The formate dehydrogenase isolated from the aerobe Methylobacterium sp. RXM is a molybdenum-containing protein.
The formate dehydrogenase (FDH) isolated from cells of Methylobacterium sp. RXM grown on molybdenum-containing mineral medium using methanol as carbon source, was partially purified (at least 90% pure as revealed by SDS-PAGE). The enzyme is unstable under oxygen and all the purification steps were conducted under strict anaerobic conditions. The molecular mass is 75 kDa (gel exclusion 300 kDa). The enzyme was characterized in terms of the kinetic parameters towards different substrates and electron acceptors, pH and temperature dependence and the effect of a wide range of compounds in the enzymatic activity. The EPR spectra of the dithionite reduced sample show, at low temperature (below 20 K), two rhombic EPR signals due to two distinct [Fe-S] centres (centre I at g-values 2.023, 1.951 and 1.933, and centre II at g-values 2.054 and 1.913). At high temperature (around 100 K) another rhombic EPR signal is optimally observed at g-values 2.002, 1.987 and 1.959 and attributed to the molybdenum site. The EPR signals assigned to the iron-sulfur centres show a strong analogy with the aldehyde oxido-reductase from Desulfovibrio gigas known to contain a Mo-pterin and two [2Fe-2S] centres and whose crystallographic structure was recently resolved.
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Departamento de Química and Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal.
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D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis*; D013053:Spectrophotometry; D013816:Thermodynamics
D007506:Iron-Sulfur Proteins; D008982:Molybdenum; D005560:Formate Dehydrogenases