Ascorbate Peroxidase 2 (APX2) of Chlamydomonas Binds Copper and Modulates the Copper Insertion into Plastocyanin.

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called "ascorbate peroxidase-related" (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from 1H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae.

Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives.

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors.

Hydrogen peroxide (H2O2) acts as a signalling molecule by oxidising cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H2O2-based oxidation to a multitude of target proteins. Moreover, it is becoming clear that peroxiredoxins fulfil their function in organised microdomains, where not all interactors are covalently bound. However, most studies aimed at identifying peroxiredoxin interactors were based on methods that only detect covalently linked partners. Here, we explore the applicability of two thiol-disulphide independent in-cell trapping methodological approaches in combination with mass spectrometry for the identification of interaction partners of peroxiredoxin 2 (Prdx2). The first is biotin-dependent proximity-labelling (BioID) with a biotin ligase A (BirA*)-fused Prdx2, which has never been applied on redox-active proteins. The second is crosslinker co-immunoprecipitation with an N-terminally His-tagged Prdx2. During the initial characterisation of the tagged Prdx2 constructs, we found that the His-tag, but not BirA*, compromises the peroxidase and signalling activities of Prdx2. Further, the Prdx2 interactors identified with each approach showed little overlap. We therefore concluded that BioID is a more reliable method than crosslinker co-immunoprecipitation. After a stringent mass spec data filtering, BioID identified 13 interactors under elevated H2O2 conditions, including subunit five of the COP9 signalosome complex (CSN5). The Prdx2:CSN5 interaction was further confirmed in a proximity ligation assay. Taken together, our results demonstrate that BioID can be used as a method for the identification of interactors of Prdxs, and that caution should be exercised when interpreting protein-protein interaction results using tagged Prdxs.

Prdx1 Interacts with ASK1 upon Exposure to H2O2 and Independently of a Scaffolding Protein.

Hydrogen peroxide (H2O2) is a key redox signaling molecule that selectively oxidizes cysteines on proteins. It can accomplish this even in the presence of highly efficient and abundant H2O2 scavengers, peroxiredoxins (Prdxs), as it is the Prdxs themselves that transfer oxidative equivalents to specific protein thiols on target proteins via their redox-relay functionality. The first evidence of a mammalian cytosolic Prdx-mediated redox-relay-Prdx1 with the kinase ASK1-was presented a decade ago based on the outcome of a co-immunoprecipitation experiment. A second such redox-relay-Prdx2:STAT3-soon followed, for which further studies provided insights into its specificity, organization, and mechanism. The Prdx1:ASK1 redox-relay, however, has never undergone such a characterization. Here, we combine cellular and in vitro protein-protein interaction methods to investigate the Prdx1:ASK1 interaction more thoroughly. We show that, contrary to the Prdx2:STAT3 redox-relay, Prdx1 interacts with ASK1 at elevated H2O2 concentrations, and that this interaction can happen independently of a scaffolding protein. We also provide evidence of a Prdx2:ASK1 interaction, and demonstrate that it requires a facilitator that, however, is not annexin A2. Our results reveal that cytosolic Prdx redox-relays can be organized in different ways and yet again highlight the differentiated roles of Prdx1 and Prdx2.

Charge Interactions in a Highly Charge-Depleted Protein.

Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.

Arabidopsis APx-R Is a Plastidial Ascorbate-Independent Peroxidase Regulated by Photomorphogenesis.

Peroxidases are enzymes that catalyze the reduction of hydrogen peroxide, thus minimizing cell injury and modulating signaling pathways as response to this reactive oxygen species. Using a phylogenetic approach, we previously identified a new peroxidase family composed of a small subset of ascorbate peroxidase (APx) homologs with distinguished features, which we named ascorbate peroxidase-related (APx-R). In this study, we showed that APx-R is an ascorbate-independent heme peroxidase. Despite being annotated as a cytosolic protein in public databases, transient expression of AtAPx-R-YFP in Arabidopsis thaliana protoplasts and stable overexpression in plants showed that the protein is targeted to plastids. To characterize APx-R participation in the antioxidant metabolism, we analyzed loss-of-function mutants and AtAPx-R overexpressing lines. Molecular analysis showed that glutathione peroxidase 7 (GPx07) is specifically induced to compensate the absence of APx-R. APx-R overexpressing lines display faster germination rates, further confirming the involvement of APx-R in seed germination. The constitutive overexpression of AtAPx-R-YFP unraveled the existence of a post-translational mechanism that eliminates APx-R from most tissues, in a process coordinated with photomorphogenesis. Our results show a direct role of APx-R during germinative and post-germinative development associated with etioplasts differentiation.

Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins.

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (ß1-ß2-ß5), the immunoproteasome (ß1i-ß2i-ß5i) and the two intermediate proteasomes (ß1-ß2-ß5i and ß1i-ß2-ß5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c--myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three ß5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, ß5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three ß5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function.

Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade.

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Mining for protein S-sulfenylation in Arabidopsis uncovers redox-sensitive sites.

Hydrogen peroxide (H2O2) is an important messenger molecule for diverse cellular processes. H2O2 oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in Arabidopsis thaliana cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and Arabidopsis S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in Arabidopsis cells under H2O2 stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.

Bifunctional Chloroplastic DJ-1B from Arabidopsis thaliana is an Oxidation-Robust Holdase and a Glyoxalase Sensitive to H2O2.

Members of the DJ-1 protein family are multifunctional enzymes whose loss increases the susceptibility of the cell to oxidative stress. However, little is known about the function of the plant DJ-1 homologs. Therefore, we analyzed the effect of oxidation on the structure and function of chloroplastic AtDJ-1B and studied the phenotype of T-DNA lines lacking the protein. In vitro oxidation of AtDJ-1B with H2O2 lowers its glyoxalase activity, but has no effect on its holdase chaperone function. Remarkably, upon oxidation, the thermostability of AtDJ-1B increases with no significant alteration of the overall secondary structure. Moreover, we found that AtDJ-1B transcript levels are invariable, and loss of AtDJ-1B does not affect plant viability, growth and stress response. All in all, two discrete functions of AtDJ-1B respond differently to H2O2, and AtDJ-1B is not essential for plant development under stress.

Redox-regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H-site flexibility.

Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X-ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH-binding site (G-site) and a hydrophobic co-substrate-binding site (H-site). At elevated H2 O2 concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H-site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox-regulated enzyme.

Structural snapshots of OxyR reveal the peroxidatic mechanism of H2O2 sensing.

Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2 One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2 In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.

An essential thioredoxin is involved in the control of the cell cycle in the bacterium Caulobacter crescentus.

Thioredoxins (Trxs) are antioxidant proteins that are conserved among all species. These proteins have been extensively studied and perform reducing reactions on a broad range of substrates. Here, we identified Caulobacter crescentus Trx1 (CCNA_03653; CcTrx1) as an oxidoreductase that is involved in the cell cycle progression of this model bacterium and is required to sustain life. Intriguingly, the abundance of CcTrx1 varies throughout the C. crescentus cell cycle: although the expression of CcTrx1 is induced in stalked cells, right before DNA replication initiation, CcTrx1 is actively degraded by the ClpXP protease in predivisional cells. Importantly, we demonstrated that regulation of the abundance of CcTrx1 is crucial for cell growth and survival as modulating CcTrx1 levels leads to cell death. Finally, we also report a comprehensive biochemical and structural characterization of this unique and essential Trx. The requirement to precisely control the abundance of CcTrx1 for cell survival underlines the importance of redox control for optimal cell cycle progression in C. crescentus.

Self-protection of cytosolic malate dehydrogenase against oxidative stress in Arabidopsis.

Plant malate dehydrogenase (MDH) isoforms are found in different cell compartments and function in key metabolic pathways. It is well known that the chloroplastic NADP-dependent MDH activities are strictly redox regulated and controlled by light. However, redox dependence of other NAD-dependent MDH isoforms have been less studied. Here, we show by in vitro biochemical characterization that the major cytosolic MDH isoform (cytMDH1) is sensitive to H2O2 through sulfur oxidation of cysteines and methionines. CytMDH1 oxidation affects the kinetics, secondary structure, and thermodynamic stability of cytMDH1. Moreover, MS analyses and comparison of crystal structures between the reduced and H2O2-treated cytMDH1 further show that thioredoxin-reversible homodimerization of cytMDH1 through Cys330 disulfide formation protects the protein from overoxidation. Consistently, we found that cytosolic thioredoxins interact specifically with cytMDH in a yeast two-hybrid system. Importantly, we also show that cytosolic and chloroplastic, but not mitochondrial NAD-MDH activities are sensitive to H2O2 stress in Arabidopsis. NAD-MDH activities decreased both in a catalase2 mutant and in an NADP-thioredoxin reductase mutant, emphasizing the importance of the thioredoxin-reducing system to protect MDH from oxidation in vivo. We propose that the redox switch of the MDH activity contributes to adapt the cell metabolism to environmental constraints.

Disulfide bond formation protects Arabidopsis thaliana glutathione transferase tau 23 from oxidative damage.

BACKGROUND: Glutathione transferases play an important role as detoxifying enzymes. In A. thaliana, elevated levels of reactive oxygen species (ROS), provoked during biotic and abiotic stress, influence the activity of GSTU23. The aim of this study is to determine the impact of oxidative stress on the function and structure of GSTU23. METHODS: The impact of oxidation on the function of GSTU23 was studied using a glutathione transferase biochemical assay and mass spectrometry. With kinetics, circular dichroism and thermodynamics, we compared reduced with oxidized GSTU23. X-ray crystal structures of GSTU23 visualize the impact of oxidation on methionines and cysteines. RESULTS: In the presence of 100µM H2O2, oxidation of the methionine side-chain to a sulfoxide is the prominent post-translational modification, which can be reduced by C. diphtheriae MsrA and MsrB. However, increasing the level to 200µM H2O2 results in a reversible intramolecular disulfide between Cys65-Cys110, which is substrate for glutaredoxin. Under these oxidizing conditions, GSTU23 undergoes a structural change and forms a more favourable enzyme-substrate complex to overcome kcat decrease. CONCLUSIONS AND SIGNIFICANCE: At lower H2O2 levels (100µM), GSTU23 forms methionine sulfoxides. Specifically, oxidation of Met14, located near the catalytic Ser13, could interfere with both GSH binding and catalytic activation. At higher H2O2 levels (200µM), the Cys65-Cys110 disulfide bond protects other cysteines and also methionines from overoxidation. This study shows the impact of oxidative stress on GSTU23 regulated by methionine sulfoxide reductases and glutaredoxin, and the mechanisms involved in maintaining its catalytic functionality under oxidizing conditions.

Erratum: Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis.

This corrects the article DOI: 10.1038/srep42494.

The antibacterial prodrug activator Rv2466c is a mycothiol-dependent reductase in the oxidative stress response of Mycobacterium tuberculosis.

The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis.

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.