TY - JOUR A1 - Hall, James A1 - Reschke, Stefan A1 - Cao, Hongnan A1 - Leimkühler, Silke A1 - Hille, Russ T1 - The reductive half-reaction of xanthine dehydrogenase from rhodobacter capsulatus the role of GLU(232) in catalysis JF - The journal of biological chemistry N2 - Background: Kinetic characterization of wild-type xanthine dehydrogenase and variants. Results: Comparison of the pH dependence of both k(red) and k(red)/K-d, as well as k(cat) and k(cat)/K-m. Conclusion: Ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine. Significance: Examining the contributions of Glu(232) to catalysis is essential for understanding the mechanism of xanthine dehydrogenase. The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k(red), the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k(red) and k(red)/K-d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK(a) of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. KW - Enzyme Kinetics KW - Glutamate KW - Glutamine KW - Isotope Effect KW - Ultraviolet-visible Spectroscopy (UV-visible Spectroscopy) KW - Xanthine KW - Xanthine Dehydrogenase KW - Xanthine Oxidase KW - pH Dependence Y1 - 2014 U6 - https://doi.org/10.1074/jbc.M114.603456 SN - 0021-9258 SN - 1083-351X VL - 289 IS - 46 SP - 32121 EP - 32130 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda 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 -