title: The state of reduction of molybdopterin in xanthine oxidase and sulfite oxidase.
abstract: Methods have been devised to examine the spectral properties and state of reduction of the pterin ring of molybdopterin (MPT) in milk xanthine oxidase and the Mo-containing domain of rat liver sulfite oxidase. The absorption spectrum of the native pterin was visualized by difference spectroscopy of each protein, denatured anaerobically in 6 M guanidine hydrochloride (GdnHCl), versus a sample containing the respective apoprotein and other necessary components. The state of reduction of MPT was also probed using 2,6-dichlorobenzenoneindophenol (DCIP) to measure reducing equivalents/MPT, after anaerobic denaturation of the protein in GdnHCl in the presence or absence of Hg2+. In the case of xanthine oxidase the data indicate that the terminal sulfide ligand of Mo causes the reduction of a native dihydro form of MPT to the tetrahydro level. This reduction does not occur if Hg2+ is added prior to denaturation of the protein. Based on its observed behavior, the native MPT in the Mo cofactor of xanthine oxidase is postulated to exist as a quinonoid dihydropterin. Quantitation of DCIP reduction by MPT of Mo fragment of sulfite oxidase showed a two-electron oxidation of MPT, even when the Mo fragment was denatured in the presence of Hg2+ to prevent internal reduction reactions due to sulfhydryls or sulfide. Difference spectra of DCIP-treated versus untreated Mo fragment showed that MPT had been fully oxidized. These data indicate that the native MPT in sulfite oxidase must be a dihydro isomer different from that in xanthine oxidase.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: S
authors.initials: S
authors.lastname: Gardlik
authors.forename: K V
authors.initials: KV
authors.lastname: Rajagopalan
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D003067:Coenzymes; D005260:Female; D008099:Liver / Q000201:enzymology; D008667:Metalloproteins / Q000378:metabolism*; D008892:Milk / Q000201:enzymology; D015394:Molecular Structure; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism*; D011621:Pteridines / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry; D014969:Xanthine Oxidase / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011621:Pteridines; D014969:Xanthine Oxidase; C022261:molybdopterin