge columns. These results indicate that the photosynthetic, unlike the respiratory, F₁-ATPases have fasterk _off constants for two of the Mg-dependent nucleotide binding sites. This could be the reason for the tenfold lower Mg²⁺ than Ca²⁺-ATPase activity observed with native RrF₁, as with -depleted, activated CF₁. An almost complete conversion of both RrF₁ and CF₁ from Ca²⁺- to Mg²⁺-dependent ATPases is obtained upon addition of octylglucoside, at concentrations below its CMC, to the ATPase assay medium. Thus, octylglucoside seems to affect directly the RrF₁ and CF₁ divalent cation binding site(s), in addition to its proposed role in relieving their inhibition by free Mg²⁺ ions. The RrF₁-ATPase activity is 30-fold more sensitive than CF₁ to efrapeptin, and completely resistant to either inhibition or stimulation by the CF₁ effector, tentoxin. Octylglucoside decreases the inhibition by efrapeptin and tentoxin, but exposes on CF₁ a low-affinity, stimulatory site for tentoxin.Abbreviations: CF₁, EcF₁, MF₁, and TF₁, the soluble F₁-ATPase from chloroplasts, PE. coli, mitochondria,R. rubrum, and the thermophilic bacterium PS3, respectively: AMP-PNP, adenylyl-, -imidodiphosphate; CMC, critical micellar concentration; DTT, dithiothreitol, LDAO, lauryl dimethylamine oxide.Dedicated to Professor Achim Trebst in honor of this 65th birthday.     

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

Purification and properties of a F420-nonreactive,membrane-bound hydrogenase from Methanosarcina strain Gö1

The distribution of the F₄₂₀-reactive and F₄₂₀-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F₄₂₀-nonreactive enzyme. The membrane-bound F₄₂₀-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H₂ oxidized · min^-1 · mg protein^-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S^2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F₄₂₀-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F₄₂₀-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations transmembrane electrochemical gradient of H^- - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - Mb. Methanobacterium - PMSF phenylmethyl-sulfonylfluoride - Cl₃AcOH trichloroacetic acid     

Two N 5, N 10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme

It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H₂-forming N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F₄₂₀-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH₄)₂SO₄, 2 M Na₂HPO₄, 1.5 M K₂HPO₄, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V _max rather than the K _m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH₄)₂SO₄ the V _max was 4000 U/mg (k _cat=2400 s^-1) and the K _m for N ⁵,N ¹⁰-methylenetetrahydromethanopterin and for coenzyme F₄₂₀ were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K₂HPO₄ at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F₄₂₀-dependent N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H₄MPT tetrahydromethanopterin - F₄₂₀ coenzyme F₄₂₀ - CH₂=H₄MPT N ⁵,N ¹⁰-methylenetrahydromethanopterin - CHH₄MPT⁺ N ⁵,N ¹⁰-methenyltetrahydromethanopterin - methylene-H₄MPT dehydrogenase N ⁵,N ¹⁰-methylenetetrahydromethanopterin dehydrogenase - Mops N-morpholinopropane sulfonic acid - Tricine N-[Tris(hydroxymethyl)-methyl]glycine - 1 U = 1 mol/min     

ATP Synthases With Novel Rotor Subunits: New Insights into Structure,Function and Evolution of ATPases

ATPases with unusual membrane-embedded rotor subunits were found in both F₁F₀ and A₁A₀ ATP synthases. The rotor subunit c of A₁A₀ ATPases is, in most cases, similar to subunit c from F₀. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases. A duplicated c subunit with only one ion-translocating group was found along with “normal” F₀ c subunits in the Na⁺ F₁F₀ ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.     

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics

Background

The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases.

Methods

The inhibition mechanism of macrolides was explored on swine heart mitochondrial F₁F_O-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction.

Results

When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I₁ and I₂ pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI₁I₂), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT ≥ 1 μM. The TBT-driven enzyme desensitization was reversed by DTE.

Conclusions

The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of F_O. The oxidation of highly conserved thiols in the ATP synthase c-ring of F_O weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT.

General significance

The findings, by elucidating the macrolide inhibitory mechanism on F_O, indirectly cast light on the F₁F_O torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.     

Vanadyl as a Probe of the Function of the F1-ATPase-Mg2+ Cofactor

The Mg²⁺ dependent asymmetry of the F₁-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 ⁵¹V-hyperfine parameters derived from EPR spectra of VO²⁺ bound to specific sites on F₁ provide a direct probe of the metal ligands. Site-directed mutations of metal ligand residues cause measurable changes in the ⁵¹V-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 MF₁E197 become ligands in lieu of the hydroxyl groups.     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

Impact of aluminium,fluoride and fluoroaluminate complex on ATPase activity ofNostoc linckia andChlorella vulgaris

This study demonstrates a pH-dependent inhibition of Mg²⁺- and Ca²⁺- ATPase activities ofNostoc linckia andChlorella vulgaris exposed to AlCl₃, AlF₃, NaF and AlCl₃ + NaF together. AlF₃ and the combination of AlCl₃ + NaF were more inhibitory to both the enzymes as compared with AlCl₃ and NaF. Toxicity of the test compounds increased with increasing acidity. Interaction of AlCl₃ + NaF was additive onN. linckia andC. vulgaris, respectively, at pH 7.5 and 6.8, and synergistic at pH 6.0 and 4.5. In the presence of 60 and 100 m PO₄ ^3- an increased NaF concentration (in the AlCl₃ + NaF combination) was required to produce the same degree of inhibition in ATP synthesis and ATPase activity. Toxicity of fluoroaluminate was reduced in the presence of EDTA and citrate. Except for beryllium to some extent, combinations of cadmium, cobalt, iron, manganese, tin and zinc with fluoride were not as effective as aluminium in inhibiting the ATPase activity. The presence of a 100 kDa protein band in SDS-PAGE of both control as well as AlCl₃ + NaF-treated samples suggested that AlF₄ ^– inhibits the ATPase activity by acting as a functional barrier without affecting the structure of the enzyme.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F₁F₀-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F₀, namely oligomycin and N,N′-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F₁, maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F₁ and F₀ domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F₀ proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F₁F₀-ATPase oligomycin sensitivity loss.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Relationship of the Escherichia coli TrkA System of Potassium Ion Uptake with the F0F1-ATPase Under Growth Conditions Without Anaerobic or Aerobic Respiration

K⁺ uptake by the Escherichia coli TrkA system is unusual in that it requires both ATP and ; a relation withH⁺ circulation through the membrane is thereforesuggested. The relationship of this system with theF₀F₁-ATPase was studied in intact cells grownunder different conditions. A significant increase of theN,N-dicyclohexylcarbodiimide(DCCD)-inhibitedH⁺ efflux through the F₀F₁ by 5 mMK⁺, but not by Na⁺ added into thepotassium-free medium was revealed only in fermenting wild-type orparent cells, that were grown under anaerobic conditions withoutanaerobic or aerobic respiration and with the production ofH₂. Such an increase disappeared in the unc or the trkA mutants that have alteredF₀F₁ or defective TrkA, respectively.This finding indicates a closed relationship between TrkA andF₀F₁, with these transport systems beingassociated in a single mechanism that functions as an ATP-drivenH⁺–K⁺-exchanging pump. ADCCD-inhibited H⁺–K⁺-exchangethrough these systems with the fixed stoichiometry of H⁺and K⁺ fluxes(2H⁺/K⁺) and a higherK⁺ gradient between the cytoplasm and the externalmedium were also found in these bacteria. They were not observed incells cultured under anaerobic conditions in the presence of nitrate orunder aerobic conditions with respiration and without production ofH₂. The role of anaerobic or aerobic respiration as adeterminant of the relationship of the TrkA with theF₀F₁ is postulated. Moreover, an increase ofDCCD-inhibited H⁺ efflux by added K⁺, aswell as the characteristics of DCCD-sensitiveH⁺–K⁺-exchange found in a parentstrain, were lost in the arcA mutant with a defectiveArc system, suggesting a repression of enzymes in respiratorypathways. In addition, K⁺ influx in the latest mutantwas not markedly changed by valinomycin or with temperature. ThearcA gene product or the Arc system is proposed to beimplicated in the regulation of the relationship between TrkAand F₀F₁.     

Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H₂:CO₂ at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F₄₂₀-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu²⁺ column. Hydrogenase II did not react with ferredoxin or F₄₂₀, whereas hydrogenase I coupled to both ferredoxin and F₄₂₀. A reconstituted soluble protein system composed of purified CO dehydrogenase, F₄₂₀-reactive hydrogenase I fraction, and ferredoxin produced H₂ from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H₂ consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H₂ consumption coupled to methanogenesis and ATP synthesis, respectively.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Introduction: A Close Look at the Vacuolar ATPase

The vacuolar ATPases (V-type ATPases) are a family of ATP-dependent ion pumps and found in two principal locations, in endomembranes and in plasma membranes. This family of ATPases is responsible for acidification of intracellulare compartments and, in certain cases, ion transport across the plasma membrane of eucaryotic cells. V-ATPases are composed of two distinct domains: a catalytic V₁ sector, in which ATP hydrolysis takes place, and the membrane-embedded sector, V₀, which functions in ion conduction. In the past decade impressive progress has been made in elucidating the properties structure, function and moleculare biology. These knowledge sheds light also on the evolution of V-ATPases and their related families of A-(A₁A₀-ATPase) and F-type (F₁F₀-ATPases)ATPases.