The improved purification scheme for nicotinic acid hydroxylase from Clostridium barkeri was developed and physico-chemical properties of the enzyme were studied. The enzyme contains 1 Mo, 1 FAD, 6-7 Fe and up to 1 dissociable Se that is coordinated to Mo. These cofactors in Se-containing and Se-deficient isozymes of nicotinic acid hydroxylase were characterized in detail. Formate dehydrogenase H from Escherichia coli contains a selenocysteine amino acid residue, and has the Mo-Se bond essential for catalysis. The enzyme was stabilized in a reduced state. Active formate treated enzyme was crystallized under strictly anaerobic conditions and preliminary crystallographic analysis was performed at 2.6 A. Mechanistic and structural methods including EPR, ENDOR, and EXAFS were applied to study NAH and FDH. The minimal structures and geometry of Mo-Se active centers of these enzymes were obtained. It was found that infection of T-cells with HIV affects cellular selenium metabolism.
