In vitro reconstitution of demolybdosulfite oxidase by molybdate.

Reconstitution of purified demolybdosulfite oxidase from rat liver has been achieved using inorganic molybdate as the source of molybdenum. The activation process has a pH optimum of 7.4 and is dependent on concentrations of molybdate and demolybdoenzyme. The reaction is inhibited by high concentrations of anions and by reduction of the demolybdoenzyme and requires incubation temperatures higher than 30 degrees. A reconstitution mechanism involving loss of tungsten and concomitant replacement with molybdenum in those demolybdo molecules which contain tungsten is supported by the following observations: (a) the extent of activation achieved by molybdate corresponds to the proportion of molecules in the preparation which contain tungsten. (b) Incubation of the demolybdoenzyme preparation at 37 degrees in the absence of molybdate results in progressive and concentration-dependent loss of ability to be reconstituted by molybdate and a corresponding but more rapid loss of tungsten from the enzyme. The reconstituted enzyme displays the molybdenum EPR signal characteristic of native enzyme and is inactivated by incubation at 42 degrees in a manner identical to native sulfite oxidase.     

A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei.

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten-grown cells, has been purified and shown to be a tungsten-iron-sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum-grown cells and shown that it is a molybdenum-iron-sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146-kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non-heme iron but no tungsten (< 0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N-furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N-terminal amino acid sequences determined for the alpha and beta subunits were identical up to residue 23, indicating that the two proteins are isoenzymes. The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.     

The Effect of the Simultaneous Addition of Molybdenum and Tungsten to the Culture Medium on the Formate Dehydrogenase Activity from Methylobacterium sp. RXM

Shake flask cultivation of the facultative methylotroph Methylobacterium sp. RXM was carried out by using a statistical experimental design to investigate the role of metal association on the formate dehydrogenase (FDH) levels. The maximal values of FDH activity were obtained for tungsten concentration up to 0.6 μM and for molybdenum concentration between 0.6 and 0.9 μM. The negative polynomial parameter (β₂) for tungsten compared with the positive polynomial parameter (β₁) for molybdenum on the FDH activity suggested that the latter metal exerts a stronger influence on the enzyme stimulation than the tungsten metal. A negative interaction between both metals was found, suggesting that tungsten and molybdenum shared an antagonistic effect on the enzyme activity. Received: 21 October 1997 / Accepted: 8 December 1997     

Why is the molybdenum-substituted tungsten-dependent formaldehyde ferredoxin oxidoreductase not active? A quantum chemical study

Formaldehyde ferredoxin oxidoreductase is a tungsten-dependent enzyme that catalyzes the oxidative degradation of formaldehyde to formic acid. The molybdenum ion can be incorporated into the active site to displace the tungsten ion, but is without activity. Density functional calculations have been employed to understand the incapacitation of the enzyme caused by molybdenum substitution. The calculations show that the enzyme with molybdenum (Mo-FOR) has higher redox potential than that with tungsten, which makes the formation of the Mo^VI=O complex endothermic by 14 kcal/mol. Following our previously suggested mechanism for this enzyme, the formaldehyde substrate oxidation was also investigated for Mo-FOR using the same quantum-mechanics-only model, except for the displacement of tungsten by molybdenum. The calculations demonstrate that formaldehyde oxidation occurs via a sequential two-step mechanism. Similarly to the tungsten-catalyzed reaction, the Mo^VI=O species performs the nucleophilic attack on the formaldehyde carbon, followed by proton transfer in concert with two-electron reduction of the metal center. The first step is rate-limiting, with a total barrier of 28.2 kcal/mol. The higher barrier is mainly due to the large energy penalty for the formation of the Mo^VI=O species.     

Activation of nit-1 nitrate reductase by W-formate dehydrogenase

Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.     

Isolation,Purification, and Characterization of Nitrate Reductase from a Salt-Tolerant <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> Yeast Strain Grown in the Presence of Tungsten

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.     

The extremely thermophilic eubacterium, Thermotoga maritima, contains a novel iron-hydrogenase whose cellular activity is dependent upon tungsten.

Thermotoga maritima is the most thermophilic eubacterium currently known and grows up to 90 degrees C by a fermentative metabolism in which H2, CO2, and organic acids are end products. It was shown that the production of H2 is catalyzed by a single hydrogenase located in the cytoplasm. The addition of tungsten to the growth medium was found to increase both the cellular concentration of the hydrogenase and its in vitro catalytic activity by up to 10-fold, but the purified enzyme did not contain tungsten. It is a homotetramer of Mr 280,000 and contains approximately 20 atoms of Fe and 18 atoms of acid-labile sulfide/monomer. Other transition metals, including nickel (and also selenium), were present in only trace amounts (less than 0.1 atoms/monomer). The hydrogenase was unstable at both 4 and 23 degrees C, even under anaerobic conditions, but no activity was lost in anaerobic buffer containing glycerol and dithiothreitol. Under these conditions the enzyme was also quite thermostable (t50% approximately 1 h at 90 degrees C) but extremely sensitive to irreversible inactivation by O2 (t50% approximately 10 s in air). The optimum pH ranges for H2 evolution and H2 oxidation were 8.6-9.5 and greater than or equal to 10.4, respectively, and the optimum temperature for catalytic activity was above 95 degrees C. In contrast to mesophilic Fe hydrogenases, the T. maritima enzyme had very low H2 evolution activity, did not use T. maritima ferredoxin as an electron donor for H2 evolution, was inhibited by acetylene but not by nitrite, and exhibited EPR signals typical of [2Fe-2S]1+ clusters. Moreover, the oxidized enzyme did not exhibit the rhombic EPR signal that is characteristic of the catalytic iron-sulfur cluster of mesophilic Fe hydrogenases. These data suggest that T. maritima hydrogenase has a different FeS site and/or mechanism for catalyzing H2 production. The potential role of tungsten in regulating the activity of this enzyme is discussed.     

Tungsten-dependent formaldehyde ferredoxin oxidoreductase: reaction mechanism from quantum chemical calculations

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a tungsten-dependent enzyme that catalyzes the oxidation of formaldehyde to formic acid. In the present study, quantum chemical calculations are used to elucidate the reaction mechanism of this enzyme. Several possible mechanistic scenarios are investigated with a large model of the active site designed on the basis of the X-ray crystal structure of the native enzyme. Based on the calculations, we propose a new mechanism in which the formaldehyde substrate binds directly to the tungsten ion. W^VI=O then performs a nucleophilic attack on the formaldehyde carbon to form a tetrahedral intermediate. In the second step, which is calculated to be rate limiting, a proton is transferred to the second-shell Glu308 residue, coupled with a two-electron reduction of the tungsten ion. The calculated barriers for the mechanism are energetically very feasible and in relatively good agreement with experimental rate constants. Three other second-shell mechanisms, including one previously proposed based on experimental findings, are considered but ruled out because of their high barriers.     

Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium

Nitrogenase was isolated and purified from wild-type and a tungsten-resistant mutant (LM2) of Azotobacter vinelandii strain OP derepressed on medium containing 1-10 mM W. While the enzyme from the wild-type strain contained the polypeptides of the conventional enzyme, metal analysis of component 1 demonstrated the existence of one atom each of molybdenum and tungsten. Furthermore, the ESR spectrum of this protein contained three signals, two of which originated from S = 3/2 spin states. One of these signals is nearly identical to that of the conventional MoFe-protein while the other is hypothesized to originate from a W-containing cofactor. In spite of the presence of W, the substrate reduction pattern of this enzyme is the same as that of the conventional enzyme.     

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE(-/-) mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE(-/-) mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE(-/-) mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE(-/-) mice on WD.     

The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway

The anaerobic archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative-type metabolism in which H2, CO2, and organic acids are end products. The growth of this organism is stimulated by tungsten, and, from it, a novel, red-colored, tungsten-iron-sulfur protein, abbreviated RTP, has been purified (Mukund, S., and Adams, M. W. W. (1990) J. Biol. Chem. 265, 11508-11516). RTP (Mr approximately 85,000) contained approximately 1W, 7Fe, and 5 acid-labile sulfide atoms/molecule and exhibited unique EPR properties. The physiological function of the protein, however, was unknown. We show here that RTP is an inactive form of an aldehyde ferredoxin oxidoreductase (AOR). The active enzyme was obtained by rapid purification under anaerobic conditions using buffers containing dithiothreitol and glycerol. AOR catalyzed the oxidation of a range of aliphatic aldehydes with an optimum temperature for activity above 90 degrees C, but it did not oxidize glucose or glyceraldehyde 3-phosphate, nor reduce NAD(P), and its activity was independent of CoA. The active (AOR) and inactive (RTP) forms of the enzyme were indistinguishable in their contents of metals and acid-labile sulfide and in their EPR properties. The latter are though to originate from two nonidentical and spin-coupled iron-sulfur clusters, whereas the tungsten in this enzyme, which was not detectable by EPR, appears to be present as a novel pterin cofactor. Inhibition and activation studies indicated that AOR contains a catalytically essential W-SH group that is not present in RTP, the inactive form. AOR is a new type of aldehyde-oxidizing enzyme and is the first aldehyde oxidoreductase to be purified from an archaebacterium or a nonactogenic anaerobic bacterium. Its physiological role in P. furiosus is proposed as the oxidation of glyceraldehyde to glycerate in a unique, partially nonphosphorylated, glycolytic pathway that generates acetyl-CoA from glucose without the participation of nicotinamide nucleotides.     

Mechanism of growth inhibition by tungsten in Acidithiobacillus ferrooxidans

Cell growth of three hundred iron-oxidizing bacteria isolated from natural environments was inhibited strongly by 0.05 mM, and completely by 0.2 mM of sodium tungstate (Na2WO4), respectively. Since no great difference in the level of tungsten inhibition was observed among the 300 strains tested, the mechanism of inhibition by Na2WO4 was studied with Acidithiobacillus ferrooxidans strain AP19-3. When resting cells of AP19-3 were incubated in 0.1 M beta-alanine-SO4(2-) buffer (pH 3.0) with 0.1 mM Na2WO4 for 1 h, the amount of tungsten bound to the cells was 12 microg/mg protein. The optimum pH for tungsten binding to the resting cells was 2 to approximately 3. Approximately 2 times more tungsten bound to the cells at pH 3.0 than at pH 6.0. The tungsten binding was specifically inhibited by sodium molybdenum. However, copper, nickel, cadmium, zinc, manganese, cobalt, and vanadate did not disturb tungsten binding to the resting cells. The iron-oxidizing activity of AP19-3 was inhibited 24, 62, and 77% by 1, 5, and 10 mM of Na2WO4, respectively. Among the components of iron oxidation enzyme system, iron:cytochrome c oxidoreductase activity was not inhibited by 10 mM of Na2WO4. In contrast, the activity of cytochrome c oxidase purified highly from the strain was inhibited 50 and 72%, respectively, by 0.05 and 0.1 mM of Na2WO4. The amounts of tungsten bound to plasma membrane, cytosol fraction, and a purified cytochrome c oxidase were 8, 0.5, and 191 microg/mg protein, respectively. From the results, the growth inhibition by Na2WO4 observed in A. ferrooxidans is explained as follows: tungsten binds to cytochrome c oxidase in plasma membranes and inhibits cytochrome c oxidase activity, and as a results, the generation of energy needed for cell growth from the oxidation of Fe2+ is stopped.     

Novel bacterial molybdenum and tungsten enzymes: three-dimensional structure, spectroscopy, and reaction mechanism

The molybdenum enzymes 4-hydroxybenzoyl-CoA reductase and pyrogallol-phloroglucinol transhydroxylase and the tungsten enzyme acetylene hydratase catalyze reductive dehydroxylation reactions, i.e., transhydroxylation between phenolic residues and the addition of water to a triple bond. Such activities are unusual for this class of enzymes, which carry either a mononuclear Mo or W center. Crystallization and subsequent structural analysis by high-resolution X-ray crystallography has helped to resolve the reaction centers of these enzymes to a degree that allows us to understand the interaction of the enzyme and the respective substrate(s) in detail, and to develop a concept for the respective reaction mechanism, at least in two cases.     

185W-labelled nitrate reductase from Chlorella

By adding ¹⁸⁵W-tungstate to a Chlorella culture, it has beenpossible to incorporate this metal into the nitrate reductasecomplex. The W-labelled enzyme was completely inactive as nitratereductase, but maintained unaffected its diaphorase activity.In vivo incorporation of tungsten into the enzyme was competitivelyhindered by molybdenum. ¹ This work was supported by a grant from the Instituto de EstudiosNucleares, J.E.N., Spain. (Received July 6, 1971; )     

Characterization of molybdenum cofactor from Escherichia coli.

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.     

Effect of cyanophage N-1 infection on the synthesis and stability ofNostoc muscorum nitrate reductase

The control operative on the nitrate reductase enzyme system of host cyanobacteriumNostoc muscorum was studied after being infected with the cyanophage N-1. Phage infection lifted the host nitrate reductase activity level via accelerating the enzyme synthesis. It was found that the phage-mediated increase in the molybdenum cofactor synthesis was a major contributing factor for apparent elevated nitrate reductase level of the host. This process was inhibited in the presence of erythromycin and tungsten, the inhibitors of protein synthesis and new nitrate reductase synthesis respectively. While the preformed nitrate reductase of healthy cyanobacterium was inhibited by hydrogen peroxide, an oxidizing photosynthetic product, the same enzyme of infected cells remained virtually insensitive to this inhibitor. These data suggest involvement of new nitrate reductase synthesis and its resistance to oxidative inactivation as joint factors controlling the characteristic high enzyme level of host cyanobacterium.     

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.     

“CLASPing” tungsten's effects on microtubules with “PINs”

Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed.     

Electron paramagnetic resonance of the tungsten derivative of rat liver sulfite oxidase.

Sulfite oxidase purified from livers of tungsten-treated rats has been used for EPR studies of tungsten substituted at the molybdenum site of the enzyme in a fraction of the molecules. The EPR signal of W(V) in sulfite oxidase is quite similar to that of Mo(V) in its line shape and in its sensitivity to the presence of anions such as phosphate and fluoride. Hyperfine interaction with a dissociable proton is also observed in both signals. The pH-dependent alteration in line shape exhibited by the Mo(V) EPR signal of the rat liver enzyme. Incomplete reduction of the tungsten center at pH 9 is indicated by attenuated signal intensity at this pH. The W(V) signal has g values lower than those of the Mo(V) signal, has a much broader resonance envelope, and is much less readily saturated by increasing microwave power. Kinetic studies on the reduction of the heme and tungsten centers of sulfite oxidase have shown that reduction of de-molybdo forms of sulfite oxidase by sulfite is catalyzed by the residual traces of native molybdenum-containing molecules. Reduction is accomplished by electron transfer involving intermolecular heme-heme interaction. The W(V) signal is generated only after all the heme centers are reduced. The rate and extent of heme reduction at pH 9 are the same as at pH 7. Studies on the reoxidation of W(V) and reduced heme by O2 and by cytochrome c suggest that the cytochrome b5 of sulfite oxidase is the site of electron transfer to cytochrome c, whereas oxidase activity is the property of the molybdenum center. It appears that the tungsten center in sulfite oxidase is incapable of oxidizing sulfite.