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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

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Abstract

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, α=93±3 kDa and β=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 d1 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.

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Abbreviations

AOR:

aldehyde oxidoreductase

FDH:

formate dehydrogenase

NAP:

periplasmic nitrate reductase

SRB:

sulfate-reducing bacteria

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Acknowledgements

This work was supported by Fundação para a Ciência e Tecnologia (Portugal) POCTI/BME/36152/99 and CAI+D-UNL (Argentina). MJF thanks the British Council for financial support for travel to Portugal.

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Author notes

  1. Maria J. Feio

    Present address: Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Av. Américo Vespucio s/n, 41092, Seville, Spain

Authors and Affiliations

  1. REQUIMTE, Departamento de Química, Centro de Química Fisica e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal

    Carlos D. Brondino, Jorge Caldeira, Maria J. Almendra, Jose J. G. Moura & Isabel Moura

  2. Departamento de Física, Facultad de Bioquímica y Ciencias Biologicas, Universidad Nacional del Litoral, 3000, Santa Fe, Argentina

    Carlos D. Brondino & Mario C. G. Passeggi

  3. Instituto de Desarrollo Tecnologico (UNL-CONICET), 3000, Santa Fe, Argentina

    Mario C. G. Passeggi

  4. School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth, PO1 2DT, UK

    Maria J. Feio

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  1. Carlos D. Brondino
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Correspondence to Isabel Moura.

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Brondino, C.D., Passeggi, M.C.G., Caldeira, J. et al. 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. J Biol Inorg Chem 9, 145–151 (2004). https://doi.org/10.1007/s00775-003-0506-z

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