Electron paramagnetic resonance studies of the tungsten-containing formate dehydrogenase from (original) (raw)
Related papers
Biochemical and Biophysical Research Communications, 1987
The redox centers in the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum were examined by potentiometric titration and electron paramagnetic resonance spectroscopy. At low temperature two overlapping iron-sulfur signals which correlated with enzymatic activity were observed with formal potentials near-400 mV vs. SHE. Based on their temperature dependences, one signal is assigned to a reduced Fe2S2 cluster and one to a reduced Fe4S4 cluster. Quantitation of signal intensity suggests two Fe2S2 and two Fe4S4 clusters per formate dehydrogenase molecule. Another signal (g= 2.101, 1.980, 1.950) present in low concentrations at more negative potentials was observable up to 200' K and is not attributed to any iron-sulfur cluster. The possible orgin of this signal is analyzed using ligand field theory, and the redox behavior is considered with respect to possible ligation at the active site. 0 1987 Academic Press, 1°C. Formate dehydrogenases (FDH) catalyze the reversible interconversion of formate and CO2 in numerous plants and bacteria (for a review see 1). Most are molybdenum enzymes, but a few require tungsten for activity and constitute the only known occurrence of a third transition series element in metalloenzymes. For each molybdenum-containing FDH (MO-FDH) examined by either an assay involving nit-l Neurosnora crassa mutants(2) or the fluorescence of oxidatively degraded protein (3) evidence has been found for a non-nitrogenase type molybdenum-containing cofactor, called "MO-CO", in which a pterin is thought to coordinate to the MO. In the presence of MoO42-, acidified samples of the tungsten-containing FDH (W-FDH) from Clostridium thermoaceticum also activated the u nitrate reductase indicating the presence of a tungsten analogue of the MO-CO(~), which is consistent with the fluorescence results of Yamamoto et al. (5).
Journal of Biological Inorganic Chemistry, 2004
We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126±2 kDa composed of two subunits, a=93±3 kDa and b=32±2 kDa, which contains 6±1 Fe/molecule, 0.4±0.1 Mo/molecule, 0.3±0.1 W/molecule, and 1.3±0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d 1 configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme. Keywords Electron paramagnetic resonance AE Formate dehydrogenase AE Magnetic interactions AE Molybdenum-containing enzymes AE Tungsten-containing enzymes Abbreviations AOR aldehyde oxidoreductase AE FDH formate dehydrogenase AE NAP periplasmic nitrate reductase AE SRB sulfate-reducing bacteria
Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus
Journal of Bacteriology, 2010
The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope 99 Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten-and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.
Journal of The American Chemical Society, 1996
The complex [Et 4 N] 2 [W VI O 2 (mnt) 2 ] (1), [Et 4 N] 2 [W IV O(mnt) 2 ] (2), and [Et 4 N] 2 [W VI O(S 2 )(mnt) 2 ] (3) (mnt 2-) 1,2-dicyanoethylenedithiolate) have been synthesized as possible models for the tungsten cofactor of inactive red tungsten protein (RTP) and the active aldehyde ferredoxin oxidoreductase (AOR) of the hyperthermophilic archaeon Pyrococcus furiosus. The [Ph 4 P] + salt of the complex anion of 1‚2H 2 O crystallizes in space group Pbcn, with a ) 20.526(3) Å, b ) 15.791(3) Å, c ) 17.641(3) Å, and Z ) 4. The W VI O 2 S 4 core of [Ph 4 P] 2 [W VI O 2 (mnt) 2 ]‚2H 2 O has distorted octahedral geometry with cis dioxo groups. 2 crystallizes in space group P2 1 2 1 2, with a ) 14.78(3) Å, b
Biological Chemistry, 2000
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.
Biochemistry, 1999
An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism DesulfoVibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [R (92 kDa) and (29 kDa) subunits] and contains 7 ( 1 Fe/protein and 0.9 ( 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 ( 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with 57 Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d 1 configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions. † This work was supported by Grants PRAXIS 2/2.2/QUI/3/94 (I.M.), PRAXIS 2/2.
Archives of Biochemistry and Biophysics, 2002
Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b 557-and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 lmol NO À 3 consumed/min/nmol enzyme and with a K NO À 3 m app of 8 lM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000 (MobA À strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 lmol NO À 3 consumed/min/nmol enzyme and with a K NO À 3 m app of 12 lM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin-and heme-containing domains. Ó 2002 Elsevier Science (USA). All rights reserved. NAD(P)H:nitrate reductase (NR 1 ; EC 1.6.6.1-2) catalyzes the rate-limiting and regulated step, the twoelectron reduction of NO À 3 to NO À 2 , in the pathway of inorganic nitrogen assimilation [1-3]. The enzyme has been isolated from a variety of sources including algae [4], fungi [5], yeast [6], and higher plants [7] and in all
Structure, 2002
Gene Sequence and the 1.8 Å Crystal Structure of the Tungsten-Containing Formate Dehydrogenase from Desulfovibrio gigas dehydrogenase isolated from D. desulfuricans ATCC 27774 is homologous to DvMo-FDH and was characterized by EPR and Mö ssbauer spectroscopies [4]. A monoheme cytochrome, c 553 , was identified as the physiological partner for DvMo-FDH [3, 5]. The physiological donor of DgW-FDH is also a monoheme cytochrome that was recently purified (our unpublished data). Departamento de Química FCT DgW-FDH consists of two subunits of 977 and 214 amino acids, and it belongs to the DMSO reductase Universidade Nova de Lisboa 2829-516 Caparica (DMSOR) family of enzymes, one of the four classes into which molybdopterin-containing enzymes have been Portugal 2 Max-Planck-Institut fü r Biochemie classified [6-9]. This family is considerably broad, and, besides DMSOR [10, 11], it includes enzymes such as Am Klopferspitz 18a D-82152 Martinsried dissimilatory nitrate reductases (NAP) [12] and formate dehydrogenases. The members of this family have two Germany molybdopterins (MGD cofactor) in the coordination sphere of Mo (or W), which is also bound to an amino acid side chain. This amino acid changes with the enzy-Summary matic functionality and is a serine residue in DMSOR, a cysteine in NAP, and a selenocysteine in FDHs [2, 13].