BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Purifications and Characterizations of a Ferredoxin and Its Related 2-Oxoacid:Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon, Sulfolobus solfataricus P1

  • Park, Young-Jun (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Yoo, Chul-Bae (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Choi, Soo-Young (Department of Biomedical Sciences and Institute for Bioscience and Biotechnology, Hallym University) ;
  • Lee, Hee-Bong (Department of Biochemistry, College of Natural Sciences, Kangwon National University)
  • Received : 2005.09.14
  • Accepted : 2005.10.07
  • Published : 2006.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of $11,180{\pm}50$ Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa $\alpha$ and 34 kDa $\beta$ subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at $55^{\circ}C$, pH 7.0. Maximum activity was observed at $70^{\circ}C$ and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with $K_m$ and $k_{cat}$ values of $163\;{\mu}M$ and $452\;min^{-1}$, respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

Keywords

References

  1. Aasa, R. and Vanngard, T. (1975) EPR signal intensity and powder shapes: a re-examination. J. Magn. Reson. 19, 308-315
  2. Aono, S., Bryant, F. O. and Adams, M. W. W. (1989) A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus. J. Bacteriol. 171, 3433-3439 https://doi.org/10.1128/jb.171.6.3433-3439.1989
  3. Beinert, H. (1990) Recent developments in the field of iron-sulfur proteins. FASEB J. 4, 2483-2491 https://doi.org/10.1096/fasebj.4.8.2185975
  4. Beinert, H., Kennedy, M. C. and Stout, C. D. (1996) Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem. Rev. 96, 2335-2374 https://doi.org/10.1021/cr950040z
  5. Blamey, J. M. and Adams, M. W. W. (1993) Purification and characterization of pyruvate ferredoxin oxidoreductase from the hyperthermophilic archeon Pyrococcus furiosus. Biochim. Biophys. Acta. 1161, 19-27 https://doi.org/10.1016/0167-4838(93)90190-3
  6. Blamey, J. M., Chiong, M., López, C. and Smith, E. T. (2000) Purification and characterization of ferredoxin from hyperthermophilic Pyrococcus woesei. Anaerobe 6, 285-290 https://doi.org/10.1006/anae.2000.0351
  7. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  8. Breton, J. L., Duff, J. L. C., Butt, J. N., Armstrong, F. A., George, S. J., Petillot, Y., Forest, E., Schafer, G. and Thompson, A. J. (1995) Identification of the iron-sulfur clusters in a ferredoxin from the archaeon Sulfolobus acidocaldarius. Evidence for a reduced [3Fe-4S] cluster with pH-dependent electronic properties. Eur. J. Biochem. 233, 937-946 https://doi.org/10.1111/j.1432-1033.1995.937_3.x
  9. Brock, T. D., Brock, K. M., Belly, R. T. and Weiss, R. L. (1972) Sulfolobus: a new genus of sulfur oxidizing bacteria living at low pH and high temperature. Arch. Microbiol. 84, 54-68
  10. Broderick, J. B., Duderstadt, R. E., Fernandez, D. C., Wojtuszewski, K., Henshaw, T. F. and Johnson, M. K. (1997) Pyruvate formate-lyase activating enzyme is an iron-sulfur protein. J. Am. Chem. Soc. 119, 7396-7397 https://doi.org/10.1021/ja9711425
  11. Brostedt, E. and Nordlund, S. (1991) Purification and partial characterization of a pyruvate oxidoreductase from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen fixing conditions. Biochem. J. 279, 155-158 https://doi.org/10.1042/bj2790155
  12. Daas, P. J., Hagen, W. R., Keltjens, J. T. and Vogels, G. D. (1994) Characterization and determination of the redox properties of the 2[4Fe-4S] ferredoxin from Methanosarcina barkeri strain MS. FEBS Lett. 356, 342-344 https://doi.org/10.1016/0014-5793(94)01313-6
  13. Fujii, T., Moriyama, H., Takenaka, A., Tanaka, N., Wakagi, T. and Oshima, T. (1991) Crystallization and preliminary X-ray studies on Sulfolobus acidocaldarius ferredoxin. J. Biochem. (Tokyo) 110, 472-473 https://doi.org/10.1093/oxfordjournals.jbchem.a123605
  14. Gomes, C. M., Faria, A., Carita, J. C., Mendes, J., Regalla, M., Chicau, P., Huber, H., Stetter, K. O. and Teixeira, M. (1998) Di-cluster, seven-iron ferredoxins from hyperthermophilic Sulfolobales. JBIC 3, 499-507 https://doi.org/10.1007/s007750050260
  15. Grandoni, J. A., Switzer, R. L., Makaroff, C. A. and Zalkin, H. (1989) Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo. J. Biol. Chem. 264, 6058-6064
  16. Green, A. J., Munro, A. W., Cheesman, M. R., Reid, G. A., Wachenfeldt, C. and Chapman, S. K. (2003) Expression, purification and characterization of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI. J. Inorg. Biochem. 93, 92-99 https://doi.org/10.1016/S0162-0134(02)00456-7
  17. Grinberg, A. V., Hannemann, F., Schiffler, B., Muller, J., Heinemann, U. and Bernhardt, R. (2000) Adrenodoxin: Structure, stability, and electron transfer properties. Proteins 40, 590-612 https://doi.org/10.1002/1097-0134(20000901)40:4<590::AID-PROT50>3.0.CO;2-P
  18. Hagen, W. R., Dunham, W. R., Johnson, M. K. and Fee, J. A. (1985) Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin. Possible new fingerprints for three iron clusters. Biochim. Biophys. Acta 828, 369-374 https://doi.org/10.1016/0167-4838(85)90318-8
  19. Hentze, W. and Kunh, L. C. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc. Natl. Acad. Sci. USA 93, 8175-8182 https://doi.org/10.1073/pnas.93.16.8175
  20. Howard, J. B. and Rees, D. C. (1996) Structural basis of biological nitrogen fixation. Chem. Rev. 96, 2965-2982 https://doi.org/10.1021/cr9500545
  21. Iwasaki, T. and Oshima, T. (2001) Ferredoxin and related enzymes from Sulfolobus; in Methods in Enzymology 334, Adams, M. W. W. (ed.), pp. 3-22, Academic Press, New York, USA
  22. Iwasaki, T., Suzuki, T., Kon, T., Imai, T., Urushiyama, A., Ohmori, D. and Oshima, T. (1997) Novel zinc-containing ferredoxin family in thermoacidophilic archaea. J. Biol. Chem. 272, 3453-3458 https://doi.org/10.1074/jbc.272.6.3453
  23. Iwasaki, T., Wakagi, T., Isogani, Y., Tanaka, K., Lizuka, T. and Oshima, T. (1994) Functional and evolutionary implications of a [3Fe-4S] cluster of the dicluster-type ferredoxin from the thermoacidophilic archaeon, Sulfolobus sp. strain 7. J. Biol. Chem. 269, 29444-29450
  24. Johnson, M. K. (1994) Iron-Sulfur Proteins; in Encyclopedia of Inorganic Chemisty, R. B. King (Ed.), pp. 1896-1915, Wiley- Interscience, New York, USA
  25. Kerscher, L., Nowitzki, S. and Oesterhelt, D. (1982) Thermophilic archaebacteria contain bacterial-type ferredoxins acting as electron acceptors of 2-oxoacid:ferredoxin oxidoreductases. Eur. J. Biochem. 128, 223-230 https://doi.org/10.1111/j.1432-1033.1982.tb06955.x
  26. Kerscher, L. and Oesterhelt, D. (1981) Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. Eur. J. Biochem. 116, 587-594 https://doi.org/10.1111/j.1432-1033.1981.tb05376.x
  27. Kletzin, A. and Adams, M. W. W. (1996) Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga martima. J. Bacteriol. 178, 248-257 https://doi.org/10.1128/jb.178.1.248-257.1996
  28. Koike, M. and Koike, K. (1976) Structure, assembly and function of mammalian ${\alpha}$-keto acid dehydrogenase complexes. Adv. Biophys. 9, 187-227
  29. Kounosu, A., Li, Z., Cosper, N. J., Shokes, J. E., Scott, R. A., Imai, T., Urushiyama, A. and Iwasaki, T. (2004) Engineering a three-cystein, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus. J. Biol. Chem., 279, 12519-12528 https://doi.org/10.1074/jbc.M305923200
  30. Kunow, J., Linder, D. and Thauer, R. K. (1995) Pyruvate:ferredoxin oxidoreductase from the sulfate-reducing Achaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments. Arch. Microbiol. 163, 21- 28
  31. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680- 685 https://doi.org/10.1038/227680a0
  32. Matsubara, H. and Saecki, K. (1992) Structural and functional diversity of ferredoxins and related proteins. Adv. Inorg. Chem. 38, 223-280 https://doi.org/10.1016/S0898-8838(08)60065-3
  33. Meinecke, B., Bertram, J. and Gottachalk, G. (1989) Purification and characterization of the puruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum. Arch. Microbiol. 152, 244- 250 https://doi.org/10.1007/BF00409658
  34. Michaels, M. L., Pham, L., Nghiem, Y., Cruz, C. and Miller, J. H. (1990) MutY, an adenine glycosylase active on G-A mispairs, has homology to endonuclease III. Nucleic Acids Res. 18, 3841-3845 https://doi.org/10.1093/nar/18.13.3841
  35. Minami, Y., Wakabayashi, S., Wada, K., Matsubara, H., Kerscher, L. and Oesterhelt, D. (1985) J. Biochem. (Tokyo) 97, 745-751 https://doi.org/10.1093/oxfordjournals.jbchem.a135114
  36. Mortenson, L. E., Valentine, R. C. and Carnahan, J. E. (1962) An electron transport factor from Clostridium pasteurianum. Biochem. Biophys. Res. Commun. 7, 448-452 https://doi.org/10.1016/0006-291X(62)90333-9
  37. Nakajima, Y., Fujiwara, T. and Fukumori, Y. (1998) Purification and characterization of a [3Fe-4S][4Fe-4S] type ferredoxin from hyperthermophilic archeon, Pyrobaculum islandicum. J. Biochem. 23, 521-527
  38. Ohsawa, K. and Ebata, N. (1983) Silver stain for detecting 10- femtogram quantities of protein after polyacrylamide gel electrophoresis. Anal. Biochem. 135, 409-415 https://doi.org/10.1016/0003-2697(83)90703-0
  39. Rosa, M. D., Gambacorta, A. and Bu'lock, J. D. (1975) Extremely thermophilic acidophilic bacteria convergent with Sulfolobus acidocaldarius. J. Gen. Microbiol. 86, 156-164 https://doi.org/10.1099/00221287-86-1-156
  40. She, Q., Singh, R. K., Confalonieri, F., Zivanovic, Y., Allard, G., Awayez, M. J., Chan Weiher, C. C. Y., Clausen, I. G., Curtis, B. A., Moors, A. D., Erauso, G., Fletcher, C., Gordon, P. M. K., Heikamp, D. J. I., Jeffries, A. C., Kozera, C. J., Medina, N., Peng, X., Thi-Ngoc, H. P., Redder, P., Schenk, M. E., Theriault, C., Tolstrup, N., Charlebois, R. L., Doolittle, W. F., Duguet, M., Gaasterland, T., Garrett, R. A., Ragan, M. A., Sensen, C. W. and Oost, V. D. J. (2001) The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc. Natl. Acad. Sci. USA 98, 7835-7840 https://doi.org/10.1073/pnas.141222098
  41. Teixeira, M., Batista, R., Campos, A. P., Gomes, C., Mendes, J., Pacheco, I., Anemuller, S. and Hagen, W. R. (1995) A seveniron ferredoxin from the thermoacidophilic archaeon Desulfurolobus ambivalens. Eur. J. Biochem. 227, 322-327 https://doi.org/10.1111/j.1432-1033.1995.tb20392.x
  42. Thauer, R. K. and Schonheit, P. (1982) Iron-sulfur complexs of ferredoxin as a storage form of iron in Clostridium pasteurianum; in Iron-Sulfur Proteins. T. G. Spiro (Ed.), pp. 329-341, Wiley-Interscience, New York, USA
  43. Wakagi, T., Fujii, T. and Oshima, T. (1996) Molecular cloning, sequencing, and heterologous expression of a novel zinccontaining ferredoxin gene from a thermoacidophilic archeon Sulfolobus sp. strain 7. Biochem. Biophys. Res. Commun. 225, 489-493 https://doi.org/10.1006/bbrc.1996.1200
  44. Williams, K., Lowe, P. N. and Leadlay, P. F. (1987) Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoan Trichomonas vaginalis. Biochem. J. 246, 529-536 https://doi.org/10.1042/bj2460529
  45. Zhang, Q., Iwasaki, T., Wakagi, T. and Oshima, T. (1996) 2- Oxoacid:ferredoxin oxidoreductase from the thermoacidophilic archaeon, Sulfolobus sp. strain 7. J. Biochem. 120, 587-599 https://doi.org/10.1093/oxfordjournals.jbchem.a021454
  46. Zillig, W., Stetter, K. O., Wunderl, S., Schulz, W., Priess, H. and Scholz, I. (1980) The Sulfolobus 'Caldariella' group: taxonomy on the basis of the structure of DNA-dependent RNA poly-merase. Arch. Microbiol. 125, 259-269 https://doi.org/10.1007/BF00446886

Cited by

  1. Alpha-ketoglutarate oxidoreductase, an essential salvage enzyme of energy metabolism, in coccoid form of Helicobacter pylori vol.376, pp.1, 2008, https://doi.org/10.1016/j.bbrc.2008.08.078
  2. Proteomics and comparative genomics ofNitrososphaera viennensisreveal the core genome and adaptations of archaeal ammonia oxidizers vol.113, pp.49, 2016, https://doi.org/10.1073/pnas.1601212113
  3. pH-Dependent surface-enhanced resonance Raman scattering of yeast iso-1-cytochrome c adsorbed on silver nanoparticle surfaces under denaturing conditions at pH < 3 vol.42, pp.4, 2009, https://doi.org/10.5483/BMBRep.2009.42.4.223
  4. Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens vol.1837, pp.7, 2014, https://doi.org/10.1016/j.bbabio.2013.12.002
  5. The genome of the ammonia-oxidizingCandidatusNitrososphaera gargensis: insights into metabolic versatility and environmental adaptations vol.14, pp.12, 2012, https://doi.org/10.1111/j.1462-2920.2012.02893.x