Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria.
We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126+/-2 kDa composed of two subunits, alpha=93+/-3 kDa and beta=32+/-2 kDa, which contains 6+/-1 Fe/molecule, 0.4+/-0.1 Mo/molecule, 0.3+/-0.1 W/molecule, and 1.3+/-0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d(1) configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.
Brondino
Carlos D
CD
REQUIMTE, Departamento de Química, Centro de Química Fisica e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
Passeggi
Mario C G
MC
Caldeira
Jorge
J
Almendra
Maria J
MJ
Feio
Maria J
MJ
Moura
Jose J G
JJ
Moura
Isabel
I
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003901:Desulfovibrio / Q000737:chemistry / Q000201:enzymology* / Q000254:growth & development; D004574:Electromagnetic Fields; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry*; D008970:Molecular Weight; D008982:Molybdenum / Q000378:metabolism*; D010084:Oxidation-Reduction; D011622:Pterins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013431:Sulfates / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry*; D014414:Tungsten / Q000378:metabolism*
D011622:Pterins; D013431:Sulfates; D013455:Sulfur; D008982:Molybdenum; D007501:Iron; D005560:Formate Dehydrogenases; D014414:Tungsten