Skip to main content

Advertisement

SpringerLink
Log in

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum

  • Original Article
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

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 μM ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

AOR:

Aldehyde:ferredoxin oxidoreductase

DEAE:

(Diethylamino)ethyl

EPPS:

N-(2-Hydroxyethyl)piperazine-N′-propanesulfonic acid

EPR:

Electron paramagnetic resonance

FOR:

Formaldehyde:ferredoxin oxidoreductase

GAPOR:

Glyceraldehyde-3-phosphate:ferredoxin oxidoreductase

LC:

Liquid chromatography

MS:

Mass spectrometry

ORF:

Open reading frame

PVDF:

Poly(vinylidene difluoride)

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tris:

Tris(hydroxymethyl)aminomethane

References

  1. Kletzin A, Adams MWW (1996) FEMS Microbiol Rev 18:5–63

    Google Scholar 

  2. Kisker C, Schindelin H, Rees DC (1997) Annu Rev Biochem 66:233–267

    Article  CAS  PubMed  Google Scholar 

  3. Roy R, Adams MWW (2002) Met Ions Biol Syst 39:673–697

    Google Scholar 

  4. Trautwein T, Krauss F, Lottspeich F, Simon H (1994) Eur J Biochem 222:1025–1032

    Google Scholar 

  5. Mukund S, Adams MWW (1991) J Biol Chem 266:14208–14216

    Google Scholar 

  6. Mukund S (1995) Department of Biochemistry, PhD thesis, University of Georgia, Athens, Georgia

  7. Heider J, Kesen M, Adams MWW (1995) J Bacteriol 177:4757–4764

    Google Scholar 

  8. Strobl G, Feicht R, White H, Lottspeich F, Simon H (1992) Biol Chem 373:123–132

    Google Scholar 

  9. White H, Feicht R, Huber C, Lottspeich F, Simon H (1991) Biol Chem 372:999–1005

    Google Scholar 

  10. Rauh D, Graentzdoerffer A, Granderath K, Andreesen JR, Pich A (2004) Eur J Biochem 271:212–219

    Google Scholar 

  11. Hensgens CMH, Hagen WR, Hansen TA (1995) J Bacteriol 177:6195–6200

    CAS  PubMed  Google Scholar 

  12. Chan M K, Mukund S, Kletzin A, Adams MWW, Rees DC (1995) Science 267:1463–1469

    Google Scholar 

  13. Volkl P, Huber R, Drobner E, Rachel R, Burggraf S, Trincone A, Stetter KO (1993) Appl Environ Microbiol 59:2918–2926

    CAS  PubMed  Google Scholar 

  14. Afshar S, Kim C, Monbouquette HG, Schröder I (1998) Appl Environ Microbiol 64:3004–3008

    Google Scholar 

  15. Oost Jvd, Schut G, Kengen SWM, Hagen WR, Thomm M, Vos WMd (1998) J Biol Chem 273:28149–28154

    Google Scholar 

  16. Ma K, Hutchins A, Sung S-JS, Adams MWW (1997) Proc Natl Acad Sci 94:9608–9613

    Google Scholar 

  17. Nakajima Y, Fujiware T, Fukumori Y (1998) J Biochem (Tokyo) 123:521–527

    Google Scholar 

  18. Vries SD, Strampraad MJF, Lu S, Moënne-Loccoz P, Schröder I (2003) J Biol Chem 278:35861–35868

    Google Scholar 

  19. Bensadoun A, Weinstein D (1976) Anal Biochem 70:241–250

    Google Scholar 

  20. Hennessy DJ, Reid GR, Smith FE, Thompson SL (1984) Can J Chem 62:721–724

    Google Scholar 

  21. Hagedoorn PL, Van ’t Slot P, Van Leeuwen HP, Hagen WR (2001) Anal Biochem 297:71–78

    Google Scholar 

  22. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Anal Chem 68:850–858

    Article  CAS  PubMed  Google Scholar 

  23. Hagen WR (1989) Eur J Biochem 182:523–530

    Google Scholar 

  24. Pierik AJ, Hagen WR, Redeker JS, Wolbert RB, Boersma M, Verhagen MFJM, Grande HJ, Veeger C, Mutsaers PHA, Sands RH, Dunham WR (1992) Eur J Biochem 209:63–72

    Google Scholar 

  25. Arendsen AF, De Vocht M, Bulsink YBM, Hagen WR (1996) J Biol Inorg Chem 1:292–296

    Google Scholar 

  26. Hagen WR, Hearshen DO, Sands RH, Dunham WR (1985) J Magn Reson 61:220–232

    Google Scholar 

  27. Hagen WR (1992) Adv Inorg Chem 38:165–222

    Google Scholar 

  28. Hickey AJ, Conway de Macario E, Macario AJL (2002) Crit Rev Biochem Mol Biol 37:199–258

    Google Scholar 

  29. Soppa J (1999) Mol Microbiol 31:1295–1305

    Google Scholar 

  30. Reiter WD, Palm P, Zillig W (1988) Nucleic Acids Res 16:2445–2459

    Google Scholar 

  31. Iwasaki T, Wakagi T, Isogai Y, Tanaka K, Iizuka T, Oshima T (1994) J Biol Chem 269:29444–29450

    Google Scholar 

  32. Battistuzzi G, Borsari M, Ferretti S, Luchinat C, Sola M (1995) Arch Biochem Biophys 320:149–154

    Google Scholar 

  33. Kardinahl S, Schmidt C L, Hansen T, Anemueller S, Petersen A, Schaefer G (1999) Eur J Biochem 260:540–548

    Google Scholar 

  34. Lucas S, Toffin L, Zivanovic Y, Charlier D, Moussard H, Forterre P, Prieur D, Erauso G (2002) Appl Environ Microbiol 68:5528–5536

    Google Scholar 

  35. Wahyudi AT, Takeyama H, Okamura Y, Fukuda Y, Matsunaga T (2003) Biochem Biophys Res Commun 303:223–229

    Google Scholar 

  36. Hu Y, Faham S, Roy R, Adams MWW, Rees DC (1999) J Mol Biol 286:899–914

    Google Scholar 

  37. Lambert C, Leonard N, De Bolle X, Depiereux E (2002) Bioinformatics 18:1250–1256

    Google Scholar 

  38. Koehler BP, Mukund S, Conover RC, Dhawan IK, Roy R, Adams MWW, Johnson MK (1996) J Am Chem Soc 118:12391–12405

    Google Scholar 

  39. Hagen WR, van den Berg WA, van dongen WMAM, Reijerse EJ, van Kan PJM (1998) J Chem Soc Faraday Trans 94:2969–2973

    Google Scholar 

  40. Johnson MK, Rees DC, Adams MWW (1996) Chem Rev 96:2817–2839

    Article  CAS  PubMed  Google Scholar 

  41. Dhawan IK, Roy R, Koehler BP, Mukund S, Adams MWW, Johnson MK (2000) J Biol Inorg Chem 5:313–327

    Google Scholar 

  42. Hagedoorn PL, Freije JR, Hagen WR (1999) FEBS Lett 462:66–70

    Google Scholar 

  43. Huber C, Caldeira J, Jongejan JA, Simon H (1994) Arch Microbiol 162:303–309

    Google Scholar 

  44. Hagedoorn PL, Hagen WR, Stewart LJ, Docrat A, Bailey S, Garner CD (2003) FEBS Lett 555:606–610

    Google Scholar 

  45. Schut GJ, Menon AL, Adams MWW (2001) Methods Enzymol 331:144–158

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands

    Peter L. Hagedoorn, Tianhong Chen, Simon de Vries & Wilfred R. Hagen

  2. Department of Microbiology, Immunology and Molecular Genetics, 1602 Molecular Sciences Building, UCLA, Los Angeles, CA, 90095-1489, USA

    Imke Schröder

  3. FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ, Amsterdam, The Netherlands

    Sander R. Piersma

Authors
  1. Peter L. Hagedoorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tianhong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Imke Schröder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sander R. Piersma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Simon de Vries
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wilfred R. Hagen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter L. Hagedoorn.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hagedoorn, P.L., Chen, T., Schröder, I. et al. Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum. J Biol Inorg Chem 10, 259–269 (2005). https://doi.org/10.1007/s00775-005-0637-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-005-0637-5

Keywords

Search

Navigation