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  - Havelius, Kajsa G. V.
A1  - Reschke, Stefan
A1  - Horn, Sebastian
A1  - Doerlng, Alexander
A1  - Niks, Dimitri
A1  - Hille, Russ
A1  - Schulzke, Carola
A1  - Leimkühler, Silke
A1  - Haumann, Michael
T1  - Structure of the molybdenum site in YedY, a sulfite oxidase homologue from escherichia coli
JF  - Inorganic chemistry
N2  - YedY from Escherichia coil is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of similar to 2.4 angstrom), and one (axial) oxo ligand (Mo=O, similar to 1.7 angstrom). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (similar to 2.1 angstrom) or possibly to a shorter M-O(-) bond. Yet another oxygen (or nitrogen) at a similar to 2.6 angstrom distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a similar to 2.6 angstrom distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism.
Y1  - 2011
U6  - https://doi.org/10.1021/ic101291j
SN  - 0020-1669
VL  - 50
IS  - 3
SP  - 741
EP  - 748
PB  - American Chemical Society
CY  - Washington
ER  -