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  - 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  -