Photo-inactivation of System II centers by carbonyl cyanide m-chlorophenylhydrazone in Chlorella pyrenoidosa

In the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (4 · 10^?6 M), the S₂ and S₃ dark decays are accelerated and become biphasic with a first half-time of 0.6 s. The first fast phase of the decays does not correspond to a simple reduction of S₂, S₃ back to S₀, S₁ (i.e. to an acceleration of the deactivation reaction), but to a decrease in the number of oxygen-evolving System II centers. This photo-inactivation produced by CCCP is rapidly reversible in the dark.     

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P₆₈₀, and the first quinone electron acceptor, Q_A, were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S₂Q_A⁻ and S₂Q_B⁻ states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S₂Q_A⁻ and S₂Q_B⁻ states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q_A/Q_A⁻ relative to that observed in the presence of DCMU, charge recombination from the S₂Q_A⁻ state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P₆₈₀⁺Phe⁻ radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P₆₈₀^* from ¹[P₆₈₀⁺Ph⁻] and direct recombination of the ³[P₆₈₀⁺Ph⁻] and ¹[P₆₈₀⁺Ph⁻] radical states, respectively. An additional non-radiative pathway involves direct recombination of P₆₈₀⁺Q_A⁻. The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of ΔG(P₆₈₀^* ↔ P₆₈₀⁺Phe⁻) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of ΔG(P₆₈₀⁺Phe⁻ ↔ P₆₈₀⁺Q_A⁻) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).     

Enhancement of rural domestic sewage treatment performance, and assessment of microbial community diversity and structure using tower vermifiltration

The performance of a novel three-stage vermifiltration (VF) system using the earthworm, Eisenia fetida, for rural domestic wastewater treatment was studied during a 131-day period. The average removal efficiencies of the tower VF planted with Penstemon campanulatus were as follows: chemical oxygen demand, 81.3%; ammonium, 98%; total nitrogen, 60.2%; total phosphorus, 98.4%; total nitrogen, mainly in the form of nitrate. Soils played an important role in removing the organic matter. The three-sectional design with increasing oxygen demand concentration in the effluents, and the distribution of certain oxides in the padding were likely beneficial for ammonium and phosphorus removal, respectively. The microbial community profiles revealed that band patterns varied more or less in various matrices of each stage at different sampling times, while the presence of earthworms intensified the bacterial diversity in soils. Retrieved sequences recovered from the media in VF primarily belonged to unknown bacterium and Bacilli of Firmicutes.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

A framework for describing topological frustration in models of protein folding

In a natively folded protein of moderate or larger size, the protein backbone may weave through itself in complex ways, raising questions about what sequence of events might have to occur in order for the protein to reach its native configuration from the unfolded state. A mathematical framework is presented here for describing the notion of a topological folding barrier, which occurs when a protein chain must pass through a hole or opening, formed by other regions of the protein structure. Different folding pathways encounter different numbers of such barriers and therefore different degrees of frustration. A dynamic programming algorithm finds the optimal theoretical folding path and minimal degree of frustration for a protein based on its natively folded configuration. Calculations over a database of protein structures provide insights into questions such as whether the path of minimal frustration might tend to favor folding from one or from many sites of folding nucleation, or whether proteins favor folding around the N terminus, thereby providing support for the hypothesis that proteins fold co-translationally. The computational methods are applied to a multi-disulfide bonded protein, with computational findings that are consistent with the experimentally observed folding pathway. Attention is drawn to certain complex protein folds for which the computational method suggests there may be a preferred site of nucleation or where folding is likely to proceed through a relatively well-defined pathway or intermediate. The computational analyses lead to testable models for protein folding.     

Chimeric phosphofructokinases involving exchange of the N- and C-terminal halves of mammalian isozymes: implications for ligand binding sites

Two phosphofructokinase (PFK) chimeras were constructed by exchanging the N- and C-terminal halves of the mammalian M- and C-type isozymes, to investigate the contribution of each terminus to the catalytic site and the fructose-2,6-P(2)/fructose-1,6-P(2) allosteric site. The homogeneously-purified chimeric enzymes organized into tetramers, and exhibited kinetic properties for fructose-6-P and MgATP similar to those of the native enzyme that furnished the N-terminal domain in each case, whereas their fructose-2,6-P(2) activatory characteristics coincided with those of the isozyme that provided the C-terminal half. This reflected the role of each domain in the formation of the corresponding binding site. Grafting the N-terminus of PFK-M onto the C-terminus of the fructose-1,6-P(2) insensitive PFK-C restored transduction of this signal to the catalytic site, which significance is also discussed.     

Half channels mediating H transport and the mechanism of gating in the Fo sector of Escherichia coli F1Fo ATP synthase

H⁺-transporting F₁F_o ATP synthase catalyzes the synthesis of ATP via coupled rotary motors within F_o and F₁. H⁺ transport at the subunit a–c interface in trans-membranous F_o drives rotation of the c-ring within the membrane, with subunit c being bound in a complex with the γ and ε subunits extending from the membrane. Finally, the rotation of subunit γ within the α₃β₃ sector of F₁ mechanically drives ATP synthesis within the catalytic sites. In this review, we propose and provide evidence supporting the route of proton transfer via half channels from one side of the membrane to the other, and the mechanism of gating H⁺ binding to and release from Asp61 of subunit c, via conformational movements of Arg210 in subunit a. We propose that protons are gated from the inside of a four-helix bundle at the periplasmic side of subunit a to drive protonation of cAsp61, and that this gating movement is facilitated by the swiveling of trans-membrane helices (TMHs) 4 and 5 at the site of interaction with cAsp61 on the periphery of the c-ring. Proton release to the cytoplasmic half channel is facilitated by the movement of aArg210 as a consequence of this proposed helical swiveling. Finally, release from the cytoplasmic half channel is mediated by residues in a complex of interacting extra-membraneous loops formed between TMHs of both subunits a and c. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.     

Renaturation of the reduced bovine pancreatic trypsin inhibitor

Refolding of the reduced pancreatic trypsin inhibitor has been investigated using thiol-disulphide exchange with various disulphide reagents to regenerate the three disulphide bonds. Essentially quantitative renaturation was routinely achieved. The refolded inhibitor was indistinguishable from the original protein in interaction with trypsin and chymotrypsin, electrophoretic mobility, and nature of disulphide bonds.The kinetics of refolding using oxidized dithiothreitol to form the disulphide bonds have been studied in some detail. The renaturation reaction is usually of second-order, being first-order in both inhibitor and disulphide reagent concentrations. A short lag period in the appearance of inhibitor activity and the inhibition of the rate, but not the extent, of renaturation by low levels of reduced dithiothreitol suggest the accumulation of metastable intermediates. In addition, heterogeneity of the refolding reaction is apparent at high concentrations of disulphide reagent, with a fraction of the material being only slowly renatured.     

The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.     

Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (> 5 mg/ml)) is associated with Photosystem I, since it supports phosphorylation with a P/e₂ value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e₂ value routinely observed is about 0.2. Bovine serum albumin (1–2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even in the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.     

Synthesis of a fluorogenic mucopolysaccharide by chondrocytes in cell culture with 4-methylumbelliferyl β-d-xyloside

Culture of chondrocytes in the presence of 4-methylumbelliferyl β-d-xyloside resulted in a synthesis of protein-free, fluorogenic chondroitin sulfate which was heterogeneous on DEAE-cellulose chromatography. Degradation of the major chromatographic fraction with chondroitinase-ABC yielded, in addition to a large quantity of Δ⁴-glucuronic acid-containing disaccharides, two flurogenic oligosaccharides of different size. Quantitative analysis showed that Δ⁴-glucuronic acid, galactose, xylose, and 4-methylumbelliferone were present in the small oligosaccharide fragment in a molar ratio of 1:2:1:1. Since these analytical data are analogous to those reported for glycopeptides derived from proteochondroitin sulfates, it may be suggested that 4-methylumbelliferyl β-d-xyloside replaces the need for xylosyl protein core in the normal synthesis of proteochondroitin sulfate with a resultant production of the unusual polysaccharide bearing the added xyloside at the reducing end.     

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.     

Arginine vasotocin neuronal phenotypes, telencephalic fiber varicosities, and social behavior in butterflyfishes (Chaetodontidae): potential similarities to birds and mammals

The neuropeptide arginine vasopressin (AVP) influences many social behaviors through its action in the forebrain of mammals. However, the function of the homologous arginine vasotocin (AVT) in the forebrain of fishes, specifically the telencephalon remains unresolved. We tested whether the density of AVT-immunoreactive (-ir) fiber varicosities, somata size or number of AVT-ir neuronal phenotypes within the forebrain were predictive of social behavior in reproductive males of seven species of butterflyfishes (family Chaetodontidae) in four phylogenetic clades. Similar to other fishes, the aggressive (often territorial) species in most cases had larger AVT-ir cells within the gigantocellular preoptic cell group. Linear discriminant function analyses demonstrated that the density of AVT-ir varicosities within homologous telencephalic nuclei to those important for social behavior in mammals and birds were predictive of aggressive behavior, social affiliations, and mating system. Of note, the density of AVT-ir varicosities within the ventral nucleus of the ventral telencephalon, thought to be homologous to the septum of other vertebrates, was the strongest predictor of aggressive behavior, social affiliation, and mating system. These results are consistent with the postulate that AVT within the telencephalon of fishes plays an important role in social behavior and may function in a similar manner to that of AVT / AVP in birds and mammals despite having cell populations solely within the preoptic area.     

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. III. A dibromothymoquinone-insensitive phosphorylation reaction associated with Photosystem II

Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP⁺. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the $\text{P}\text{e}{}_2$ ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.