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-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/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 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. 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 cluster-containing 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.
Gladyshev
V N
VN
Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Boyington
J C
JC
Khangulov
S V
SV
Grahame
D A
DA
Stadtman
T C
TC
Sun
P D
PD
D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000648:ultrastructure; D005615:Freezing; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000737:chemistry / Q000648:ultrastructure; D008982:Molybdenum; D010084:Oxidation-Reduction; D012643:Selenium; D013057:Spectrum Analysis
D008667:Metalloproteins; D008982:Molybdenum; D005560:Formate Dehydrogenases; D012643:Selenium