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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1209418810
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu12094188102021-08-03T05:53:34Z Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough) Wolfe, Bonnie Michelle <p>Desulfoviridin (DV), a multimeric dissimilatory sulfite reductase (M<sub>r</sub> ~ 226 kDa) from <i>Desulfovibrio vulgaris</i> (Hildenborough), catalyzes the 6-electron reduction of sulfite to sulfide. <i>Desulfovibrio vulgaris</i> (Hildenborough) is a sulfate-reducing bacterium. We have shown that the enzyme contains two [Fe<sub>4</sub>S<sub>4</sub>] clusters and high-spin pentacoordinate sirohemes. This stands in contrast to the monomeric assimilatory sulfite reductase (SiR) from the same bacterium (M<sub>r</sub> ~24 kDa), which contains a [Fe<sub>4</sub>S<sub>4</sub>] cluster coupled to a hexacoordinate low-spin siroheme.</p><p>The assimilatory SiR reduces sulfite to sulfide completely, but the reduction ofsulfite by DV is accompanied by the production of side products [thiosulfate (S<sub>2</sub>O<sub>3</sub><sup>2-</sup>)and trithionate (S<sub>3</sub>O<sub>6</sub><sup>2-</sup>)] <i>in vitro</i>.</p><p>Conditions for the rigorous purification of desulfoviridin have been established. Afinal purification by fast protein liquid chromatography yields at least three distinct bands that each exhibit the characteristic absorption spectrum of desulfoviridin. Two of these are extensively characterized by amino acid analysis, isoelectric focusing, polyacrylamide gel electrophoresis, and formulation of the prosthetic centers. Each contains two pairs of [Fe<sub>4</sub>S<sub>4</sub>] and siroheme units. These results stand in marked contrast to work claiming significant demetallation of siroheme, excess iron content, and the presence of Fe<sub>6</sub>S<sub>6</sub> clusters. These proposals are critically assessed in light of our results and other published work. Steady state kinetic parameters have been determined: k<sub>cat</sub> (SO<sub>3</sub><sup>2-</sup>) = 0.31 mol SO<sub>3</sub><sup>2-</sup> s<sup>-1</sup>mole heme<sup>-1</sup>, K<sub>m</sub> = 0.06 mM; k<sub>cat</sub> (NO<sub>2</sub><sup>-</sup>) = 0.038 mol NO<sub>2</sub><sup>-</sup> s<sup>-1</sup>mole heme<sup>-1</sup>, K<sub>m</sub> = 0.028 mM; k<sub>cat</sub> (NH<sub>2</sub>OH) = 29 mol NH<sub>2</sub>OH s<sup>-1</sup> mole heme<sup>-1</sup>, K<sub>m</sub> = 48 mM. These are compared to similar enzymes and implications for the mechanism are discussed. Comparisons of the thermal stability of the two enzymes are made and preliminary thermodynamic parameters are determined based upon the steady-state kinetic values.</p> 1999 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1209418810 http://rave.ohiolink.edu/etdc/view?acc_num=osu1209418810 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
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NDLTD
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English
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NDLTD
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| author |
Wolfe, Bonnie Michelle
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Wolfe, Bonnie Michelle
Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough)
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| author_facet |
Wolfe, Bonnie Michelle
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| author_sort |
Wolfe, Bonnie Michelle
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| title |
Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough)
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| title_short |
Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough)
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| title_full |
Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough)
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| title_fullStr |
Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough)
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| title_full_unstemmed |
Purification, characterization and kinetic studies of Desulfoviridin : a dissimilatory sufite reductase from Desulfovibrio vulgaris (Hildenborough)
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| title_sort |
purification, characterization and kinetic studies of desulfoviridin : a dissimilatory sufite reductase from desulfovibrio vulgaris (hildenborough)
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| publisher |
The Ohio State University / OhioLINK
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| publishDate |
1999
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http://rave.ohiolink.edu/etdc/view?acc_num=osu1209418810
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| work_keys_str_mv |
AT wolfebonniemichelle purificationcharacterizationandkineticstudiesofdesulfoviridinadissimilatorysufitereductasefromdesulfovibriovulgarishildenborough
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1719427272082980864
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