Nicotinic acid hydroxylase from Clostridium barkeri: electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme.
Nicotinic acid hydroxylase from Clostridium barkeri contains selenium in an unidentified form that is dissociated as a low molecular weight compound upon denaturation of the enzyme. Other cofactors of this enzyme are molybdopterin, FAD, and iron-sulfur clusters. In the current study, we show that the enzyme, as isolated, exhibits a stable Mo(V) electron paramagnetic resonance (EPR) signal ("resting" signal) and that this signal is correlated with the selenium content and nicotinate hydroxylase activity of the enzyme. Substitution of 77Se for normal selenium isotope abundance results in splitting of the Mo(V) EPR signal of the native protein without affecting the iron signals of the FeS clusters. The Mo(V) EPR signal and nicotinic acid hydroxylase activity of enzyme isolated from cells grown in selenium-deficient medium are barely detectable. In contrast, the EPR signals of the FeS clusters, the electronic absorption spectrum, the NADPH oxidase activity, and the chromatographic behavior are changed little and are typical of active selenium-containing enzyme. An EPR signal indicative of the presence of molybdenum in the selenium-deficient enzyme also is exhibited. From these results, we conclude that a dissociable selenium moiety is coordinated directly with molybdenum in the molybdopterin cofactor and, moreover, this selenium is essential for nicotinic acid hydroxylase activity.
Gladyshev
V N
VN
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Khangulov
S V
SV
Stadtman
T C
TC
D001426:Bacterial Proteins / Q000737:chemistry; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006358:Hot Temperature; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008970:Molecular Weight; D008982:Molybdenum / Q000737:chemistry*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D012643:Selenium / Q000737:chemistry*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008982:Molybdenum; C021862:nicotinate dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012643:Selenium