Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cells

Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts.     

The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques

Nisin, a peptide antibiotic, efficiently kills bacteria through a unique mechanism which includes inhibition of cell wall biosynthesis and pore formation in cytoplasmic membranes. Both mechanisms are based on interaction with the cell wall precursor lipid II which is simultaneously used as target and pore constituent. We combined two biosensor techniques to investigate the nisin activity with respect to membrane binding and pore formation in real time. Quartz crystal microbalance (QCM) allows the detection of nisin binding kinetics. The presence of 0.1 mol% lipid II strongly increased nisin binding affinity to DOPC (k_D 2.68 × 10^− 7 M vs. 1.03 × 10^− 6 M) by a higher association rate. Differences were less pronounced while using negatively charged DOPG membranes. However, lipid II does not influence the absolute amount of bound nisin. Cyclic voltammetry (CV) data confirmed that in presence of 0.1 mol% lipid II, nanomolar nisin concentrations were sufficient to form pores, while micromolar concentrations were necessary in absence of lipid II. Both techniques suggested unspecific destruction of pure DOPG membranes by micromolar nisin concentrations which were prevented by lipid II. This model membrane stabilization by lipid II was confirmed by atomic force microscopy. Combined CV and QCM are valuable to interpret the role of lipid II in nisin activity.     

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V_th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V_th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V_th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V_th ∼ 250 mV) followed by a noticeable destabilization (V_th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V_th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10^− 3 proteins/μm². In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.     

A dye-binding assay for measurement of the binding of Cu(II) to proteins

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to α-synuclein of ∼1 × 10⁹ M⁻¹ was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).     

Plasma membrane-porating domain in poliovirus 2B protein. A short peptide mimics viroporin activity

Picornavirus 2B, a non-structural protein required for effective viral replication, has been implicated in cell membrane permeabilization during the late phases of infection. Here, we have approached the molecular mechanism of this process by assessing the pore-forming activity of an overlapping peptide library that spanned the complete 2B sequence. At non-cytopathic concentrations, only the P3 peptide, spanning 2B residues 35-55, effectively assembled hydrophilic pores that allowed diffusion of low molecular mass solutes across the cell plasma membrane (IC₅₀ ≈ 4 × 10⁻⁷ M) and boundary liposome bilayers (starting at peptide to lipid molar ratios > 1:10⁴). Circular dichroism data were consistent with its capacity to fold as a helix in a membrane-like environment. Furthermore, addition of this peptide to a sealed plasma-membrane model, consisting of retinal rod outer segments patch-clamped in a whole-cell configuration, induced ion channel activity within seconds at concentrations as low as 10⁻⁸ M. Thus, we have established a “one-helix” 2B version that possesses the intrinsic pore-forming activity required to directly and effectively permeabilize the cell plasma membrane. We conclude that 2B viroporin can be classified as a genuine pore-forming toxin of viral origin, which is produced intracellularly at certain times post infection.     

Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix–helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of − 4.61 ± 0.04 kcal/mol at bicelle conditions where the sampling of random helix–helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of > 1 kcal/mol was obtained from helix–helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500 ± 100 s^− 1 and 2.1 ± 0.1 s^− 1, respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen–Manganese Transporter MntC

MntC is a metal-binding protein component of the Mn^2 +-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2 Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn^2 +-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn^2 +-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium–hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn^2 +.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

Conformational Dynamics in the Selectivity Filter of KcsA in Response to Potassium Ion Concentration

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 μM. Potassium ion titration studies reveal that the site-specific K_d for K⁺ binding at the key pore residue Val76 is on the order of ∼ 7 μM and that a relatively high sample hydration is necessary to observe the low-K⁺ conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K⁺ and low-K⁺ states are in slow exchange on the NMR timescale (k_ex < 500 s^− 1). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K⁺ state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M⁻¹ s⁻¹ and 0.34 ± 0.15 s⁻¹, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min⁻¹ at pH 6.0.     

The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C Virus Helicase NS3h Is Dependent on Both Sugar and Base Moieties

The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3′-to-5′ superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s^− 1 (oligo(dT)), 35 nt s^− 1 (oligo(dU)), and 42 nt s^− 1 (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K_0.5 for NS3h translocation at a saturating ATP concentration are as follows: 3.3 ± 0.4 μM nucleotide (poly(dT)), 27 ± 2 μM nucleotide (poly(dU)), and 36 ± 2 μM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS⁰ are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (∼ 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.     

Thermodynamics of GTP and GDP Binding to Bacterial Initiation Factor 2 Suggests Two Types of Structural Transitions

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 °C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 °C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (− 868 ± 25 and − 577 ± 23 cal mol^− 1 K^− 1, respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the γ-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.     

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.