ABSTRACT
The sediments in the Grenland fjords in southern Norway are heavily contaminated by large emissions of dioxins and mercury from historic industrial activities. As a possible in situ remediation option, thin-layer sediment surface capping with powdered activated carbon (AC) mixed with clay was applied at two large test sites (10,000 and 40,000 m2) at 30-m and 95-m depths, respectively, in 2009. This paper describes the long-term biological effects of the AC treatment on marine benthic communities up to 4 years after treatment. Our results show that the capping with AC strongly reduced the benthic species diversity, abundance, and biomass by up to 90%. Vital functions in the benthic ecosystem such as particle reworking and bioirrigation of the sediment were also reduced, analyzed by using novel bioturbation and bioirrigation indices (BPc, BIPc, and IPc). Much of the initial effects observed after 1 and 14 months were still present after 49 months, indicating that the effects are long-lasting. These long-lasting negative ecological effects should be carefully considered before decisions are made on sediment remediation with powdered AC, especially in large areas, since important ecosystem functions can be impaired.
Subject(s)
Charcoal , Estuaries , Ecosystem , Geologic Sediments , NorwayABSTRACT
The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their ß-barrel domain in response to the fluctuating acidity of the thylakoid lumen.
Subject(s)
Chlamydomonas reinhardtii/metabolism , Spinacia oleracea/metabolism , Thylakoids/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Photosystem II Protein Complex/metabolismABSTRACT
Vyacheslav Vasilevich (V.V.) Klimov (or Slava, as most of us called him) was born on January 12, 1945 and passed away on May 9, 2017. He began his scientific career at the Bach Institute of Biochemistry of the USSR Academy of Sciences (Akademy Nauk (AN) SSSR), Moscow, Russia, and then, he was associated with the Institute of Photosynthesis, Pushchino, Moscow Region, for about 50 years. He worked in the field of biochemistry and biophysics of photosynthesis. He is known for his studies on the molecular organization of photosystem II (PSII). He was an eminent scientist in the field of photobiology, a well-respected professor, and, above all, an outstanding researcher. Further, he was one of the founding members of the Institute of Photosynthesis in Pushchino, Russia. To most, Slava Klimov was a great human being. He was one of the pioneers of research on the understanding of the mechanism of light energy conversion and of water oxidation in photosynthesis. Slava had many collaborations all over the world, and he is (and will be) very much missed by the scientific community and friends in Russia as well as around the World. We present here a brief biography and some comments on his research in photosynthesis. We remember him as a friendly and enthusiastic person who had an unflagging curiosity and energy to conduct outstanding research in many aspects of photosynthesis, especially that related to PSII.
Subject(s)
Biochemistry/history , Biophysics/history , History, 20th Century , History, 21st Century , HumansABSTRACT
A field experiment with thin-layer capping was conducted in the Grenland fjords, Norway, for remediation in situ of mercury and dioxin-contaminated sediments. Experimental fields at 30 and 95 m depth were capped with (i) powdered activated carbon (AC) mixed with clay (AC+cla`y), (ii) clay, and (iii) crushed limestone. Ecological effects on the benthic community and species-feeding guilds were studied 1 and 14 months after capping, and a total of 158 species were included in the analyses. The results show that clay and limestone had only minor effects on the benthic community, while AC+clay caused severe perturbations. AC+clay reduced the abundance, biomass, and number of species by up to 90% at both 30 and 95 m depth, and few indications of recovery were found during the period of this investigation. The negative effects of AC+clay were observed on a wide range of species with different feeding strategies, although the suspension feeding brittle star Amphiura filiformis was particularly affected. Even though activated carbon is effective in reducing sediment-to-water fluxes of dioxins and other organic pollutants, this study shows that capping with powdered AC can lead to substantial disturbances to the benthic community.
Subject(s)
Charcoal , Estuaries , Aluminum Silicates , Animals , Aquatic Organisms , Biomass , Clay , Environmental Restoration and Remediation , Food Chain , Geologic Sediments , Norway , Population DynamicsABSTRACT
National recommendations in Sweden recommend a safety distance of 3 meter (m) between mobile phones and medical-electrical (ME) equipment in hospitals. A questionnaire was used to investigate how often mobile phones were reported to interfere with ME products in clinical practice across Sweden. The results confirmed that ME equipment can be affected by mobile phone use but, the risk of the patient's outcome being affected were minimal; no cases were identified which led to injury or death. In conclusion, the results support recommendations for a general safety distance of 0.5 m between mobile phones and ME equipment in care environments.
Subject(s)
Cell Phone/instrumentation , Electromagnetic Fields , Equipment and Supplies, Hospital , Hospitals/standards , Humans , SwedenABSTRACT
Three types of thin-layer caps with activated carbon (AC) were tested in situ in experimental plots (10 × 10 m) in Trondheim harbor, Norway, using AC + clay, AC-only or AC + sand. One year after capping, intact sediment cores were collected from the amended plots for ex situ surveys of the capping efficiency in reducing the PAH and PCB aqueous concentrations and bioaccumulation by the polychaete Hediste diversicolor and the clam Abra nitida. Reduced pore water concentrations were observed in all AC treatments. The capping efficiency was in general AC + clay > AC-only > AC + sand. AC + clay reduced bioaccumulation of PAH and PCB congeners between 40% and 87% in the worms and between 67% and 97% in the clams. Sediment capped with AC-only also led to reduced bioaccumulation of PCBs, while AC + sand showed no reduction in bioaccumulation. Thus the best thin-layer capping method in this study was AC mixed with clay.
Subject(s)
Bivalvia/metabolism , Charcoal/chemistry , Environmental Restoration and Remediation , Polychaeta/metabolism , Polychlorinated Biphenyls/metabolism , Polycyclic Aromatic Hydrocarbons/metabolism , Water Pollutants, Chemical/metabolism , Animals , Environmental Monitoring , Geologic Sediments/chemistry , Norway , SeasonsABSTRACT
In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 (-) increases PSII activity by acting as a mobile acceptor of the protons produced by PSII. In the green alga Chlamydomonas reinhardtii, a luminal carbonic anhydrase, CrCAH3, was suggested to improve proton removal from PSII, possibly by rapid reformation of HCO3 (-) from CO2. In this study, we investigated the interplay between PSII and CrCAH3 by membrane inlet mass spectrometry and x-ray crystallography. Membrane inlet mass spectrometry measurements showed that CrCAH3 was most active at the slightly acidic pH values prevalent in the thylakoid lumen under illumination. Two crystal structures of CrCAH3 in complex with either acetazolamide or phosphate ions were determined at 2.6- and 2.7-Å resolution, respectively. CrCAH3 is a dimer at pH 4.1 that is stabilized by swapping of the N-terminal arms, a feature not previously observed in α-type carbonic anhydrases. The structure contains a disulfide bond, and redox titration of CrCAH3 function with dithiothreitol suggested a possible redox regulation of the enzyme. The stimulating effect of CrCAH3 and CO2/HCO3 (-) on PSII activity was demonstrated by comparing the flash-induced oxygen evolution pattern of wild-type and CrCAH3-less PSII preparations. We showed that CrCAH3 has unique structural features that allow this enzyme to maximize PSII activity at low pH and CO2 concentration.
Subject(s)
Carbonic Anhydrases/chemistry , Carbonic Anhydrases/metabolism , Chlamydomonas reinhardtii/enzymology , Photosystem II Protein Complex/metabolism , Carbonic Anhydrase Inhibitors/pharmacology , Catalytic Domain , Crystallography, X-Ray , Cysteine/metabolism , Disulfides/metabolism , Hydrogen-Ion Concentration , Mass Spectrometry , Mutation , Oxidation-Reduction/drug effects , Oxygen/metabolism , Protein Structure, SecondaryABSTRACT
Cyanobacteria, algae, and plants oxidize water to the O2 we breathe, and consume CO2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO2/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast's lumen, resulting in a light-driven production of O2 and CO2. Depletion of from the media leads, in the absence of added buffers, to a reversible down-regulation of O2 production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO2/ in photosynthetic water oxidation.
Subject(s)
Bicarbonates/metabolism , Photosynthesis , Protons , Water/metabolism , Carbon Dioxide/metabolism , Carbon Isotopes , Mass Spectrometry , Models, Biological , Online Systems , Oxidation-Reduction , Oxygen/metabolism , Oxygen Isotopes , Photosystem II Protein Complex/metabolism , Time FactorsABSTRACT
Over 40 years ago, Joliot et al. (Photochem Photobiol 10:309-329, 1969) designed and employed an elegant and highly sensitive electrochemical technique capable of measuring O2 evolved by photosystem II (PSII) in response to trains of single turn-over light flashes. The measurement and analysis of flash-induced oxygen evolution patterns (FIOPs) has since proven to be a powerful method for probing the turnover efficiency of PSII. Stemler et al. (Proc Natl Acad Sci USA 71(12):4679-4683, 1974), in Govindjee's lab, were the first to study the effect of "bicarbonate" on FIOPs by adding the competitive inhibitor acetate. Here, we extend this earlier work by performing FIOPs experiments at various, strictly controlled inorganic carbon (Ci) levels without addition of any inhibitors. For this, we placed a Joliot-type bare platinum electrode inside a N2-filled glove-box (containing 10-20 ppm CO2) and reduced the Ci concentration simply by washing the samples in Ci-depleted media. FIOPs of spinach thylakoids were recorded either at 20-times reduced levels of Ci or at ambient Ci conditions (390 ppm CO2). Numerical analysis of the FIOPs within an extended Kok model reveals that under Ci-depleted conditions the miss probability is discernibly larger (by 2-3 %) than at ambient conditions, and that the addition of 5 mM HCO3 (-) to the Ci-depleted thylakoids largely restores the original miss parameter. Since a "mild" Ci-depletion procedure was employed, we discuss our data with respect to a possible function of free or weakly bound HCO3 (-) at the water-splitting side of PSII.
Subject(s)
Carbon/metabolism , Inorganic Chemicals/metabolism , Photosynthesis , Spinacia oleracea/metabolism , Water/metabolism , Buffers , Darkness , Oxidation-Reduction , Oxygen/metabolism , Thylakoids/metabolismABSTRACT
BACKGROUND: A tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16-20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Foot-and-Mouth Disease Virus/genetics , Foot-and-Mouth Disease Virus/metabolism , Viral Proteins/biosynthesis , Viral Proteins/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Carbonic Anhydrases/genetics , Carbonic Anhydrases/metabolism , Chloroplasts/genetics , Chloroplasts/metabolism , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Monomeric GTP-Binding Proteins/biosynthesis , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Mutation , Peptides/genetics , Peptides/metabolism , Plastids/metabolism , Polyproteins/genetics , Polyproteins/metabolism , Protein Transport , R-SNARE Proteins/genetics , R-SNARE Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
The efficiency of thin-layer capping in reducing sediment-to-water fluxes and bioaccumulation of polychlorinated dibenzo-p-dioxins and dibenzofurans, hexachlorobenzene, and octachlorostyrene was investigated in a boxcosm experiment. The influence of cap thickness (0.5-5 cm) and different cap materials was tested using a three-factor experimental design. The cap materials consisted of a passive material (coarse or fine limestone or a marine clay) and an active material (activated carbon (AC) or kraft lignin) to sequester the contaminants. The cap thickness and the type of active material were significant factors, whereas no statistically significant effects of the type of passive material were observed. Sediment-to-water fluxes and bioaccumulation by the two test species, the surface-dwelling Nassarius nitidus and the deep-burrowing Nereis spp., decreased with increased cap thickness and with addition of active material. Activated carbon was more efficient than lignin, and a ~90% reduction of fluxes and bioaccumulation was achieved with 3 cm caps with 3.3% AC. Small increases in fluxes with increased survival of Nereis spp. indicated that bioturbation by Nereis spp. affected the fluxes.
Subject(s)
Benzofurans/metabolism , Dioxins/metabolism , Geologic Sediments , Invertebrates/metabolism , Water Pollutants, Chemical/metabolism , Aluminum Silicates/chemistry , Animals , Benzofurans/chemistry , Calcium Carbonate/chemistry , Carbon/chemistry , Clay , Dioxins/chemistry , Environmental Restoration and Remediation/methods , Geologic Sediments/chemistry , Hydrophobic and Hydrophilic Interactions , Lignin/chemistry , Water Pollutants, Chemical/chemistryABSTRACT
The ß-class carbonic anhydrases (ß-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the ß-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of ß-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa ß-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa ß-CA structure is similar to other ß-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion--a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa ß-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and ß-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3)(-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.
Subject(s)
Acetazolamide/chemistry , Anions/chemistry , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/chemistry , Chlorophyta/metabolism , Buffers , Carbonic Anhydrase Inhibitors/pharmacology , Catalysis , Catalytic Domain , Crystallography, X-Ray/methods , Dimerization , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Conformation , Protein Binding , Protein Conformation , Static Electricity , Tyrosine/chemistryABSTRACT
Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler. Electron transfer from excited chlorophyll to ANT is impossible due to the very low reduction potential of ANT and therefore we propose that toxic concentrations of ANT increase the thylakoid membrane permeability and thereby function as an uncoupler, enhancing electron transport in vitro. Hence, its unspecific interference with photosynthetic membranes in vitro suggests that the inhibitory effect observed on intact cell photosynthesis is caused by uncoupling of phosphorylation.
Subject(s)
Anthracenes/toxicity , Chlamydomonas reinhardtii/drug effects , Photosynthesis/drug effects , Photosystem II Protein Complex/metabolism , Thylakoids/metabolism , Water Pollutants, Chemical/toxicity , Chlamydomonas reinhardtii/metabolism , Photosystem II Protein Complex/isolation & purification , Thylakoids/drug effectsABSTRACT
BACKGROUND: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. METHODOLOGY/PRINCIPAL FINDINGS: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. CONCLUSIONS/SIGNIFICANCE: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/enzymology , Carbonic Anhydrases/metabolism , Chloroplasts/enzymology , Protein Processing, Post-Translational , Amino Acid Sequence , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Binding Sites , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/genetics , Chloroplasts/metabolism , Disulfides/chemistry , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/metabolism , Glycosylation , Models, Molecular , Molecular Sequence Data , Polysaccharides/metabolism , Protein Conformation , Protein Folding , Protein TransportABSTRACT
In situ amendment of contaminated sediments using activated carbon (AC) is a recent remediation technique, where the strong sorption of contaminants to added AC reduces their release from sediments and uptake into organisms. The current study describes a marine underwater field pilot study in Trondheim harbor, Norway, in which powdered AC alone or in combination with sand or clay was tested as a thin-layer capping material for polycyclic aromatic hydrocarbon (PAH)-contaminated sediment. Several novel elements were included, such as measuring PAH fluxes, no active mixing of AC into the sediment, and the testing of new manners of placing a thin AC cap on sediment, such as AC+clay and AC+sand combinations. Innovative chemical and biological monitoring methods were deployed to test capping effectiveness. In situ sediment-to-water PAH fluxes were measured using recently developed benthic flux chambers. Compared to the reference field, AC capping reduced fluxes by a factor of 2-10. Pore water PAH concentration profiles were measured in situ using a new passive sampler technique, and yielded a reduction factor of 2-3 compared to the reference field. The benthic macrofauna composition and biodiversity were affected by the AC amendments, AC + clay having a lower impact on the benthic taxa than AC-only or AC + sand. In addition, AC + clay gave the highest AC recoveries (60% vs 30% for AC-only and AC + sand) and strongest reductions in sediment-to-water PAH fluxes and porewater concentrations. Thus, application of an AC-clay mixture is recommended as the optimal choice of the currently tested thin-layer capping methods for PAHs, and more research on optimizing its implementation is needed.
Subject(s)
Biodiversity , Charcoal/chemistry , Environmental Pollutants/analysis , Environmental Restoration and Remediation/methods , Geologic Sediments/analysis , Invertebrates , Polycyclic Aromatic Hydrocarbons/analysis , Adsorption , Analysis of Variance , Animals , Norway , Video RecordingABSTRACT
Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO(2)-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO(2). The biggest differences were typically observed 3 h after transfer to low-CO(2) conditions. Therefore, we made a careful metabolomic examination at the 3-h time point, comparing low-CO(2) treatment to high-CO(2) control. Five metabolites involved in photorespiration, 11 amino acids, and one lipid were increased, while six amino acids and, interestingly, 21 lipids were significantly lower. Our conclusion is that the metabolic pattern during early induction of the carbon-concentrating mechanism fit a model where photorespiration is increasing.
Subject(s)
Acclimatization/drug effects , Carbon Dioxide/pharmacology , Chlamydomonas reinhardtii/drug effects , Chlamydomonas reinhardtii/metabolism , Metabolome/drug effects , Metabolomics/methods , Chlamydomonas reinhardtii/growth & development , Kinetics , Models, Biological , Oxygen/metabolism , Photosynthesis/drug effects , Time FactorsABSTRACT
PsbO protein is an important constituent of the water-oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond. The PsbO protein purified from a pET32 system that includes thioredoxin fusion is properly folded and functionally active. Urea unfolding experiments imply that the reduction of the single disulfide bridge decreases stability of the protein. Analysis of inter-residue contact density through the PsbO molecule shows that Cys51 is located in a cluster with high contact density. Reduction of the Cys28-Cys51 bond is proposed to perturb the packing interactions in this cluster and destabilize the protein as a whole. Taken together, our results give evidence that PsbO exists in solution as a compact highly ordered structure, provided that the disulfide bridge is not reduced.
Subject(s)
Disulfides , Photosystem II Protein Complex/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Photosystem II Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , Spinacia oleracea/chemistry , Thioredoxins/metabolismABSTRACT
Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.
Subject(s)
Carbonic Anhydrases/metabolism , Chlamydomonas reinhardtii/metabolism , Oxygen/metabolism , Photosystem II Protein Complex/metabolism , Animals , Bicarbonates/metabolism , Carbonic Anhydrases/genetics , Chlorophyll/metabolism , Mutation , Protons , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.
Subject(s)
Photosystem II Protein Complex/chemistry , Photosystem II Protein Complex/metabolism , Protons , Water/chemistry , Models, Chemical , Models, Molecular , Oxidation-ReductionABSTRACT
Active extracellular carbonic anhydrases (CAs) were found in the alkaliphilic stromatolite-forming cyanobacterium Microcoleus chthonoplastes. Enzyme activity was detected in intact cells and in the cell envelope fraction. Western blot analysis of polypeptides from the cell envelope suggested the presence of at least two polypeptides cross-reacting with antibodies against both alpha and beta classes of CA. Immunocytochemical analysis revealed putative alpha-CA localized in the glycocalyx. This alpha-CA has a molecular mass of about 34 kDa and a pI of 3.5. External CAs showed two peaks of activity at around pH 10 and 7.5. The possible involvement of extracellular CAs of M. chthonoplastes in photosynthetic assimilation of inorganic carbon and its relationship to CaCO(3) deposition during mineralization of cyanobacterial cells are discussed.