Coordination of selenium to molybdenum in formate dehydrogenase H from \u3ci\u3eEscherichia coli\u3c/i\u3e

Formate dehydrogenase H from Escherichia col contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center, and a selenocysteine residue (SeCys-140 in the polypeptide chain) that is essential for catalytic activity. Here we show that addition of formate to the native enzyme induces a signal typical of Mo(V) species. This signal is detected by electron pm etc resonance (EPR) spectroscopy. Substitution of 77Se for natural isotope abundance Se leads to transformation of this signal, indicating a direct coordination of Se with Mo. Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and exhibits a different EPR signal. Since deternation of the Se content of wild-type enzyme indicates 1 gram atom per mol, we conclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo. The amino acd sequence flanking the selenocysteine residue in formate dehydrogenase H is s r to a conserved sequence found in several other prokaryotic molybdopterin-dependent enzymes. In most of these other enzymes a cysteine residue, or in a few cases a serine or a selenocysteine residue, occurs in the position corresonding to SeCys-140 of formate dehydrogeme H. By analogy with formate dehydrogenase H in these other enzymes, at least one of the ilgands to Mo should be provided by an amino acid residue of the protein. This igand could be the Se of a selenocysteine residue, sulfur of a cysteine residue, or, in the case of a serine residue, oxygen

Crystal Structure of Formate Dehydrogenase H: Catalysis Involving Mo, Molybdopterin, Selenocysteine, and an Fe\u3csub\u3e4\u3c/sub\u3eS\u3csub\u3e4\u3c/sub\u3e Cluster

Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo( VI), Fe4S4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV ), Fe4S4(red)] form have been determined, revealing a four-domain ab structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer

Characterization of Crystalline Formate Dehydrogenase H from \u3ci\u3eEscherichia coli\u3c/i\u3e

The selenocysteine-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 400. The crystals diffract to 2.6 Å resolution 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 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