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. 1994 Jun 1;300(Pt 2):413–418. doi: 10.1042/bj3000413

Recombinant expression of the fdxD gene of Rhodobacter capsulatus and characterization of its product, a [2Fe-2S] ferredoxin.

J Armengaud 1, C Meyer 1, Y Jouanneau 1
PMCID: PMC1138178  PMID: 8002946

Abstract

A gene called fdxD that could potentially code for a ferredoxin has recently been identified upstream of the nitrogenase structural genes in Rhodobacter capsulatus [Willison, Pierrard and Hübner (1993) Gene 133, 39-46]. In the present study, the fdxD gene product has been overproduced in Escherichia coli in a soluble form. The recombinant protein, pink in colour, was purified to homogeneity, and biochemically characterized as a new ferredoxin. It represents the fifth ferredoxin so far identified in R. capsulatus and was designated FdV. Its N-terminal sequence is identical with that of the native ferredoxin isolated from R. capsulatus. U.v-visible-absorption spectra as well as results of c.d. and e.p.r. spectroscopy demonstrated that the fdxD product contained a [2Fe-2S] cluster correctly assembled and incorporated into the polypeptide. Although similar to plant-type ferredoxins, FdV appeared poorly competent in the photo-reduction of NADP+. On the basis of in vitro assays, FdV cannot serve as an electron donor for nitrogenase. The lack of reactivity of FdV in either of these assays may primarily be due to its relatively high mid-point redox potential (E'o = -220 mV, pH 7.5).

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Selected References

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  1. Beinert H. Recent developments in the field of iron-sulfur proteins. FASEB J. 1990 May;4(8):2483–2491. doi: 10.1096/fasebj.4.8.2185975. [DOI] [PubMed] [Google Scholar]
  2. Bourdineaud J. P., Howard S. P., Pages J. M., Bernadac A., Leroy G., Bruschi M., Lazdunski C. Cytoplasmic and periplasmic expression of a synthetic gene for ferredoxin in Escherichia coli. Biochimie. 1990 Jun-Jul;72(6-7):407–415. doi: 10.1016/0300-9084(90)90065-o. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Britt R. D., Sauer K., Klein M. P., Knaff D. B., Kriauciunas A., Yu C. A., Yu L., Malkin R. Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two histidine ligands to the Rieske Fe-S centers of the cytochrome b6f complex of spinach and the cytochrome bc1 complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26, and bovine heart mitochondria. Biochemistry. 1991 Feb 19;30(7):1892–1901. doi: 10.1021/bi00221a023. [DOI] [PubMed] [Google Scholar]
  5. Cammack R. New developments in iron-sulphur proteins. Nature. 1980 Jul 31;286(5772):442–442. doi: 10.1038/286442a0. [DOI] [PubMed] [Google Scholar]
  6. Carithers R. P., Yoch D. C., Arnon D. I. Isolation and characterization of bound ion-sulfur proteins from bacterial photosynthetic membranes. II. Succinate dehydrogenase from Rhodospirillum rubrum chromatophores. J Biol Chem. 1977 Nov 10;252(21):7461–7467. [PubMed] [Google Scholar]
  7. Coghlan V. M., Vickery L. E. Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Feb;86(3):835–839. doi: 10.1073/pnas.86.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elliott J. I., Ljungdahl L. G. Isolation and characterization of an Fe,-S8 ferredoxin (ferredoxin II) from Clostridium thermoaceticum. J Bacteriol. 1982 Jul;151(1):328–333. doi: 10.1128/jb.151.1.328-333.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fujinaga J., Meyer J. Cloning and expression in Escherichia coli of the gene encoding the [2Fe-2S] ferredoxin from Clostridium pasteurianum. Biochem Biophys Res Commun. 1993 May 14;192(3):1115–1122. doi: 10.1006/bbrc.1993.1532. [DOI] [PubMed] [Google Scholar]
  10. Grabau C., Schatt E., Jouanneau Y., Vignais P. M. A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli. J Biol Chem. 1991 Feb 15;266(5):3294–3299. [PubMed] [Google Scholar]
  11. Gurbiel R. J., Ohnishi T., Robertson D. E., Daldal F., Hoffman B. M. Q-band ENDOR spectra of the Rieske protein from Rhodobactor capsulatus ubiquinol-cytochrome c oxidoreductase show two histidines coordinated to the [2Fe-2S] cluster. Biochemistry. 1991 Dec 10;30(49):11579–11584. doi: 10.1021/bi00113a013. [DOI] [PubMed] [Google Scholar]
  12. Haaker H., Veeger C. Involvement of the cytoplasmic membrane in nitrogen fixation by Azotobacter vinelandii. Eur J Biochem. 1977 Jul 1;77(1):1–10. doi: 10.1111/j.1432-1033.1977.tb11634.x. [DOI] [PubMed] [Google Scholar]
  13. Hase T., Mizutani S., Mukohata Y. Expression of Maize Ferredoxin cDNA in Escherichia coli: Comparison of Photosynthetic and Nonphotosynthetic Ferredoxin Isoproteins and their Chimeric Molecule. Plant Physiol. 1991 Dec;97(4):1395–1401. doi: 10.1104/pp.97.4.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hurley J. K., Salamon Z., Meyer T. E., Fitch J. C., Cusanovich M. A., Markley J. L., Cheng H., Xia B., Chae Y. K., Medina M. Amino acid residues in Anabaena ferredoxin crucial to interaction with ferredoxin-NADP+ reductase: site-directed mutagenesis and laser flash photolysis. Biochemistry. 1993 Sep 14;32(36):9346–9354. doi: 10.1021/bi00087a013. [DOI] [PubMed] [Google Scholar]
  15. Jouanneau Y., Duport C., Meyer C., Gaillard J. Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus. Biochem J. 1992 Aug 15;286(Pt 1):269–273. doi: 10.1042/bj2860269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jouanneau Y., Meyer C., Gaillard J., Forest E., Gagnon J. Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus. J Biol Chem. 1993 May 15;268(14):10636–10644. [PubMed] [Google Scholar]
  17. Jouanneau Y., Meyer C., Gaillard J., Vignais P. M. Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus. Biochem Biophys Res Commun. 1990 Aug 31;171(1):273–279. doi: 10.1016/0006-291x(90)91388-9. [DOI] [PubMed] [Google Scholar]
  18. Kimura T., Suzuki K. Components of the electron transport system in adrenal steroid hydroxylase. Isolation and properties of non-heme iron protein (adrenodoxin). J Biol Chem. 1967 Feb 10;242(3):485–491. [PubMed] [Google Scholar]
  19. Knoell H. E., Knappe J. Escherichia coli ferredoxin, an iron-sulfur protein of the adrenodoxin type. Eur J Biochem. 1974 Dec 16;50(1):245–252. doi: 10.1111/j.1432-1033.1974.tb03893.x. [DOI] [PubMed] [Google Scholar]
  20. Matsubara H., Hase T., Wakabayashi S., Wada K. Structural and evolution of chloroplast- and bacterial-type ferredoxins. UCLA Forum Med Sci. 1979;(21):245–266. doi: 10.1016/b978-0-12-643150-6.50022-1. [DOI] [PubMed] [Google Scholar]
  21. Meyer J., Moulis J. M., Lutz M. Structural differences between [2Fe-2S] clusters in spinach ferredoxin and in the "red paramagnetic protein" from Clostridium pasteurianum. A resonance Raman study. Biochem Biophys Res Commun. 1984 Mar 30;119(3):828–835. doi: 10.1016/0006-291x(84)90848-9. [DOI] [PubMed] [Google Scholar]
  22. Meyer T. E., Cusanovich M. A. Structure, function and distribution of soluble bacterial redox proteins. Biochim Biophys Acta. 1989 Jun 23;975(1):1–28. doi: 10.1016/s0005-2728(89)80196-3. [DOI] [PubMed] [Google Scholar]
  23. Moulis J. M., Meyer J. Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum. Biochemistry. 1982 Sep 14;21(19):4762–4771. doi: 10.1021/bi00262a037. [DOI] [PubMed] [Google Scholar]
  24. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  25. O'Keefe D. P., Gibson K. J., Emptage M. H., Lenstra R., Romesser J. A., Litle P. J., Omer C. A. Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus. Biochemistry. 1991 Jan 15;30(2):447–455. doi: 10.1021/bi00216a021. [DOI] [PubMed] [Google Scholar]
  26. Ploug M., Jensen A. L., Barkholt V. Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3. Anal Biochem. 1989 Aug 15;181(1):33–39. doi: 10.1016/0003-2697(89)90390-4. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schatt E., Jouanneau Y., Vignais P. M. Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol. 1989 Nov;171(11):6218–6226. doi: 10.1128/jb.171.11.6218-6226.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  30. Shethna Y. I., DerVartanian D. V., Beinert H. Non heme (iron-sulfur) proteins of Azotobacter vinelandii. Biochem Biophys Res Commun. 1968 Jun 28;31(6):862–868. doi: 10.1016/0006-291x(68)90531-7. [DOI] [PubMed] [Google Scholar]
  31. Stephens P. J., Jensen G. M., Devlin F. J., Morgan T. V., Stout C. D., Martin A. E., Burgess B. K. Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I. Biochemistry. 1991 Apr 2;30(13):3200–3209. doi: 10.1021/bi00227a007. [DOI] [PubMed] [Google Scholar]
  32. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  33. Ta D. T., Vickery L. E. Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli. J Biol Chem. 1992 Jun 5;267(16):11120–11125. [PubMed] [Google Scholar]
  34. Vignais P. M., Colbeau A., Willison J. C., Jouanneau Y. Hydrogenase, nitrogenase, and hydrogen metabolism in the photosynthetic bacteria. Adv Microb Physiol. 1985;26:155–234. doi: 10.1016/s0065-2911(08)60397-5. [DOI] [PubMed] [Google Scholar]
  35. Willison J. C., Pierrard J., Hübner P. Sequence and transcript analysis of the nitrogenase structural gene operon (nifHDK) of Rhodobacter capsulatus: evidence for intramolecular processing of nifHDK mRNA. Gene. 1993 Oct 29;133(1):39–46. doi: 10.1016/0378-1119(93)90222-o. [DOI] [PubMed] [Google Scholar]
  36. Yakunin A. F., Gennaro G., Hallenbeck P. C. Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol. 1993 Nov;175(21):6775–6780. doi: 10.1128/jb.175.21.6775-6780.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yakunin A. F., Hallenbeck P. C., Troshina OYu, Gogotov I. N. Purification and properties of a bacterial-type ferredoxin from the nitrogen-fixing cyanobacterium Anabaena variabilis ATCC29413. Biochim Biophys Acta. 1993 May 13;1163(2):124–130. doi: 10.1016/0167-4838(93)90173-o. [DOI] [PubMed] [Google Scholar]
  38. Yoch D. C., Carithers R. P., Arnon D. I. Isolation and characterization of bound ion-sulfur proteins from bacterial photosynthetic membranes. I. Ferredoxins III and IV from Rhodospirillum rubrum chromatophores. J Biol Chem. 1977 Nov 10;252(21):7453–7460. [PubMed] [Google Scholar]

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