title: Characterization of chloride-depleted human sulfite oxidase by electron paramagnetic resonance spectroscopy: experimental evidence for the role of anions in product release.
abstract: The Mo(V) state of the molybdoenzyme sulfite oxidase (SO) is paramagnetic and can be studied by electron paramagnetic resonance (EPR) spectroscopy. Vertebrate SO at pH <7 and >9 exhibits characteristic EPR spectra that correspond to two structurally different forms of the Mo(V) active center termed the low-pH (lpH) and high-pH (hpH) forms, respectively. Both EPR forms have an exchangeable equatorial OH ligand, but its orientation in the two forms is different. It has been hypothesized that the formation of the lpH species is dependent on the presence of chloride. In this work, we have prepared and purified samples of the wild type and various mutants of human SO that are depleted of chloride. These samples do not exhibit the typical lpH EPR spectrum at low pH but rather exhibit spectra that are characteristic of the blocked species that contains an exchangeable equatorial sulfate ligand. Addition of chloride to these samples results in the disappearance of the blocked species and the formation of the lpH species. Similarly, if chloride is added before sulfite, the lpH species is formed instead of the blocked one. Qualitatively similar results were observed for samples of sulfite-oxidizing enzymes from other organisms that were previously reported to form a blocked species at low pH. However, the depletion of chloride has no effect upon the formation of the hpH species.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: Asha
authors.initials: A
authors.lastname: Rajapakshe
authors.forename: Kayunta
authors.initials: K
authors.lastname: Johnson-Winters
authors.forename: Anna R
authors.initials: AR
authors.lastname: Nordstrom
authors.forename: Kimberly T
authors.initials: KT
authors.lastname: Meyers
authors.forename: Safia
authors.initials: S
authors.lastname: Emesh
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Astashkin
authors.forename: John H
authors.initials: JH
authors.lastname: Enemark
mesh_terms: D000838:Anions; D002712:Chlorides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006801:Humans; D050876:Sulfite Oxidase / Q000737:chemistry*
chemical_list: D000838:Anions; D002712:Chlorides; D050876:Sulfite Oxidase