Rotational mobility and domain flexibility of membrane-bound bacterial coupling factor as detected with the triplet probe eosin-isothiocyanate

The membrane-bound coupling factor (BF₁) of chromatophores from the photosynthetic bacerium Rhodopseudomonas sphaeroides was covalently labeled with the triplet probe eosin-isothiocyanate. The labeled enzyme was isolated and functionally reconstituted into depleted chromatophores from the same bacterium. ATPase and ATP synthase activities of the reconstituted vesicles were strongly dependent on the labeling conditions, decreasing at increasing load of eosin molecules per BF₁. When labeling was carried out in the dark and in the presence of ATP, one molecule of eosin isothiocyanate was bound per BF₁ and the activities catalyzed by the reconstituted and labeled enzyme were as high as in untreated chromatophores. Upon light energization of the chromatophore membrane, a large conformational change of BF₁ could be detected by using the triplet probe as a spectroscopic tool. The domain flexibility and rotational mobility of the reconstituted coupling enzyme were directly related to the enhancement of the ATPase activity induced by light. Both the light-stimulated ATPase activity and conformational changes could be prevented by addition of ADP or oligomycin and affected to the same extent by uncouplers and inhibitors of electron transport. Moreover, the detected conformational changes were reversible in time, appearing with a half-time of 10 ms upon illumination of the chromatophores, and disappearing with a half-time of 70 ms in the dark. The results obtained prove the feasibility of the spectroscopic technique in detecting conformational changes of the membrane-bound BF₁, similarly to what already has been observed for chloroplast coupling factor (Wagner, R. and Junge, W. (1980) FEBS Lett. 114, 327–333), and add to the possibility of characterizing, by this method, energy transduction at a molecular level.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

The peptides of chloroplast membranes: I. The soluble coupling factor (Ca2+-ATPase)

The chloroplast coupling factor (CF₁) was analyzed by gel electrophoresis in SDS and found to contain two major bands in equal amounts with mobilities corresponding to molecular weights of 62,000 and 57,000 and three minor bands of molecular weights 38,000, 21,000, and 14,000. The peptides were present in comparable amounts in many different preparations of the protein and, therefore, were thought not to be tightly bound contaminants. The interaction between these five peptides was shown to be noncovalent.Incubation of the enzyme with trypsin, under conditions which activate the latent ATPase, was found to cause selective digestion of the five peptides; the 62,000 M_r peptide was the most susceptible to digestion, while the 57,000 M_r peptide was most stable to trypsin. When chloroplast membranes were exposed to trypsin in the light to activate the postillumination Mg²⁺-dependent ATPase activity, EDTA extraction solubilized a protein fraction which contained the normal CF₁ peptide pattern. Also, the membranes, when solubilized and chromatographed on SDS-gels did not show the disappearance of any band.The ATPase activity of the protein was highly susceptible to ionic strength, being 50% inhibited by monovalent salts at a concentration of 0.05 m.     

Theoretical studies on DNA-binding, DNA-photocleavage and spectral properties of Co(III) complexes [Co(phen)2(L)] (L = pip, hpip, hnaip)

Theoretical studies on the DNA-binding, DNA-photocleavage and spectral properties of Co(III) polypyridyl complexes [Co(phen)₂(L)]³⁺ (L = pip, hpip, hnaip) have been carried out, using the density functional theory (DFT), Hartree-Fock (HF) and configuration interaction singles (CIS) methods. The optimized geometric structures of these Co(III) complexes in aqueous solution are more close to experimental data than those in vacuo at the B3LYP/LanL2DZ level. Based on the optimized geometric structures in solution, the electronic structures of these Co(III) complexes were analyzed and the trend in the DNA-binding constants (K_b) was reasonably explained. In particular, via the analysis of natural charges of the complexes in ground state and excited state, it is very interesting to find the following: under UV or visible light irradiation, the Co(Ш) polypyridyl complexes undergo an intra-molecular electron transfer from S₀ state to T₁ state, and the positive charges on the main-ligand in the T₁ state are greatly increased, so as to form a radical cation with strong oxidation ability. Meanwhile, the change in geometry of the complexes under light irradiation also helps to the radical cation easily approaching and further oxidating DNA-base-pairs. These results offer the theoretical explanation for the photo-induced oxidation-reduction mechanism which was experimentally proposed on DNA-photocleavage by Co(Ш) polypyridyl complexes. In addition, the electronic absorption spectra of these complexes were calculated and simulated in aqueous solution using the time dependent DFT (TDDFT) method, in satisfying agreement with experimental results, and the properties of experimental absorption bands have been theoretically explained in detail.     

Limited proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solublized coupling factor with chymotrypsin rapidly produced an A′-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the ATPase enzyme. Secondary chymotryptic cleavage yielded an A″-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at ?20°C in the presence of 4 mM MgCl₂ with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4°C in the presence or absence of MgCl₂ both decreased ATPase activity and generated A′-type alpha subunit species. Since presence of phenylmethylsulfonyl fluoride prevented these changes, an unknown protease was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparently located within the same small segment of alpha subunit polypeptide chain.     

Photoaffinity labeling of soluble chloroplast adenosine 5′-triphosphatase with 3′-O-(4-benzoyl)benzoyl ADP

3′-O-(4-benzoyl)benzoyl ADP (BzADP) acts as a reversible inhibitor of the chloroplast coupling factor 1 ATPase (CF₁) when incubated with the enzyme in the dark. The V_max of ATP hydrolysis is decreased and the kinetics of the reaction are altered from noncooperative to cooperative with respect to ATP. Photoactivation of the benzophenone group in BzADP by irradiation with ultraviolet light (366 nm) results in the covalent binding of BzADP to the enzyme and inactivation of its enzymic activity. Polyacrylamide gel electrophoresis of CF₁-ATPase in the presence of sodium dodecyl sulfate shows that the analog is bound primarily to the enzyme's β subunit. Complete inactivation of the activated CF₁-ATPase occurs upon covalent binding of 2.45 mol BzADP/mol CF₁. Binding of BzADP and inactivation of the ATPase are prevented if ADP, but not ATP, is present during the photoactivation step. The presence of Ca²⁺ during irradiation enhances the rate of BzADP covalent binding as well as the rate of inactivation of the enzyme.     

Synthesis and characterization of the cobalt(III) complexes of two pendant-arm cross-bridged cyclams

The cobalt(III) complexes of 4,11-diacetato-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (1), [Co(1)]PF₆, and 4,11-diacetamido-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (2), [Co(2)][PF₆]₃, have been synthesized and characterized. The crystal structure of [Co(1)]PF₆ consists of an octahedral cobalt(III) cation coordinated to all four ligand nitrogen donors in the macrobicycle’s cavity, as well as to the deprotonated carboxylate oxygen atoms of both pendant arms. Analytical and spectroscopic data indicates that the ligand in [Co(2)][PF₆]₃ is not deprotonated, suggesting coordination through the amide carbonyl oxygens. Study of the electronic spectra of these novel complexes and comparison with data from related cobalt(III) complexes characterizes the ligands as strong field with Δ₀=24,040 and Δ₀=24,250 cm⁻¹ for 1 and 2, respectively. Cyclic voltammograms were obtained for both complexes with large variations observed due to the differences in ligand charge and coordination.     

Lateral Distribution of the Cytochrome b(6)/f and Coupling Factor ATP Synthetase Complexes of Chloroplast Thylakoid Membranes

We have visualized directly the distribution of the cytochrome b₆/f and coupling factor ATP synthetase complexes in thylakoid membranes of embedded, thin-sectioned, intact chloroplasts by using rabbit antibodies directed against each complex, followed by ferritin-conjugated goat anti- (rabbit immunoglobulin G) antibodies. The labeling patterns indicate that in spinach (Spinacia oleracea) chloroplasts the cytochrome b₆/f complex is distributed laterally throughout both stacked grana and unstacked stroma membrane regions, whereas the coupling factor ATP synthetase complex is found exclusively in stroma thylakoids and in the marginal and end membranes of grana.     

Magnetic properties of six-coordinated high-spin cobalt(II) complexes: Theoretical background and its application

In this contribution we study and analyse the influence of the different parameters involved in the magnetic susceptibility of six-coordinated high-spin Co(II) complexes. We propose an empirical expression to fit the magnetic susceptibility of polycrystalline samples of mononuclear Co(II) complexes with an axial distortion, the variable parameters being Δ (axial distortion), α (orbital reduction factor) and λ (spin–orbit coupling). This expression avoids solving the 12 × 12 matrix associated to the distortion of the ⁴T_1g term. In order to take into account the magnetic coupling (J) in the polynuclear Co(II) complexes, a perturbational approach is proposed to describe their magnetic susceptibility in the whole temperature range (2–300 K) as a function of J, Δ, α and λ. This approach is valid in the limit of the weak magnetic coupling as compared to the spin–orbit coupling, |J/λ| < 0.1. The model allows the treatment of each cobalt(II) ion in axial symmetry as an effective spin S_eff = 1/2. That causes a drastic reduction of the matrix size of the polynuclear compounds from 12ⁿ × 12ⁿ to 2ⁿ × 2ⁿ, n being the number of Co(II) ions in the complex. The main advantage of the model is to make possible the fit of the magnetic susceptibility data of those polynuclear Co(II) complexes whose high nuclearity involved intractable matrices.     

Inhibition of energy-transducing functions of chloroplasts by spegazzinine.

The effects of spegazzinine, a dihydroindole alkaloid, on various energy-transducing functions of chloroplasts were studied. The following observations were made, (i) Spegazzinine inhibited both cyclic and noncyclic photophosphorylation in isolated spinach chloroplast. The I₅₀ value was about 80 μm. Over a concentration range which gave marked inhibition of ÀTP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids, while the fraction of electron transport stimulated by coupled phosphorylation was reduced to the basal level by spegazzinine. (ii) The regulatory effect of low concentrations of ATP on proton movements and electron transport was diminished by the alkaloid, (iii) Spegazzinine also inhibited with similar efficiency the ATPase activities of membrane-bound coupling factor 1 (CF₁) and of purified CF₁. One mole of spegazzinine per mole of CF₁ seemed to be required to inhibit the ATPase activity, (iv) The allosteric effect of ADP on ATPase activity was not affected by spegazzinine. (v) On the basis of these results it is concluded that spegazzinine acts as an energy transfer inhibitor of hotophosphorylation and that its site of action may be at or near the catalytic site of ATPase.     

Synthesis, characterization, electrochemical and spectroscopic investigation of cobalt(III) Schiff base complexes with axial amine ligands: The layered crystal structure of [Co(salophen)(4-picoline)2]ClO4 · CH2Cl2

Cobalt(III) complexes with potentially tetradentate salophen (H₂salophen = N,N′-bis(salicylidene)-1,2-phenylenediamine) as equatorial ligand and with different axial amine ligands (NH₃, cyclohexylamine, aniline, 4-picoline and pyridine) were synthesized and characterized by IR, ¹H NMR, elemental analysis. Electronic spectra and electrochemical properties of the complexes were studied in DMF solutions. The lowest energy transitions, which occur between 464.8 and 477 nm, are attributed mainly to the intraligand charge transfer, confirmed by Zindo/S electronic structure calculations. The reduction potentials of Co(III)/Co(II) are more affected than those of Co(II)/Co(I) by the axial amine ligands. The crystal structure of the [Co^III(salophen)(4- picoline)₂]ClO₄ · CH₂Cl₂ was determined, indicating that the cobalt(III) center is six coordinated surrounded by the tetradentate salophen ligand and two 4-picoline ligands. The crystal packing of the complex shows a layered structure, in which the perchlorate counter ions and also the lattice solvent molecules are intercalated between the bc planes of the complex cations.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate ³²P_i. ³²P_i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or ³²P_i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

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.     

Effect of removal or modification of subunit polypeptides on the coupling factor and hydrolytic activities of the Ca2+ and Mg2+-activated adenosine triphosphatase of Escherichia coli

The effect of removal or modification of the polypeptide subunits (α, β γ, δ, and ?) of the Ca²⁺ and Mg²⁺-activated ATPase of Escherichia coli was investigated. Removal of the δ-polypeptide, although giving some decrease in ATPase activity, resulted in complete loss of coupling activity, where coupling activity was measured by the restoration of the energy-dependent transhydrogenase activity of ATPase-stripped respiratory particles. Modification of the γ-polypeptide, as found in the ATPase of an energy transfer coupling mutant (etc-15), resulted in diminution of the ATPase and coupling activities. The diminished coupling activity could be overcome by using more of the enzyme which suggested that this enzyme may not be able to bind to the membrane as firmly as the enzyme from the wild type.     

DNA photocleavage activity of cobalt(III) polypyridyl complexes containing dpq ligand

Four cobalt(III) polypyridyl complexes, [Co(phen)_3−n(dpq)_n]³⁺ (phen = 1,10-phenanthroline, dpq = dipyrido[3,2-f:2′,3′-h]-quinoxaline) (n = 0, 1, 2, and 3) were synthesized and the influences of the dpq ligand on the photophysical properties, electrochemical properties, DNA binding affinities, as well as photonuclease activities of the complexes, were examined in detail. The presence of dpq ligand increases the DNA binding affinities of the corresponding complexes remarkably with respect to [Co(phen)₃]³⁺. With the sequential substitution of phen ligand by dpq ligand, the ¹O₂ quantum yields of the corresponding complexes are enhanced greatly. As a result, the photonuclease activities follow the order of [Co(dpq)₃]³⁺ > [Co(phen)(dpq)₂]³⁺ > [Co(phen)₂(dpq)]³⁺ ? [Co(phen)₃]³⁺. It was found all the examined complexes can generate OH upon UV irradiation, and OH is also involved in DNA photocleavage as reactive oxygen species.     

Preparation of a highly active ATPase of the mesophilic cyanobacterium Spirulina maxima

In this work, we report new studies on the ATPase attached to the photosynthetic membranes of the mesophilic cyanobacterium Spirulina maxima. This enzyme does not display persistent latency as had been previously reported for the ATPase of Spirulina platensis. The enzyme is readily activated by the careful application of methods currently used to activate chloroplast CF₁. Photosynthetic membranes of Spirulina maxima show a Mg²⁺-dependent ATPase activity of 195±25 mol Pi (mg chl)^–1 h^–1 after a light plus dithiothreitol (DDT) treatment. Methanol treatment of these membranes elicits Mg²⁺-dependent ATPase activity of 222±18 mol Pi (mg chl)^–1 h^–1.Here, we also describe the purification of the soluble coupling factor AF₁ of Spirulina maxima. This enzyme is unique among mesophilic cyanobacterial F₁ preparations in regard to its high specific Ca²⁺-dependent ATPase activity after heat treatment (14.75±1.91 mol Pi (mg prot)^–1 min^–1) and its room temperature stability.Abbreviations AF₁ cyanobacterial coupling factor - DTT dithiothreitol - DEAE cellulose diethylaminoethyl cellulose - DCCD N,N'-dicyclohexylcarbodiimide - EDTA ethylenediamine tetracetic, sodium salt - PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulfate - PMSF phenylmethylsulfonyl fluoride - MV methylviologen - SDS sodium dodecylsulfate - TPSnCl triphenyltin chloride - Tris tris (hydroxymethyl) aminomethane - tricine N Tris (hydroxymethyl) methylglycine - BAEE N-benzoyl-L-arginine ethyl ester     

Folding strategy to prepare Co(II)-substituted metallo-beta-lactamase L1

In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-β-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.8 Eq of cobalt, yielded a UV-Vis spectrum consistent with 5-coordinate Co(II), and exhibited a k_cat of 63 s⁻¹ and K_m of 20 μM when using nitrocefin as the substrate. Pre-steady-state fluorescence and UV-Vis studies demonstrated that refolded, Co(II)-substituted L1 uses the same kinetic mechanism as Zn(II)-containing L1, in which a reaction intermediate is formed when using nitrocefin as substrate. The described refolding strategy can be used to prepare other Co(II)-substituted Zn(II)-metalloenzymes, particularly those that contain a solvent-exposable disulfide, which often causes oxidation of Co(II) to Co(III).     

New metal complexes of N2S2 tetradentate ligands: Synthesis and spectral studies

New tetradentate ligands 2-(2-mercaptoethylthio)-N-(pyridin-2-ylmethyl)acetamide H₂L¹ and 2-chloro-2-(2-mercaptoethylthio)-N-(pyridin-2-ylmethyl)acetamide H₂L² were synthesised from the reaction of 2-aminomethanepyridine with 1,4-dithian-2-one and 3-chloro-1,4-dithian-2-one, respectively. Monomeric complexes of these ligands, of general formulae K[Cr^III(Lⁿ)Cl₂], K₂[Mn^II(Lⁿ)Cl₂] and [M(Lⁿ)] (M = Fe(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) or Hg(II); n = 1, 2) are reported. The mode of bonding and overall geometry of the complexes were determined through IR, UV-Vis, NMR and mass spectral studies, magnetic moment measurements, elemental analysis, metal content and conductance. These studies revealed octahedral geometries for the Cr(III), Mn(II) complexes, square planar for Ni(II) and Cu(II) complexes and tetrahedral for the Fe(II), Co(II), Zn(II), Cd(II) and Hg(II) complexes. The study of complex formation via molar ratio in DMF solution has been investigated and results were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1).     

Cobalt and iron complexes of chiral C1- and C2-terpyridines: Synthesis, characterization and use in catalytic asymmetric cyclopropanation of styrenes

Optically pure C₁- and C₂-terpyridine ligands (L) form cobalt(II) and iron(II) complexes of formula [Co(L)Cl₂] and [Fe(L)Cl₂], respectively, and Iron(III) complexes of formulas [Fe(L)Cl₃]. Structures of three new chiral cobalt(II) and one iron(III) complexes were analysed using X-ray crystal structure analysis. These complexes were shown to be precursor of efficient catalyst for cyclopropanation. Reaction with AgOTf converted the complex to active catalyst, which gave enantioselectivities of up to 76% ee for the trans-isomers and 83% ee for the cis-isomers of styrene cyclopropanes with ethyl diazoacetate. Hammett studies showed the active species for both cobalt and iron complexes to have a non-linear relationship to σ_p constant.     

Purification and characterization of a new ribopolynucleotide synthesizing enzyme from Escherichia coli.

A new type of ribopolynucleotide-synthesizing enzyme was found both on cytoplasmic membranes and in protein-DNA complexes isolated from Escherichia coli. The enzyme was purified by exploiting a specific, reversible enzyme aggregation with ATP and spermidine. The purified enzyme (more than 90% pure) was free from other enzymatic activities such as ATPase and polynucleotide phosphorylase. The enzyme (molecular weight 270,000 ± 15%) contains two kinds of polypeptide chain (molecular weights 91,000 ± 10%, and 45,000 ± 10%) and these polypeptides are not common with those of DNA-dependent RNA polymerase. The enzyme catalyses the synthesis of ribopolynucleotides from nucleoside triphosphates in the presence of 1 mm-MgCl₂. Rifampicin and streptolydigin do not affect the enzyme reaction.