molybdopterin-guanine-dinucleotide and formic-acid

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene ( fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe(4)/S(4) cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

Topics: Amino Acid Sequence; Chromatography; DNA, Bacterial; Enzyme Stability; Epsilonproteobacteria; Escherichia coli Proteins; Formate Dehydrogenases; Formates; Guanine Nucleotides; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Membrane Transport Proteins; Molecular Sequence Data; Molecular Weight; NAD; NADP; Paraquat; Protein Sorting Signals; Protein Subunits; Pterins; Selenocysteine; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Temperature

The structure of the membrane protein formate dehydrogenase-N (Fdn-N), a major component of Escherichia coli nitrate respiration, has been determined at 1.6 angstroms. The structure demonstrates 11 redox centers, including molybdopterin-guanine dinucleotides, five [4Fe-4S] clusters, two heme b groups, and a menaquinone analog. These redox centers are aligned in a single chain, which extends almost 90 angstroms through the enzyme. The menaquinone reduction site associated with a possible proton pathway was also characterized. This structure provides critical insights into the proton motive force generation by redox loop, a common mechanism among a wide range of respiratory enzymes.

Topics: Binding Sites; Catalysis; Catalytic Domain; Cell Membrane; Crystallography, X-Ray; Electron Transport; Escherichia coli; Formate Dehydrogenases; Formates; Guanine Nucleotides; Hydrogen Bonding; Iron-Sulfur Proteins; Membrane Potentials; Models, Molecular; Nitrate Reductases; Oxidation-Reduction; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Proton-Motive Force; Protons; Pterins; Vitamin K 2

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 alphabeta 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.

Topics: Binding Sites; Carbon Dioxide; Catalysis; Crystallography, X-Ray; Electron Transport; Escherichia coli; Ferrous Compounds; Formate Dehydrogenases; Formates; Guanine Nucleotides; Hydrogen Bonding; Hydrogenase; Ligands; Models, Molecular; Molecular Sequence Data; Molybdenum; Multienzyme Complexes; Nitrites; Oxidation-Reduction; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Protons; Pterins; Selenocysteine