Applied Microbiology and Biotechnology

, Volume 63, Issue 3, pp 315–321 | Cite as

Hydrogenases in sulfate-reducing bacteria function as chromium reductase

  • B. Chardin
  • M.-T. Giudici-Orticoni
  • G. De Luca
  • B. Guigliarelli
  • M. BruschiEmail author
Original Paper


The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H2S produced by the bacteria, and enzymatically, by polyhemic cytochromes c 3. We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K m towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.


Electron Paramagnetic Resonance Electron Paramagnetic Resonance Spectrum Reductase Activity Electron Paramagnetic Resonance Signal Methylviologen 
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This work was supported by grants from the Fifth Program for RTD (EVK1-CT 1999-00033), from 99N33/0010 ECODEV-CNRS, from ADEME (France), and from BRGM (France). B. Chardin gratefully acknowledges receipt of a CIFRE research studentship from S.E.I Environnement et Procédés Industriels. We also thank Dr. A. Dolla for providing DvH strain hyd 100, Dr. C. Michel for her involvement in this work and Dr. A. Cornish-Bowden for his critical reading of the manuscript.


  1. Assfalg M, Bertini I, Bruschi M, Michel C, Turano P (2002) The metal reductase activity of some multiheme cytochrome c:chromium (III) by cytochrome c 7. Proc Natl Acad Sci USA 99:9750–9754CrossRefPubMedGoogle Scholar
  2. Aubert C, Lojou E, Bianco P, Rousset M, Durand MC, Bruschi M, Dolla A (1998) The Desulforomonas acetoxidans triheme cytochrome c 7 produced in Desulfovibrio desulfuricans retains its metal reductase activity. Appl Environ Microbiol 64:1308–1312PubMedGoogle Scholar
  3. Bauchop T, Elsden SR (1960) The growth of micro-organisms in relation to their energy supply. J Gen Microbiol 23:457–469Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Brugna M, Giudici-Orticoni M-T, Spinelli S, Brown K, Tegoni M, Bruschi M (1998) Kinetics and interaction studies between cytochrome c 3 and Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough. Proteins 33:590–600CrossRefPubMedGoogle Scholar
  6. Brugna-Guiral M, Tron P, Nitschke W, Stetter K-O, Burlat B, Guigliarelli B, Bruschi M, Giudici-Orticoni M-T (2003) [NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aelicos: properties, function, and phylogenetics. Extremophiles 7:145–157PubMedGoogle Scholar
  7. Bruschi M, Hatchikian EC, Le Gall J, Moura JJG, Xavier AV (1976) Purification, characterisation and biological activity of 3 forms of ferredoxin from the sulfate-reducing bacteria Desulfovibrio gigas. Biochim Biophys Acta 449:275–284PubMedGoogle Scholar
  8. Cammack R, Patil D, Hatchikian E, Fernandez V (1987) Nickel and iron-sulphur centres in Desulfovibrio gigas hydrogenase: ESP spectra, redox properties and interactions. Biochim Biophys Acta 912:98–109Google Scholar
  9. Chardin B, Dolla A, Chaspoul F, Fardeau ML, Gallice P, Bruschi M (2002) Bioremediation of chromate: thermodynamic analysis of the effects of Cr(VI) on sulfate-reducing bacteria. Appl Microbiol Biotechnol 60:352–360CrossRefPubMedGoogle Scholar
  10. De Luca G, de Philip P, Dermoun Z, Rousset M, Verméglio A (2001) Reduction of technetium(VII) by Desulfovibrio fructosovorans is mediated by the nickel-iron hydrogenase. Appl Environ Microbiol 67:4583–4587CrossRefPubMedGoogle Scholar
  11. Eary LE, Ral D (1988) Chromate removal from aqueous wastes by reduction with ferrous ion. Environ Sci Technol 22:972–977Google Scholar
  12. Florens L, Bianco P, Haladjian J, Bruschi M, Protasevich S, Makarov A (1995) Thermal stability of the polyheme cytochrome c 3 superfamily. FEBS Lett 373:280–284CrossRefPubMedGoogle Scholar
  13. Hatchikian CE, Traore AS, Fernandez VM, Cammack R. (1990) Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans. Eur J Biochem 187:635–643PubMedGoogle Scholar
  14. Lloyd JR, Cole JA, Mackasie LE (1997) Reduction and removal of heptavalent technetium from solution by Escherichia coli. J Bacteriol 179:2014–2021PubMedGoogle Scholar
  15. Lloyd JR, Nolting HF, Sole VA, Bosecker K, Mackasie LE (1998) Technetium reduction and precipitation by sulfate reducing bacteria. Geomicrobiol J 15:43–56Google Scholar
  16. Lojou EP, Bianco P (1999) Electrocatalytic reduction of bacterial cytochrome: biochemical and chemical factors influencing the catalytic process. J Electroanal Chem 471:96–104CrossRefGoogle Scholar
  17. Lojou EP, Bianco P, Bruschi M (1998) Kinetic studies on the electron transfer between bacterial c-type cytochromes and metal oxides. J Electroanal Chem 452:167–177CrossRefGoogle Scholar
  18. Lovley DR (1994) Dissimilatory metal reductions. Annu Rev Microbiol 60:726–728Google Scholar
  19. Lovley DR, Coates JR (1997) Bioremediation of metal contamination. Curr Opin Biotechnol 8:285–289PubMedGoogle Scholar
  20. Lovley DR, Phillips EJP (1994) Reduction of chromate by Desulfovibrio vulgaris and its c 3 cytochrome. Appl Environ Microbiol 60:726–728Google Scholar
  21. Macy JM, Santini JM, Pauling BV, O'Neill AH, Sly LI (2000) Two new arsenate/sulfate reducing bacteria: mechanisms of arsenate reduction. Arch Microbiol 17:49–57CrossRefGoogle Scholar
  22. Michel C, Brugna M, Aubert C, Bernadac M, Bruschi M (2001) Enzymatic reduction of chromate: comparative studies using sulfate reducing bacteria. Appl Microbiol Biotechnol 55:99–100CrossRefGoogle Scholar
  23. Nicolet Y, Piras C, Legrand P, Hatchikian CE, Fontecilla-Camps JC (1999) Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Structure 7:13–23PubMedGoogle Scholar
  24. Patil DS, Moura JJG, He SH, Teixeira M, Pickril BC, DerVartanian DV, Peck HD, Le Gall J, Huynh BH (1988) EPR detectable redox centers of the periplasmic hydrogenase from Desulfovibrio vulgaris. J Biol Chem 263:18732–18738PubMedGoogle Scholar
  25. Payne RB, Gentry DM, Rapp-Giles BJ, Casalot L, Wall JD (2002) Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c 3 mutant. Appl Environ Microbiol 68:3129–3132CrossRefPubMedGoogle Scholar
  26. Pohorelic BK, Voordouw JK, Lojou E, Dolla A, Harder J, Voordouw G (2002) Effect of deletion of genes encoding Fe-only hydrogenase of Desulfovibrio vulgaris Hildenborough on hydrogen and lactate metabolism. J Bacteriol 184:679–686PubMedGoogle Scholar
  27. Rousset M, Montet Y, Guigliarelli B, Forget N, Asso M, Bertrand P, Fontecilla-Camps JC, Hatchikian C (1998) [3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [Ni-Fe] hydrogenase by site-directed mutagenesis. Proc Natl Acad Sci USA 95:11625–11630PubMedGoogle Scholar
  28. Vignais PM, Billoud B, Meyer J (2001) Classifications and phylogeny of hydrogenases. FEMS Microbiol Rev 25:455–501PubMedGoogle Scholar
  29. Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, Fontecilla-Camps JC (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373:580–587PubMedGoogle Scholar
  30. White C, Gadd GM (1998) Reduction of metal cations and oxyanions by anaerobic and metal-resistant microorganisms: chemistry, physiology and potential for the control and bioremediation of toxic metal pollution. In: Horikoshi K, Grant WD (eds) Extremophiles: microbial life in extreme environments. Wiley, Chichester, pp 233–254Google Scholar
  31. White C, Sherman AK, Gadd GM (1998) An integrated microbial process for the bioremediation of soil contaminated with toxic metals. Nat Biotechnol 16:572–575PubMedGoogle Scholar
  32. Wildung RE, Gorby YA, Krupke KM, Hess NJ, Li SW, Plymale AE, McKinley JP, Frederikson JK (2000) Effect of electron donor and solution chemistry on produces of dissimilatory reduction of technetium by Schewanella putrefaciens. Appl Environ Microbiol 66:2451–2460CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • B. Chardin
    • 1
  • M.-T. Giudici-Orticoni
    • 1
  • G. De Luca
    • 2
  • B. Guigliarelli
    • 1
  • M. Bruschi
    • 1
    Email author
  1. 1.Unité de Bioénergétique et Ingénierie des Protéines, UPR 9036CNRS-IBSMMarseille cedex 20France
  2. 2.Laboratoire d'écologie microbienne de la rhizosphère, LEMiRCNRS-CEA DEVM DSVSt Paul le Durance CedexFrance

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