Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas.
An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mössbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.
Almendra
M J
MJ
Departamento de Química (Centro de Química Fina e Biotecnologia), Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal.
Brondino
C D
CD
Gavel
O
O
Pereira
A S
AS
Tavares
P
P
Bursakov
S
S
Duarte
R
R
Caldeira
J
J
Moura
J J
JJ
Moura
I
I
D000332:Aerobiosis; D002850:Chromatography, Gel; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005560:Formate Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006150:Guanine Nucleotides / Q000032:analysis; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011622:Pterins / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D014414:Tungsten / Q000737:chemistry*
D006150:Guanine Nucleotides; D008667:Metalloproteins; D011622:Pterins; C063761:molybdopterin guanine dinucleotide; D005560:Formate Dehydrogenases; D014414:Tungsten