title: Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum.
abstract: A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S](2+/1+) cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g(zyx) values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S](1+) cluster with S=3/2 and zero field splitting parameters D=7.5 cm(-1) and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at -306 and -445 mV. In the presence of 55 microM ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands. p.l.hagedoorn@tnw.tudelft.nl
authors.forename: Peter L
authors.initials: PL
authors.lastname: Hagedoorn
authors.forename: Tianhong
authors.initials: T
authors.lastname: Chen
authors.forename: Imke
authors.initials: I
authors.lastname: Schröder
authors.forename: Sander R
authors.initials: SR
authors.lastname: Piersma
authors.forename: Simon
authors.initials: S
authors.lastname: de Vries
authors.forename: Wilfred R
authors.initials: WR
authors.lastname: Hagen
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D041543:Pyrobaculum / Q000201:enzymology*; D016415:Sequence Alignment; D013057:Spectrum Analysis; D014414:Tungsten / Q000378:metabolism*
chemical_list: D000445:Aldehyde Oxidoreductases; C080010:aldehyde ferredoxin oxidoreductase; D014414:Tungsten