The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.     

The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center

The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.     

Arsenite oxidation by the heterotroph Hydrogenophaga sp. str. NT-14: the arsenite oxidase and its physiological electron acceptor

Heterotrophic arsenite oxidation by Hydrogenophaga sp. str. NT-14 is coupled to the reduction of oxygen and appears to yield energy for growth. Purification and partial characterization of the arsenite oxidase revealed that it (1). contains two heterologous subunits, AroA (86 kDa) and AroB (16 kDa), (2). has a native molecular mass of 306 kDa suggesting an alpha(3)beta(3) configuration, and (3). contains molybdenum and iron as cofactors. Although the Hydrogenophaga sp. str. NT-14 arsenite oxidase shares similarities to the arsenite oxidases purified from NT-26 and Alcaligenes faecalis, it differs with respect to activity and overall conformation. A c-551-type cytochrome was purified from Hydrogenophaga sp. str. NT-14 and appears to be the physiological electron acceptor for the arsenite oxidase. The cytochrome can also accept electrons from the purified NT-26 arsenite oxidase. A hypothetical electron transport chain for heterotrophic arsenite oxidation is proposed.     

Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.     

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite.     

Phylogenetic and phenotypic analyses of arsenic-reducing bacteria isolated from an old tin mine area in Thailand

An agar plate screening assay was used to determine whether 100 arsenic-resistant bacterial isolates, previously obtained from arsenic-contaminated soils, had the ability to transform arsenite and arsenate. Ninety-five percent of the isolates were capable of reducing arsenate on agar plates. The isolates also grew in the presence of high concentrations of arsenite, but none of the bacterial isolates oxidized arsenite to arsenate under the growth conditions tested. About 14 % (13 of 95) of the tested isolates transformed high levels of arsenate (33–70 μM) when tested using the molybdenum blue method. Partial sequence analysis of 16S rDNA genes indicated that the isolates belonged to two broad taxonomic groups: Firmicutes and Proteobacteria. Ten isolates were assigned to four species in the genus Bacillus, and three isolates belonged to two species in the genera Enterobacter and Ochrobactrum. Taken together these results indicate that phylogenetically diverse bacteria isolated from arsenic-contaminated soils in an old tin mine area in Thailand have the ability to transform arsenate to arsenite.     

Electrochemical studies of arsenite oxidase: an unusual example of a highly cooperative two-electron molybdenum center

Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center. The reduction potential for the molybdenum center, 292 mV (vs SHE) at pH 5.9 and 0 degrees C, exhibits a linear pH dependence for pH 5-10, consistent with a two-electron reduction strongly coupled to the uptake of two protons without a pK in this range. This suggests that the oxidized enzyme is best characterized as having an L(2)MoO(2) rather than L(2)MoO(OH) center in the oxidized state and that arsenite oxidase uses a "spectator oxo" effect to facilitate the oxo transfer reaction. The onset of the catalytic wave observed in the presence of substrate correlates well with the Mo(VI/IV) potential, consistent with catalytic electron transport that is limited only by turnover at the active site. The one-electron peaks for the iron-sulfur centers are difficult to observe by protein film voltammetry, but spectrophotometric titrations have been carried out to measure their reduction potentials: at pH 6.0 and 20 degrees C, that of the [3Fe-4S] center is approximately 260 mV and that of the Rieske center is approximately 130 mV.     

Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 A and 2.03 A

BACKGROUND: Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It is induced by the presence of AsO(2-) (arsenite) and functions to oxidize As(III)O(2-), which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic As(V)O(4)(3-) (arsenate) prior to methylation. RESULTS: Using a combination of multiple isomorphous replacement with anomalous scattering (MIRAS) and multiple-wavelength anomalous dispersion (MAD) methods, the crystal structure of arsenite oxidase was determined to 2.03 A in a P2(1) crystal form with two molecules in the asymmetric unit and to 1.64 A in a P1 crystal form with four molecules in the asymmetric unit. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The small subunit contains a Rieske-type [2Fe-2S] site. CONCLUSIONS: The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans, but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome bc(1) and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase.     

Respiratory arsenate reductase as a bidirectional enzyme

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.     

Turkey liver xanthine dehydrogenase: properties of the enzyme dependent on the content of functional active sites (Short Communication)

Turkey liver xanthine dehydrogenase containing the full complement of molybdenum, flavin and iron–sulphur prosthetic groups is, as normally isolated, a mixture of functional and non-functional enzyme. The latter apparently lacks the cyanolysable persulphide groups essential to the oxidation of xanthine and to interaction with arsenite. These groups are not required for the oxidation of NADH by Methylene Blue. That KI treatment effects a differential release of flavin from xanthine-prereduced and NADH-prereduced enzyme merely reflects the degree of functionality of the preparations used and may not be taken as evidence for non-equivalence of the flavin chromophores.     

p38 and Extracellular Signal‐Regulated Kinases Activations have Opposite Effects on Primary‐Cultured Rat Cerebellar Granule Neurons Exposed to Sodium Arsenite

Arsenic is a widespread environmental toxicant in the world and regarded as both a carcinogen and an anticarcinogen. The present study was designed to evaluate roles of mitogen‐activated protein kinases in sodium arsenite‐induced effects on primary‐cultured rat cerebellar granule neurons (CGNs). Results revealed a decreased viability of the cells exposed to sodium arsenite (from 0 to 50 μM) in a dose‐dependent manner. Annexin V‐fluorescein isothiocyanate assay showed that apoptosis was obviously induced by arsenite treatment. High phosphorylation expressions of p38 and extracellular signal‐regulated kinases (ERK1/2), but not of c‐Jun N‐terminal kinases were observed due to arsenite treatment by western blotting analysis. Furthermore, SB203580 (an inhibitor of p38) decreased the percentage of apoptotic cells whereas arsenite‐stimulated toxicity was enhanced by U0126 (an inhibitor of ERK1/2). Taken together, these data suggest that p38 contributes to arsenite‐induced apoptosis of rat CGNs, but ERK1/2 may involve in cell growth and survival.     

Visualisation of gradients in arsenic concentrations around individual roots of Zea mays L. using agar-immobilized bioreporter bacteria

The classical concept of arsenic transfer into plants through arsenate uptake via phosphate transporters, reduction to arsenite, complexation and compartmentation within vacuoles is challenged by recent identification of bidirectional transporters for arsenite and their potential role in plant As status regulation. Soil-based studies with chemical analysis of soil solution require root mat formation amplifying root effects on their surroundings and additionally denying investigations along individual roots differing in age and function. We tried to overcome these shortcomings by using bioreporter bacteria to visualise the spatial distribution of inorganic arsenic along roots and to characterize inorganic arsenic gradients in the rhizosphere concurrent with root age and branching. Therefore we developed an agar-based carrier element ensuring intimate contact between bioreporters and root-soil system and enabling fast and easy reporter output analysis. We show that inorganic arsenic distribution is related to root development with the highest bioreporter signal induction around lateral roots, which are known to show the highest expression of transporters responsible for bidirectional arsenite flux. Since there is so far no evidence for an arsenate efflux mechanism this is a strong indicator that we observed rather arsenite than arsenate efflux. No signal was detected along the distal region of young adventitious roots, i.e. the region of extension growth and root hair formation. The novel bioreporter assay may thus complement conventional measurements by providing information on the spatial distribution of inorganic arsenic on mm to cm-scale.     

The detection of messenger ribonucleic acid sequences in heterogeneous nuclear ribonucleic acid fractions of the oestrogen-stimulated rat uterus.

Active xanthine oxidase was labelled specifically with 33S in the cyanide-labile site of the molybdenum centre. The Very Rapid molybdenum (V) e.p.r. signal, generated from this, shows strong coupling of 33S to molybdenum, providing unambiguous evidence that, at least in the signal-giving species, this sulphur atom is a ligand of molybdenum. The structure of the signal-giving species is discussed.     

Studies by electron-paramagnetic-resonance spectroscopy on the mechanism of action of xanthine dehydrogenase from Veillonella alcalescens.

E.p.r- (electron-paramagnetic-resonance) spectroscopy was used to compare chemical environment and reactivity of molybdenum, flavin and iron-sulphur centres in the enzyme xanthine dehydrogenase from Veillonella alcalescens (Micrococcus lactilyticus) with those of the corresponding centres in milk xanthine oxidase. The dehydrogenase is frequently contaminated with small but variable amounts of a species resistant to oxidation and giving a new molybdenum (V) e.p.r. signal, "Resting I". There is also a "desulpho" form of the enzyme giving a Slow Mo(V) signal, indistinguishable from that of the milk enzyme. Molybdenum of the active enzyme behaves in a manner analogous to that of the milk enzyme, giving a Rapid Mo(V) signal on partial reduction with substrates or dithionite. Detailed comparison shows that molybdenum in each enzyme must have the same ligand atoms arranged in the same manner. As with the milk enzyme, complex-formation between reduced dehydrogenase and purine substrate molecules, presumably interacting at the normal substrate-binding site, modifies the Rapid signal, confirming that such substrates interact near molybdenum. The dehydrogenase-flavin semiquinone signal is identical with that of the oxidase but, in contrast, there is only one iron-sulphur signal. The latter gives an e.p.r. spectrum similar to that of aldehyde oxidase.     

Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli

Molybdenum insertion into the dithiolene group on the 6-alkyl side-chain of molybdopterin is a highly specific process that is catalysed by the MoeA and MogA proteins in Escherichia coli. Ligation of molybdate to molybdopterin generates the molybdenum cofactor, which can be inserted directly into molybdoenzymes binding the molybdopterin form of the molybdenum cofactor, or is further modified in bacteria to form the dinucleotide form of the molybdenum cofactor. The ability of various metals to bind tightly to sulfur-rich sites raised the question of whether other metal ions could be inserted in place of molybdenum at the dithiolene moiety of molybdopterin in molybdoenzymes. We used the heterologous expression systems of human sulfite oxidase and Rhodobacter sphaeroides dimethylsulfoxide reductase in E. coli to study the incorporation of different metal ions into the molybdopterin site of these enzymes. From the added metal-containing compounds Na(2)MoO(4), Na(2)WO(4), NaVO(3), Cu(NO(3))(2), CdSO(4) and NaAsO(2) during the growth of E. coli, only molybdate and tungstate were specifically inserted into sulfite oxidase and dimethylsulfoxide reductase. Other metals, such as copper, cadmium and arsenite, were nonspecifically inserted into sulfite oxidase, but not into dimethylsulfoxide reductase. We showed that metal insertion into molybdopterin occurs beyond the step of molybdopterin synthase and is independent of MoeA and MogA proteins. Our study shows that the activity of molybdoenzymes, such as sulfite oxidase, is inhibited by high concentrations of heavy metals in the cell, which will help to further the understanding of metal toxicity in E. coli.     

Effects of arsenite and UVA-1 radiation on calcineurin signaling

Calcineurin is a Ca(2+)-dependent serine/threonine phosphatase and the target of the immunosuppressive drugs cyclosporin and tacrolimus, which are used in transplant recipients to prevent rejection. Unfortunately, the therapeutic use of this drugs is complicated by a high incidence of skin malignancy, which has set off a number of studies into the role of calcineurin signaling in skin, particularly with respect to cell cycle control and DNA repair. Both UVA1 radiation and arsenic species are known to promote skin cancer development via production of reactive oxygen species. In light of the well-documented sensitivity of calcineurin to oxidative stress, we examined and compared the effects of UVA1 and arsenite on calcineurin signaling. In this paper, we show that physiologically relevant doses of UVA1 radiation and low micromolar concentrations of arsenite strongly inhibit calcineurin phosphatase activity in Jurkat and skin cells and decrease NFAT nuclear translocation in Jurkat cells. The effects on calcineurin signaling could be partly prevented by inhibition of NADPH oxidase in Jurkat cells or increased dismutation of superoxide in Jurkat and skin cells. In addition, both UVA1 and arsenite decreased NF-κB activity, although at lower concentrations, arsenite enhanced NF-κB activity. These data indicate that UVA1 and arsenite affect a signal transduction route of growingly acknowledged importance in skin and that calcineurin may serve as a potential link between ROS exposure and impaired tumor suppression.