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

JBIC Journal of Biological Inorganic Chemistry - 2003
Carlos D. Brondino1,2, Mario C. G. Passeggi1,3, Jorge Caldeira2, Maria J. Almendra2, Maria J. Feio4,5, Jose J. G. Moura2, Isabel Moura2
1Departamento de Física, Facultad de Bioquímica y Ciencias Biologicas, Universidad Nacional del Litoral, Santa Fe, Argentina
2REQUIMTE, Departamento de Química, Centro de Química Fisica e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal
3Instituto de Desarrollo Tecnologico (UNL-CONICET), Santa Fe, Argentina
4School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK
5Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Seville, Spain

Tóm tắt

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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JBIC Journal of Biological Inorganic Chemistry

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