CHARACTERIZATION OF CRYSTALLINE FORMATE DEHYDROGENASE-H FROM ESCHERICHIA-COLI - STABILIZATION, EPR SPECTROSCOPY, AND PRELIMINARY CRYSTALLOGRAPHIC ANALYSIS

The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-k Da component of the Escherichia coli formate-hydrogen lyase complex. T he molybdenum-coordinated selenocysteine is essential for catalytic ac tivity of the native enzyme, FDH in dilute solutions (30 mu g/ml) was rapidly inactivated at basic pH or in the presence of formate under an aerobic conditions, but at higher enzyme concentrations (greater than or equal to 3 mg/ml) the enzyme was relatively stable, The formate-red uced enzyme was extremely sensitive to air inactivation under all cond itions examined. Active formate-reduced FDH was crystallized under ana erobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 Angstrom resolution and belong to a space gr oup of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146. 1 Angstrom and c = 82.7 Angstrom. There is one monomer of FDH per crys tallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-red uced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a s ingle reduced FeS cluster was found in a crystal of reduced FDH, but n ot in a crystal of oxidized enzyme, whereas Mo(V) signal was not detec ted in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster for m upon oxidation. A procedure that combines anaerobic and cryocrystall ography has been developed that is generally applicable to crystallogr aphic studies of oxygen-sensitive enzymes. These data provide the firs t example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.