Although significant knowledge has been gained recently on the incorporation of selenium into selenoproteins, most of the emphasis of this work has been placed on enzymes that carry selenium in the form of selenocysteine. A handful of enzymes have been characterized in which selenium is present and required for activity but is not inserted during translation of the mRNA and therefore is not present as selenocysteine. Two such enzymes are nicotinic acid hydroxylase (NAH) and xanthine dehydrogenase (XD) from Clostridium. Selenium present in NAH is somewhat labile and can be removed by reducing agents, such as dithiothreitol, or by treatment with chaotropic agents, such as sodium dodecyl sulfate. In order to better understand the nature of this incorporation of selenium, we intend to isolate and characterize xanthine dehydrogenase (XD) from Clostridium purinolyticum and use this enzyme to help characterize the components necessary to incorporate selenium into the apoenzyme. Clostridium purinolyticum is known to possess a constitutive XD activity that increases when selenite is supplemented to the growth medium. Once isolated, this enzyme will be characterized with respect to subunit composition, cofactor analysis (molybdenum, Fe-S clusters, FAD), substrate specificity, enzyme kinetic analysis, and, most importantly, identification of the selenium moiety present in the active enzyme. Purifying the enzyme from cells grown in the presence of radiolabeled 75-Se in the form of selenite will allow efficient analysis of the form of selenium. Once conditions can be established for efficient removal of the selenium from the enzymatic preparation, most likely by treatment with reducing agent(s), the apoenzyme XD can then be used as a type of assay to determine the necessary components required for incorporation of selenium. This work may uncover a new delivery protein(s) or selenium donor molecule required for XD and other molybdenum hydroxylases and perhaps shed light on the delivery of selenium in the biosynthesis of selenocysteine. In addition, a novel protein, which also contained labile selenium, was uncovered during isolation of selenophosphate synthetase from the methanogen Methanococcus vannielii. The N-terminal amino acid sequence of this protein was determined and had little similarity to any protein in the Genbank database. We speculate that this protein may represent a subunit of an enzyme similar to XD in which selenium is labile and not present as selenocysteine. Another possibility exists that this protein is a selenium binding/delivery protein that binds to a selenium compound during the process of biosynthesis of selenocysteine or in the incorporation of selenium into non-selenocysteine selenoproteins, such as XD. Further isolation and study of this protein should reveal new information on the processing of selenium within the cell. - selenium, xanthine dehydrogenase, Clostridium, molybdenum hydroxylase