TY - JOUR
T1 - 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
AU - Brondino, Carlos D.
AU - Passeggi, Mario C.G.
AU - Caldeira, Jorge
AU - Almendra, Maria J.
AU - Feio, Maria J.
AU - Moura, Jose J.G.
AU - Moura, Isabel
N1 - Funding Information:
Acknowledgements This work was supported by Fundac¸ ão 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.
PY - 2004/3
Y1 - 2004/3
N2 - 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.
AB - 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.
KW - Electron paramagnetic resonance
KW - Formate dehydrogenase
KW - Magnetic interactions
KW - Molybdenum-containing enzymes
KW - Tungsten-containing enzymes
UR - http://www.scopus.com/inward/record.url?scp=1542268267&partnerID=8YFLogxK
U2 - 10.1007/s00775-003-0506-z
DO - 10.1007/s00775-003-0506-z
M3 - Article
C2 - 14669076
AN - SCOPUS:1542268267
SN - 0949-8257
VL - 9
SP - 145
EP - 151
JO - Journal of Biological Inorganic Chemistry
JF - Journal of Biological Inorganic Chemistry
IS - 2
ER -