The selenoeysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenumcoordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 μg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (≥3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 40O. The crystals diffract to 2.6 Åresolution and belong to a space group of P41212 or P43212 with unit cell dimensions a= b = 146.1 Å and c = 82.7 Å. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS clustercontaining form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.
CITATION STYLE
Gladyshev, V. N., Boyington, J. C., Khangulov, S. V., Grahame, D. A., Stadtman, T. C., & Sun, P. D. (1996). Characterization of Crystalline Formate Dehydrogenase H from Escherichia coli. Journal of Biological Chemistry, 271(14), 8095–8100. https://doi.org/10.1074/jbc.271.14.8095
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