Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts.

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

Species-dependent uptake of gadolinium in Chlamydomonas reinhardtii algae.

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) was used in this study as a valuable tool to assess the species-dependent uptake of metallopharmaceuticals into algal cells. Chlamydomonas reinhardtii algae were incubated for 24 h with four Gadolinium-based contrast agents (GBCAs) and GdCl3. A species dependency towards the uptake of the tested Gd species was observed. Using single cell-ICP-MS, a Gd signal corresponding to single cell events was detected for GdCl3 and the linear GBCAs Omniscan® (Gadodiamide, Gd-DTPA-BMA) and Magnevist® (Gadodiamide, Gd-DTPA). For the macrocyclic complexes Dotarem® (Gadoteric acid, Gd-DOTA) and Gadovist® (Gadobutrol, Gd-BT-DO3A), no such Gd signal was visible. Total Gd analysis via ICP-MS confirmed the presence of Gd in the cells only after incubation with GdCl3 and the linear GBCAs, while only small amounts of Gd were detected for the incubations with macrocyclic GBCAs. Furthermore, the results showed that more Gd is bound to cell structures or macromolecules, while smaller amounts are present in the lysate. Using hydrophilic interaction liquid chromatography (HILIC)-ICP-MS, the soluble Gd species in the lysate were analyzed to determine if the initial Gd complexes were still intact. Surprisingly, no intact GBCAs were detected in the lysates of any incubation solution, possibly due to a change in Gd speciation. Further research is needed to assess which Gd species are present in the lysate, while "free" Gd ions or adducts with cell constituents are the most likely explanation. This study highlights the need for species-dependent investigation of elements in aquatic organisms. Moreover, the uptake of linear GBCAs and their species alteration raises the question of a potential accumulation of Gd in the food chain.

The first HyDRA challenge for computational vibrational spectroscopy.

Vibrational spectroscopy in supersonic jet expansions is a powerful tool to assess molecular aggregates in close to ideal conditions for the benchmarking of quantum chemical approaches. The low temperatures achieved as well as the absence of environment effects allow for a direct comparison between computed and experimental spectra. This provides potential benchmarking data which can be revisited to hone different computational techniques, and it allows for the critical analysis of procedures under the setting of a blind challenge. In the latter case, the final result is unknown to modellers, providing an unbiased testing opportunity for quantum chemical models. In this work, we present the spectroscopic and computational results for the first HyDRA blind challenge. The latter deals with the prediction of water donor stretching vibrations in monohydrates of organic molecules. This edition features a test set of 10 systems. Experimental water donor OH vibrational wavenumbers for the vacuum-isolated monohydrates of formaldehyde, tetrahydrofuran, pyridine, tetrahydrothiophene, trifluoroethanol, methyl lactate, dimethylimidazolidinone, cyclooctanone, trifluoroacetophenone and 1-phenylcyclohexane-cis-1,2-diol are provided. The results of the challenge show promising predictive properties in both purely quantum mechanical approaches as well as regression and other machine learning strategies.

Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii.

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnect redox control with active photosynthetic electron transport and metabolism, as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

Light-independent regulation of algal photoprotection by CO2 availability.

Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

Loss of respiratory complex I subunit NDUFB10 affects complex I assembly and supercomplex formation.

The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII2 + CIVn). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P P -module is downregulated and the P D -module is completely abolished in NDUFB10 knock outs.

Remodeling of algal photosystem I through phosphorylation.

Photosystem I (PSI) with its associated light-harvesting system is the most important generator of reducing power in photosynthesis. The PSI core complex is highly conserved, whereas peripheral subunits as well as light-harvesting proteins (LHCI) reveal a dynamic plasticity. Moreover, in green alga, PSI-LHCI complexes are found as monomers, dimers, and state transition complexes, where two LHCII trimers are associated. Herein, we show light-dependent phosphorylation of PSI subunits PsaG and PsaH as well as Lhca6. Potential consequences of the dynamic phosphorylation of PsaG and PsaH are structurally analyzed and discussed in regard to the formation of the monomeric, dimeric, and LHCII-associated PSI-LHCI complexes.

Algal PETC-Pro171-Leu suppresses electron transfer in cytochrome b6f under acidic lumenal conditions.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

Algal photosystem I dimer and high-resolution model of PSI-plastocyanin complex.

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI-light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.

Hydrogen production in the presence of oxygen by Escherichia coli K-12.

Escherichia coli is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic (anoxic) conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a micro-aerobic (micro-oxic) environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under micro-aerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

Photosystem I light-harvesting proteins regulate photosynthetic electron transfer and hydrogen production.

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H2) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H2 production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H2 photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H2 photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H2 photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.

Electron transfer via cytochrome b6f complex displays sensitivity to antimycin A upon STT7 kinase activation.

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii.

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.

Using Caenorhabditis elegans to produce functional secretory proteins of parasitic nematodes.

The expression of antigens in their immunologically-active form remains a challenge, both in the analysis of regulatory pathways exploited by parasitic nematodes or in the development of vaccines. Despite the success of native proteins to induce protective immunity, recombinant proteins expressed in bacteria, yeast or insect cells offer only limited protective capacities, presumably due to incorrect folding or missing complex posttranslational modifications. The present study investigates the feasibility of using the free-living nematode Caenorhabditis elegans as an alternative expression system for proteins found in the secretome of parasitic nematodes. Exemplified by the expression of the extracellular superoxide dismutase from Haemonchus contortus (HcSODe) and the extracellular and glycosylated glutathione S-transferase from the filarial parasite Onchocerca volvulus (OvGST1), we continue our efforts to improve production and purification of recombinant proteins expressed in C. elegans. We demonstrate that sufficient quantities of functional proteins can be expressed in C. elegans for subsequent immunological and biochemical studies.

Advanced spectroscopic analysis and 15N-isotopic labelling study of nitrate and nitrite reduction to ammonia and nitrous oxide by E. coli.

Nitrate and nitrite reduction to ammonia and nitrous oxide by anaerobic E. coli batch cultures is investigated by advanced spectroscopic analytical techniques with 15N-isotopic labelling. Non-invasive, in situ analysis of the headspace is achieved using White cell FTIR and cavity-enhanced Raman (CERS) spectroscopies alongside liquid-phase Raman spectroscopy. For gas-phase analysis, White cell FTIR measures CO2, ethanol and N2O while CERS allows H2, N2 and O2 monitoring. The 6 m pathlength White cell affords trace gas detection of N2O with a noise equivalent detection limit of 60 nbar or 60 ppbv in 1 atm. Quantitative analysis is discussed for all four 14N/15N-isotopomers of N2O. Monobasic and dibasic phosphates, acetate, formate, glucose and NO3- concentrations are obtained by liquid-phase Raman spectroscopy, with a noise equivalent detection limit of 0.6 mM for NO3- at 300 s integration time. Concentrations of the phosphate anions are used to calculate the pH in situ using a modified Henderson-Hasselbalch equation. NO2- concentrations are determined by sampling for colorimetric analysis and NH4+ by basifying samples to release 14N/15N-isotopomers of NH3 for measurement in a second FTIR White cell. The reductions of 15NO3-, 15NO2-, and mixed 15NO3- and 14NO2- by anaerobic E. coli batch cultures are discussed. In a major pathway, NO3- is reduced to NH4+via NO2-, with the bulk of NO2- reduction occurring after NO3- depletion. Using isotopically labelled 15NO3-, 15NH4+ production is distinguished from background 14NH4+ in the growth medium. In a minor pathway, NO2- is reduced to N2O via the toxic radical NO. With excellent detection sensitivities, N2O serves as a monitor for trace NO2- reduction, even when cells are predominantly reducing NO3-. The analysis of N2O isotopomers reveals that for cultures supplemented with mixed 15NO3- and 14NO2- enzymatic activity to reduce 14NO2- occurs immediately, even before 15NO3- reduction begins. Optical density and pH measurements are discussed in the context of acetate, formate and CO2 production. H2 production is repressed by NO3-; but in experiments with NO2- supplementation only, CERS detects H2 produced by formate disproportionation after NO2- depletion.

Tuning the properties of hydrogen-bonded block copolymer worm gels prepared via polymerization-induced self-assembly.

Polymerization-induced self-assembly (PISA) is exploited to design hydrogen-bonded poly(stearyl methacrylate)-poly(benzyl methacrylate) [PSMA-PBzMA] worm gels in n-dodecane. Using a carboxylic acid-based RAFT agent facilitates hydrogen bonding between neighboring worms to produce much stronger physical gels than those prepared using the analogous methyl ester-based RAFT agent. Moreover, tuning the proportion of these two types of end-groups on the PSMA chains enables the storage modulus (G') of a 20% w/w worm gel to be tuned from ∼4.5 kPa up to ∼114 kPa. This is achieved via two complementary routes: (i) an in situ approach using binary mixtures of acid- and ester-capped PSMA stabilizer chains during PISA or (ii) a post-polymerization processing strategy using a thermally-induced worm-to-sphere transition to mix acid- and ester-functionalized spheres at 110 °C that fuse to form worms on cooling to 20 °C. SAXS and rheology studies of these hydrogen-bonded worm gels provide detailed insights into their inter-worm interactions and physical behavior, respectively. In the case of the carboxylic acid-functionalized worms, SAXS provides direct evidence for additional inter-worm interactions, while rheological studies confirm both a significant reduction in critical gelation concentration (from approximately 10% w/w to 2-3% w/w) and a substantial increase in critical gelation temperature (from 41 °C to 92 °C). It is remarkable that a rather subtle change in the chemical structure results in such improvements in gel strength, gelation efficiency and gel cohesion.

Functional basis of electron transport within photosynthetic complex I.

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme - the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

Structure of plant photosystem I-plastocyanin complex reveals strong hydrophobic interactions.

Photosystem I is defined as plastocyanin-ferredoxin oxidoreductase. Taking advantage of genetic engineering, kinetic analyses and cryo-EM, our data provide novel mechanistic insights into binding and electron transfer between PSI and Pc. Structural data at 2.74 Šresolution reveals strong hydrophobic interactions in the plant PSI-Pc ternary complex, leading to exclusion of water molecules from PsaA-PsaB/Pc interface once the PSI-Pc complex forms. Upon oxidation of Pc, a slight tilt of bound oxidized Pc allows water molecules to accommodate the space between Pc and PSI to drive Pc dissociation. Such a scenario is consistent with the six times larger dissociation constant of oxidized as compared with reduced Pc and mechanistically explains how this molecular machine optimized electron transfer for fast turnover.

Crystallographic analysis and phasing of iron-assimilating protein 1 (FEA1) from Chlamydomonas reinhardtii.

As an essential component of protein cofactors, iron is important for all photosynthetic organisms. In Chlamydomonas reinhardtii, iron levels are strictly controlled by proteins such as iron-assimilating protein 1 (FEA1). This periplasmic protein is expressed under conditions of iron deficiency, but its mechanisms of function remain unknown. Because FEA1 has no amino-acid similarity to protein structures in the Protein Data Bank, its crystal structure cannot be solved by molecular replacement. Here, recombinant FEA1 protein lacking the N-terminal signal sequence was successfully purified and crystals of apo FEA1 were obtained by hanging-drop vapor diffusion. Neither selenomethionine substitution nor heavy-atom derivatization was successful; therefore, the phase problem of FEA1 crystals was solved by the native sulfur SAD method using long-wavelength X-rays (2.7 Å). Laser-cutting technology was used to increase the signal-to-noise ratio and derive an initial structure. This study will lead to further structural studies of FEA1 to understand its function and its links to the iron-assimilation pathway.