PROPERTIES OF THE SELENIUM-CONTAINING AND MOLYBDENUM-CONTAINING NICOTINIC-ACID HYDROXYLASE FROM CLOSTRIDIUM-BARKERI

NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of hi gher specific activity then previously reported. NAH is most stable at alkaline pH in the presence of glycerol. The protein which consists o f four dissimilar subunits occurs in forms of different molecular mass es. There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein protomer. Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium. Mo(V), flavin radical, and two Fe2S2 clust ers could be observed with EPR spectroscopy. The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released fr om NAH as a reactive low molecular weight compound by a number of dena turing procedures. Parallel losses of Se and catalytic activity were o bserved during purification and storage of the enzyme. Addition of sod ium selenide or selenophosphate did not restore the catalytic activity of the enzyme. Instead, NAH is reversibly inactivated by these compou nds and also by sulfide. Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity. The ''as isolate d'' enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detecte d at early stages of purification. NAH exhibits a high substrate speci ficity toward electron donor substrates. The ability of a nicotinate a nalog to reduce NAH (disappearance of 2.067 signal) correlates with th e rate of oxidation of the analog in the standard assay mixture. The p roperties of NAH differentiate the enzyme from known Mo-containing hyd roxylases.