TY - JOUR A1 - Reschke, Stefan A1 - Duffus, Benjamin R. A1 - Schrapers, Peer A1 - Mebs, Stefan A1 - Teutloff, Christian A1 - Dau, Holger A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli JF - Biochemistry N2 - The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors. Y1 - 2019 U6 - https://doi.org/10.1021/acs.biochem.9b00078 SN - 0006-2960 VL - 58 IS - 17 SP - 2228 EP - 2242 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Duffus, Benjamin R. A1 - Schrapers, Peer A1 - Schuth, Nils A1 - Mebs, Stefan A1 - Dau, Holger A1 - Leimkühler, Silke A1 - Haumann, Michael T1 - Anion binding and oxidative modification at the molybdenum cofactor of formate dehydrogenase from Rhodobacter capsulatus studied by X-ray absorption spectroscopy JF - Inorganic chemistry N2 - Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O-2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo=S) and oxo (Mo=O) bonds, were observed in the presence of azide (N-3(-)) or cyanate (OCN-). Azide provided best protection against O-2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes. Y1 - 2020 U6 - https://doi.org/10.1021/acs.inorgchem.9b01613 SN - 0020-1669 SN - 1520-510X VL - 59 IS - 1 SP - 214 EP - 225 PB - American Chemical Society CY - Washington, DC ER - TY - JOUR A1 - Reschke, Stefan A1 - Sigfridsson, Kajsa G. V. A1 - Kaufmann, Paul A1 - Leidel, Nils A1 - Horn, Sebastian A1 - Gast, Klaus A1 - Schulzke, Carola A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli JF - The journal of biological chemistry N2 - The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme. Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497453 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 41 SP - 29736 EP - 29745 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Reschke, Stefan A1 - Niks, Dimitri A1 - Wilson, Heather A1 - Sigfridsson, Kajsa G. V. A1 - Haumann, Michael A1 - Rajagopalan, K. V. A1 - Hine, Russ A1 - Leimkühler, Silke T1 - Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase JF - Biochemistry N2 - Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k(cat)/K-M) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt) X-ray absorption spectroscopy revealed a Mow Se bond length of 2.51 A, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)(O2MoS2)-S-VI(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOM4Ser was altered compared to those of SOMD4Ser and SOMD4cy,(,), with g, in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo Se bond. Y1 - 2013 U6 - https://doi.org/10.1021/bi4008512 SN - 0006-2960 VL - 52 IS - 46 SP - 8295 EP - 8303 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Schrapers, Peer A1 - Hartmann, Tobias A1 - Kositzki, Ramona A1 - Dau, Holger A1 - Reschke, Stefan A1 - Schulzke, Carola A1 - Leimkühler, Silke A1 - Haumann, Michael T1 - 'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus JF - Inorganic chemistry N2 - Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH. Y1 - 2015 U6 - https://doi.org/10.1021/ic502880y SN - 0020-1669 SN - 1520-510X VL - 54 IS - 7 SP - 3260 EP - 3271 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Samuel, Prinson P. A1 - Horn, Sebastian A1 - Döring, Alexander A1 - Havelius, Kajsa G. V. A1 - Reschke, Stefan A1 - Leimkühler, Silke A1 - Haumann, Michael A1 - Schulzke, Carola T1 - A Crystallographic and Mo K-Edge XAS Study of Molybdenum Oxo Bis-,Mono-, and Non-Dithiolene Complexes - First-Sphere Coordination Geometry and Noninnocence of Ligands JF - European journal of inorganic chemistry : a journal of ChemPubSoc Europe N2 - Ten square-based pyramidal molybdenum complexes with different sulfur donor ligands, that is, a variety of dithiolenes and sulfides, were prepared, which mimic coordination motifs of the molybdenum cofactors of molybdenum-dependent oxidoreductases. The model compounds were investigated by Mo K-edge X-ray absorption spectroscopy (XAS) and (with one exception) their molecular structures were analyzed by X-ray diffraction to derive detailed information on bond lengths and geometries of the first coordination shell of molybdenum. Only small variations in Mo=O and Mo-S bond lengths and their respective coordination angles were observed for all complexes including those containing Mo(CO)(2) or Mo(mu-S)(2)Mo motifs. XAS analysis (edge energy) revealed higher relative oxidation levels in the molybdenum ion in compounds with innocent sulfur-based ligands relative to those in dithiolene complexes, which are known to exhibit noninnocence, that is, donation of substantial electron density from ligand to metal. In addition, longer average Mo-S and Mo=O bonds and consequently lower.(Mo=O) stretching frequencies in the IR spectra were observed for complexes with dithiolene-derived ligands. The results emphasize that the noninnocent character of the dithiolene ligand influences the electronic structure of the model compounds, but does not significantly affect their metal coordination geometry, which is largely determined by the Mo(IV) or (V) ion itself. The latter conclusion also holds for the molybdenum site geometries in the oxidized Mo-VI cofactor of DMSO reductase and the reduced Mo-IV cofactor of arsenite oxidase. The innocent behavior of the dithiolene molybdopterin ligands observed in the enzymes is likely to be related to cofactor-protein interactions. KW - Molybdenum KW - Enzyme models KW - X-ray absorption spectroscopy KW - Noninnocence KW - Bioinorganic chemistry Y1 - 2011 U6 - https://doi.org/10.1002/ejic.201100331 SN - 1434-1948 IS - 28 SP - 4387 EP - 4399 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Hartmann, Tobias A1 - Schrapers, Peer A1 - Utesch, Tillmann A1 - Nimtz, Manfred A1 - Rippers, Yvonne A1 - Dau, Holger A1 - Mroginski, Maria Andrea A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions JF - Biochemistry N2 - Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pK(a) of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. Y1 - 2016 U6 - https://doi.org/10.1021/acs.biochem.6b00002 SN - 0006-2960 VL - 55 SP - 2381 EP - 2389 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Correia, Marcia A. S. A1 - Otrelo-Cardoso, Ana Rita A1 - Schwuchow, Viola A1 - Clauss, Kajsa G. V. Sigfridsson A1 - Haumann, Michael A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Santos-Silva, Teresa T1 - The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes JF - ACS chemical biology N2 - The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined. Y1 - 2016 U6 - https://doi.org/10.1021/acschembio.6b00572 SN - 1554-8929 SN - 1554-8937 VL - 11 SP - 2923 EP - 2935 PB - American Chemical Society CY - Washington ER -