TY  - JOUR
A1  - Reschke, Stefan
A1  - Mebs, Stefan
A1  - Sigfridsson-Clauss, Kajsa G. V.
A1  - Kositzki, Ramona
A1  - Leimkühler, Silke
A1  - Haumann, Michael
T1  - Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy
JF  - Inorganic chemistry
N2  - Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo (VI) ion with each one MPT, Mo=O, Mo-O-, and Mo=S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK similar to 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK similar to 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A. variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.
Y1  - 2017
U6  - https://doi.org/10.1021/acs.inorgchem.6b02846
SN  - 0020-1669
SN  - 1520-510X
VL  - 56
IS  - 4
SP  - 2165
EP  - 2176
PB  - American Chemical Society
CY  - Washington
ER  - 
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  -