Zinc Center as Redox Switch—New Function for an Old Motif

Oxidative stress affects a wide variety of different cellular processes. Now, an increasing number of proteins have been identified that use the presence of reactive oxygen species or alterations in the cellular thiol–disulfide state as regulators of their protein function. This review focuses on two members of this growing group of redox-regulated proteins that utilize a cysteine-containing zinc center as the redox switch: Hsp33, the first molecular chaperone, whose ability to protect cells against stress-induced protein unfolding depends on the presence of reactive oxygen species and RsrA, the first anti-sigma factor that uses a cysteine-containing zinc center to sense and respond to cellular disulfide stress.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63205/1/ars.2006.8.835.pd

Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells

International audienceFor the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV-Vis spectroscopy. Two sizes around 20.0 ± 5.3 nm and 37.2 ± 4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidn. over a large range of temps. A high c.d. up to 1.85 ± 0.15 mA·cm- 2 was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was esp. studied as a function of the AuNP size and amt., and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power d. of 0.25 mW·cm- 2 was recorded. [on SciFinder(R)

Revisiting the pro-oxidant activity of copper: interplay of ascorbate, cysteine and glutathione

Copper (Cu) is essential for most organisms, but it can be poisonous in excess, through mechanisms such as protein aggregation, trans-metallation and oxidative stress. Latter could implicate the formation of potentially harmful Reactive Oxygen Species (ROS: O2•–, H2O2 and HO•) via the redox cycling between Cu(II)/Cu(I) states in the presence of dioxygen and physiological reducing agents such as ascorbate (AscH), cysteine (Cys) and the tripeptide glutathione (GSH). Although the reactivity of Cu with these reductants has been previously investigated, the reactions taking place in a more physiologically-relevant mixture of these biomolecules are not known. Hence, we report here on the reactivity of Cu with binary and ternary mixtures of AscH, Cys and GSH. By measuring ascorbate and thiol oxidation, as well as HO• formation, we show that Cu reacts preferentially with GSH and Cys, halting AscH oxidation and also HO• release. This could be explained by the formation of Cu-thiolate clusters with both GSH and, as we first demonstrate here, Cys. Moreover, we observed a remarkable acceleration of Cu-catalysed GSH oxidation in the presence of Cys. We provide evidence that both thiol-disulfide exchange and the generated H2O2 contribute to this effect. Based on these findings, we speculate that Cu-induced oxidative stress may be mainly driven by GSH depletion and/or protein disulfide formation rather than by HO• and envision a synergistic effect of Cys on Cu toxicity

Are Zinc-Finger Domains of Protein Kinase C Dynamic Structures That Unfold by Lipid or Redox Activation?

Protein kinase C (PKC) is activated by lipid second messengers or redox action, raising the question whether these activation modes involve the same or alternate mechanisms. Here we show that both lipid activators and oxidation target the zinc-finger domains of PKC, suggesting a unifying activation mechanism. We found that lipid agonist-binding or redox action leads to zinc release and disassembly of zinc fingers, thus triggering large-scale unfolding that underlies conversion to the active enzyme. These results suggest that PKC zinc fingers, originally considered purely structural devices, are in fact redox-sensitive flexible hinges, whose conformation is controlled both by redox conditions and lipid agonists. Antioxid. Redox Signal. 14, 757-766.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90473/1/ars-2E2010-2E3773.pd

Etude des protéines chaperons de la famille TorD dédiées à la maturation de molybdoenzymes

J'ai décrit au cours de cette thèse une nouvelle famille de protéines chaperons de plus de trente membres impliquées spécifiquement dans la maturation de molybdoenzymes chez les procaryotes. La protéine TorD d'Escherichia coli, notre modèle, est impliquée dans la maturation cytoplasmique de TorA. En effet, TorD interagit directement avec la forme cytoplasmique non mature de TorA (apoTorA). Des études par mutagenèse dirigée laisse penser qu'une région hydrophobe de TorD serait impliquée dans cette interaction. De plus, nous avons montré par des systèmes de reconstitution in vitro que la présence de TorD augmente nettement l'efficacité d'incorporation du cofacteur à molybdène dans apoTorA. TorD induirait un changement conformationnel d'apoTorA qui favorise une conformation apte à acquérir le cofacteur à molybdène. Des études in vivo et in vitro effectuées sur d'autres membres de la famille TorD ont montré que ces chaperons sont spécifiquement dédiés à leur molybdoenzyme partenaire.During my phD, I have described a new chaperone family containing more than thirty members. This family is involved in the maturation of molybdoenzymes in bacteria. The TorD protein of Escherichia coli, our model, is the specific chaperone of periplasmic molybdoenzyme TorA. I have shown that TorD is involved in cytoplasmic maturation of TorA. Indeed, TorD interacts with the cytoplasmic form of TorA (apoTorA). We have defined by directed mutagenesis a hydrophobic patch of TorD involved probably in this interaction. Moreover, I have developed an in vitro system to reconstitute the maturation step of apoTorA. This approach revealed that TorD is essential for a correct molybdenum cofactor insertion in apoTorA. The interaction TorA/TorD modifies the conformation of apoTorA probably to make it competent to receive the molybdenum cofactor. In vivo and in vitro studies on others members of the family showed that these chaperones present a high specificity toward their molybdoenzyme partners.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

The Central Role of Redox-Regulated Switch Proteins in Bacteria

International audienceBacteria possess the ability to adapt to changing environments. To enable this, cells use reversible post-translational modifications on key proteins to modulate their behavior, metabolism, defense mechanisms and adaptation of bacteria to stress. In this review, we focus on bacterial protein switches that are activated during exposure to oxidative stress. Such protein switches are triggered by either exogenous reactive oxygen species (ROS) or endogenous ROS generated as by-products of the aerobic lifestyle. Both thiol switches and metal centers have been shown to be the primary targets of ROS. Cells take advantage of such reactivity to use these reactive sites as redox sensors to detect and combat oxidative stress conditions. This in turn may induce expression of genes involved in antioxidant strategies and thus protect the proteome against stress conditions. We further describe the well-characterized mechanism of selected proteins that are regulated by redox switches. We highlight the diversity of mechanisms and functions (as well as common features) across different switches, while also presenting integrative methodologies used in discovering new members of this family. Finally, we point to future challenges in this field, both in uncovering new types of switches, as well as defining novel additional functions

Electrochemistry, AFM and PM-IRRAS spectroscopy of immobilized hydrogenase: role of a trans-membrane helix on enzyme orientation for efficient H2 oxidation

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Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus

International audienceThermus thermophilus laccase belongs to the sub-class of multicopper oxidases that is activated by the extra binding of copper to a methionine-rich domain allowing an electron pathway from the substrate to the conventional first electron acceptor, the T1 Cu. In this work, two key amino acid residues in the 1st and 2nd coordination spheres of T1 Cu are mutated in view of tuning their redox potential and investigating their influence on copper-related activity. Evolution of the kinetic parameters after copper addition highlights that both mutations play a key role influencing the enzymatic activity in distinct unexpected ways. These results clearly indicate that the methionine rich domain is not the only actor in the cuprous oxidase activity of CueO-like enzymes

DnaJ (Hsp40 Protein) Binding to Folded Substrate Impacts KplE1 Prophage Excision Efficiency

International audienceTemperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of -helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target

Involvement of a Mate Chaperone (TorD) in the Maturation Pathway of Molybdoenzyme TorA

International audienceAs many prokaryotic molybdoenzymes, the trimethylamine oxide reductase (TorA) of Escherichia coli requires the insertion of a bis(molybdopterin guanine dinucleotide)molybdenum cofactor in its catalytic site to be active and translocated to the periplasm. We show in vitro that the purified apo form of TorA was activated weakly when an appropriate bis(molybdopterin guanine dinucleotide)molybdenum source was provided, whereas addition of the TorD chaperone increased apoTorA activation up to 4-fold, allowing maturation of most of the apoprotein. We demonstrate that TorD alone is sufficient for the efficient activation of apoTorA by performing a minimal in vitro assay containing only the components for the cofactor synthesis, apoTorA and TorD. Interestingly, incubation of apoTorA with TorD before cofactor addition led to a significant increase of apoTorA activation, suggesting that TorD acts on apoTorA before cofactor insertion. This result is consistent with the fact that TorD binds to apoTorA and probably modifies its conformation in the absence of cofactor. Therefore, we propose that TorD is involved in the first step of TorA maturation to make it competent to receive the cofactor