Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C³¹ and C¹⁰¹-X-X-C¹⁰⁴; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn²⁺ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C³¹ and C¹⁰¹-X-X-C¹⁰⁴, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.     

Purification and properties of the sulfur oxygenase/reductase from the acidothermophilic archaeon, <Emphasis Type="Italic">Acidianus </Emphasis>strain S5

The sulfur oxygenase/reductase (SOR) of Acidianus strain S5 was purified and characterized after expressing the SOR gene in a recombinant strain of Escherichia coli. The N-terminal sequence of the purified SOR protein was the same as the deduced amino acid sequence from previously cloned SOR genes. Enzymatic studies indicated that the SOR catalyzed the conversion of elemental sulfur (S(o)) to sulfite, thiosulfate, and sulfide. The optimal pH and temperature were 5.0 and 70 degrees C, respectively. Comparison of this SOR and that of A. ambivalens revealed several differences between these two SORs. The most striking difference is that the SOR of Acidianus S5 had maximal activity at acidic pH. By application of anti-SOR serum and the Western blot technique, it was found that SOR proteins existed in A. brierleyi and in Acidianus S5 cells cultivated with thiosulfate as the sole energy source, indicating that SOR may also play a role in thiosulfate metabolism.     

Structural characterization of a metal-based perfusion tracer: Copper(II) pyruvaldehyde bis (N4-methylthiosemicarbazone)

Copper(II) pyruvaldehyde bis(N⁴-methylthiosemicarbazone), Cu(PTSM), has been obtained as a dark red crystalline solid from EtOH-DMSO solvent mixture and structurally characterized by x-ray crystallography. The molecule possesses the expected pseudo-square planar N₂S₂ metal coordination sphere; however, the copper center also interacts through its axial coordination site with the sulfur atom of an adjacent Cu(PTSM) molecule in the crystal lattice. The structure of this compound is compared with the structures of other metal complexes that have been proposed in the nuclear medicine literature as perfusion tracers.     

Physiologic Versatility and Growth Flexibility as the Main Characteristics of a Novel Thermoacidophilic Acidianus Strain Isolated from Copahue Geothermal Area in Argentina

A novel thermoacidophilic archaeal strain has been isolated from three geothermal acidic hot springs in Copahue, Argentina. One of the most striking characteristic of ALE1 isolate is its metabolic versatility. It grows on sulphur, tetrathionate, iron (II) and sucrose under aerobic conditions, but it can also develop under anaerobic conditions using iron (III) or sulphur as electron acceptors and sulphur or hydrogen as electron donors autotrophically. A temperature of 75 °C and a pH between 2.5 and 3.0 are strain ALE1 optimal growth conditions, but it is able to oxidise iron (II) even at pH 1.0. Cells are irregular cocci surrounded by a regularly arrayed glycoprotein layer (S-layer). Phylogenetic analysis shows that strain ALE1 belongs to the family Sulfolobaceae in the class Thermoprotei, within the phylum Crenarchaeota. Based on 16S rRNA gene sequence similarity on NCBI database, ALE1 does not have closely related relatives, neither in culture nor uncultured, which is more surprising. Its closest related species are strains of Acidianus hospitalis (91 % of sequence similarity), Acidianus infernus (90 %), Acidianus ambivalens (90 %) and Acidianus manzanensis (90 %). Its DNA base composition of 34.5 %?mol C?+?G is higher than that reported for other Acidianus species. Considering physiological and phylogenetic characteristics of strain ALE1, we considered it to represent a novel species of the genus Acidianus (candidatus “Acidianus copahuensis”). The aim of this study is to physiologically characterise this novel archaea in order to understand its role in iron and sulphur geochemical cycles in the Copahue geothermal area and to evaluate its potential applications in bioleaching and biooxidation.     

The sulfur oxygenase reductase from Acidianus ambivalens is an icosatetramer as shown by crystallization and Patterson analysis

The sulfur oxygenase reductase (SOR) is the initial enzyme in the aerobic sulfur metabolism of the thermoacidophilic and chemolithoautotrophic crenarchaeote Acidianus ambivalens. Single colorless polyhedral crystals were obtained under two crystallization conditions from SOR preparations heterologously overproduced in Escherichia coli. They belonged to space-group I4 and diffraction data were collected up to 1.7 A resolution. Their Patterson symmetry shows additional 4-, 3- and 2-fold non-crystallographic symmetry rotation axes, characteristic of the point group 432. Taking into account the molecular mass of SOR, the crystal unit cell volume, the non-crystallographic symmetry operators and previous electron microscopy studies of the SOR, it was deduced that the quaternary structure of the functionally active enzyme is an icosatetramer with 871 kDa molecular mass.     

Preparation, UV-Vis, IR and H NMR spectra, and molecular structure of the complex ion (η-carbonato)(α,α,α,α-tetrakis (o-pivalamidophenyl)porphinato)ferrate(III)

An iron(III) porphyrin complex containing a bidentate carbonato axial ligand has been synthesized for the first time and fully characterized by UV-Vis, IR and ¹H NMR spectroscopies and X-ray crystallography. Proton NMR data for the isolated species is in accordance with high-spin (S = 5/2) ferric derivative. The crystal structure of the (cryptand-222)potassium(+) (dihaptocarbonato)(α,α,α,α-terakis(o-pivalamidophenyl)(porphinato)ferrate(III) chlorobenzene has been determined. This structure confirms the high-spin-state of the carbonato derivative and shows that the coordination sphere of the iron atom is strongly affected by the asymmetric bidentate (η²) mode of the carbonato axial ligand.     

The first crystal structure of class III superoxide reductase from Treponema pallidum

Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl₂ and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)₄(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.This work is dedicated to the memory of Prof. Frank Rusnak.Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank under the accession code 1Y07.     

Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.     

The crystal structure of Deinococcus radiodurans Dps protein (DR2263) reveals the presence of a novel metal centre in the N terminus

The crystal structure of a DNA-binding protein from starved cells (Dps) (DR2263) from Deinococcus radiodurans was determined in two states: a native form, to 1.1-Å resolution, and one soaked in an iron solution, to 1.6-Å resolution. In comparison with other Dps proteins, DR2263 has an extended N-terminal extension, in both structures presented here, a novel metal binding site was identified in this N-terminal extension and was assigned to bound zinc. The zinc is tetrahedrally coordinated and the ligands, that belong to the N-terminal extension, are two histidines, one glutamate and one aspartate residue, which are unique to this protein within the Dps family. In the iron-soaked crystal structure, a total of three iron sites per monomer were found: one site corresponds to the ferroxidase centre with structural similarities to those found in other Dps family members; the two other sites are located on the two different threefold axes corresponding to small pores in the Dps sphere, which may possibly form the entrance and exit channels for iron storage.     

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.     

Hydrogen bonding to the cysteine ligand of superoxide reductase: acid–base control of the reaction intermediates

Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe²⁺ center in a [His₄Cys₁] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate, or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe³⁺–S(Cys) and S–C_β(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding involving the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O₂ ^?? were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pK _a of the first reaction intermediate, recently proposed to be an Fe²⁺–O₂ ^?? species. These data were supported by density functional theory calculations conducted on three models of the Fe²⁺–O₂ ^?? intermediate, with one, two, or no hydrogen bonds involving the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe²⁺–O₂ ^?? reaction intermediate to form an Fe³⁺–OOH species.     

The Oligomeric Assembly of the Novel Haem-Degrading Protein HbpS Is Essential for Interaction with Its Cognate Two-Component Sensor Kinase

HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2 and 1.6 Å) of octomeric HbpS crystallized in the presence and in the absence of haem and demonstrate that iron binds to surface-exposed lysine residues of an octomeric assembly. Based on an analysis of the crystal structures, we propose that the iron atom originates from the haem group and report subsequent biochemical experiments that demonstrate that HbpS possesses haem-degrading activity in vitro. Further examination of the crystal structures has identified amino acids that are essential for assembly of the octomer. The role of these residues is confirmed by biophysical experiments. Additionally, we show that while the octomeric assembly state of HbpS is not essential for haem-degrading activity, the assembly of HbpS is required for its interaction with the cognate sensor kinase, SenS. Homologs of HbpS and SenS/SenR have been identified in a number of medically and ecologically relevant bacterial species (including Vibrio cholerae, Klebsiella pneumoniae, Corynebacterium diphtheriae, Arthrobacter aurescens and Pseudomonas putida), suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to Gram-negative and Gram-positive bacteria. Thus, the data presented provide the first insight into the function of a novel protein family and an example of an iron-mediated interaction between an accessory protein and its cognate two-component sensor kinase.     

Mono- and dinuclear Fe(III) complexes with the N2O2 donor 5-chlorosalicylideneimine ligands; synthesis, X-ray structural characterization and magnetic properties

Two novel monomeric [C₁₈H₁₇Cl₃N₂O₂Fe] (1) and dimeric [C₃₈H₃₆N₄O₄Cl₆Fe₂] (2) Fe(III) tetradentate Schiff base complexes have been synthesized and their crystal structures have been determined by single crystal X-ray diffraction analysis. In complex (1) the Schiff base ligand coordinates toward one iron atom in a tetradentate mode and each iron atom is five coordinated with the coordination geometry around iron atom which can be described as a distorted square pyramid. The presence of a short (2.89 Å) non-bonding interatomic Fe···O distances between adjacent monomeric Fe(III) complexes results in the formation of a dimer. Structural analysis of compound (2) shows that the structure is a centrosymmetric dimer in which the six coordinated Fe(III) atoms are linked by μ-phenoxo bridges from one of the phenolic oxygen atoms of each Schiff base ligand to the opposite metal center. The variable-temperature (2-300 K) magnetic susceptibility (χ) data of these two compounds have been investigated. The results show that for both complexes Fe(III) centers are in the high spin configuration (S = 5/2) and indicate antiferromagnetic spin-exchange interaction between Fe(III) ions. The obtained results are briefly discussed using magnetostructural correlations developed for other class of iron(III) complexes.     

Crystal and molecular structure of racemic fac-tris-(S-methylisopropylidene-hydrazinecarbodithioato)cobalt(III), Co(NS)3, and the kinetics of its isomerization in aprotic solvents

The crystal and molecular structure of fac-tris- (S-methylisopropylidenehydrazinecarbodithioato)cobalt (III) is reported. The dark olive-green Co(NS)₃ crystallizes in the monoclinic space group P2₁/c with Z = 4 in a cell of dimensions a = 17.876(2), b = 9.792(2), c = 14.816(2) Å and β = 108.63(1)°. The coordination sphere is relatively undistorted but the cobalt atom does not lie in the center of this sphere. Kinetic studies have shown the fac to mer isomerization to have a very large (⩾100) equilibrium constant and to proceed by a trigonal twist mechanism.     

Crystal structures of the reaction intermediate and its homologue of an extradiol-cleaving catecholic dioxygenase

BphC derived from Pseudomonas sp. strain KKS102 is an extradiol-cleaving catecholic dioxygenase. This enzyme contains a non-heme iron atom and plays an important role in degrading biphenyl/polychlorinated biphenyls (PCBs) in the microbe. To elucidate detailed structures of BphC reaction intermediates, crystal structures of the substrate-free form, the BphC-substrate complex, and the BphC-substrate-NO (nitric oxide) complex were determined. These crystal structures revealed (1) the binding site of the O(2) molecule in the coordination sphere and (2) conformational changes of His194 during the catalytic reaction. On the basis of these findings, we propose a catalytic mechanism for the extradiol-cleaving catecholic dioxygenase in which His194 seems to play three distinct roles. At the early stage of the catalytic reaction, His194 appears to act as a catalytic base, which likely deprotonates the hydroxyl group of the substrate. At the next stage, the protonated His194 seems to stabilize a negative charge on the O2 molecule located in the hydrophobic O2-binding cavity. Finally, protonated His194 seems to function as a proton donor, whose existence has been proposed.     

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn₄CaO₅ cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn₄CaO₅ cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn₄CaO₅ cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S₁ state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.     

Structure and Mechanism of Iron Translocation by a Dps Protein from Microbacterium arborescens

Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.     

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site

Superoxide reductase (SOR) is a superoxide detoxification system present in some microorganisms. Its active site consists of an unusual mononuclear iron center with an FeN4S1 coordination which catalyzes the one-electron reduction of superoxide to form hydrogen peroxide. Different classes of SORs have been described depending on the presence of an additional rubredoxin-like, desulforedoxin iron center, whose function has remained unknown until now. In this work, we investigated the mechanism of the reduction of the SOR iron active site using the NADPH:flavodoxin oxidoreductase from Escherichia coli, which was previously shown to efficiently transfer electrons to the Desulfoarculus baarsii SOR. When present, the additional rubredoxin-like iron center could function as an electronic relay between cellular reductases and the iron active site for superoxide reduction. This electron transfer was mainly intermolecular, between the rubredoxin-like iron center of one SOR and the iron active site of another SOR. These data provide the first experimental evidence for a possible role of the rubredoxin-like iron center in the superoxide detoxifying activity of SOR.     

Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula

The kinetics of pyrite oxidation by Metallosphaera sedula were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content. M. sedula oxidized mineral pyrite at a greater rate than did another thermophile, Acidianus brierleyi, or a mesophile, Thiobacillus ferrooxidans. Maximum rates of coal depyritization were also greater with M. sedula, although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by M. sedula. Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.