Skip to main content
SpringerLink
Log in

Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella

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

Abstract

Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDHR55M) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053–2063, 2009). For SDHR55M only the high-pH form is observed, and samples prepared in H2 17O-enriched buffer show essentially the same 17O hyperfine interaction and nuclear quadrupole interaction parameters as SDHWT enzyme. However, the pH dependence of the EPR spectra of SDHR55Q, in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDHWT. For SDHR55Q the blocked form with bound sulfate is generated at low pH, as verified by 33S couplings observed upon reduction with 33S-labeled sulfite. This observation of bound sulfate for SDHR55Q supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156–2163, 2009). At pH ≥ 8 the high-pH form dominates for SDHR55Q.

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
Scheme 1
Fig. 7

Similar content being viewed by others

References

  1. Feng C, Tollin G, Enemark JH (2007) Biochim Biophys Acta 1774:527–539

    CAS  PubMed  Google Scholar 

  2. Rajagopalan KV (1980) In: Coughlan MP (ed) Molybdenum and molybdenum-containing enzymes. Pergamon Press, New York, pp 242–272

    Google Scholar 

  3. Enemark JH, Cosper MM (2002) Met Ions Biol Syst 39:621–654

    CAS  PubMed  Google Scholar 

  4. Kappler U, Friedrich CG, Truper HG, Dahl C (2001) Arch Microbiol 175:102–111

    Article  CAS  PubMed  Google Scholar 

  5. Kelly DP, McDonald IR, Wood AP (2000) Int J Syst Evol Microbiol 50:1797–1802

    CAS  PubMed  Google Scholar 

  6. Kisker C, Schindelin H, Pacheco A, Wehbi WA, Garrett RM, Rajagopalan KV, Enemark JH, Rees DC (1997) Cell 91:973–983

    Article  CAS  PubMed  Google Scholar 

  7. Kappler U, Bailey S (2005) J Biol Chem 280:24999–25007

    Article  CAS  PubMed  Google Scholar 

  8. Schrader N, Fischer K, Theis K, Mendel RR, Schwarz G, Kisker C (2003) Structure 11:1251–1263

    Article  CAS  PubMed  Google Scholar 

  9. George GN, Pickering IJ, Kisker C (1999) Inorg Chem 38:2539–2540

    Article  CAS  Google Scholar 

  10. Hille R (1996) Chem Rev 96:2757–2816

    Article  CAS  PubMed  Google Scholar 

  11. Bailey S, Rapson T, Johnson-Winters K, Astashkin AV, Enemark JH, Kappler U (2009) J Biol Chem 284:2053–2063

    Article  CAS  PubMed  Google Scholar 

  12. Emesh SH, Rapson TD, Rajapakshe A, Kappler U, Bernhardt PV, Tollin G, Enemark JH (2009) Biochemistry 48:2156–2163

    Article  CAS  PubMed  Google Scholar 

  13. Enemark JH, Astashkin AV, Raitsimring AM (2006) Dalton Trans 3501–3514

  14. Raitsimring AM, Pacheco A, Enemark JH (1998) J Am Chem Soc 120:11263–11278

    Article  CAS  Google Scholar 

  15. Lamy MT, Gutteridge S, Bray RC (1980) Biochem J 185:397–403

    CAS  PubMed  Google Scholar 

  16. Bray RC, Gutteridge S, Lamy MT, Wilkinson T (1983) Biochem J 211:227–236

    CAS  PubMed  Google Scholar 

  17. Astashkin AV, Raitsimring AM, Feng C, Johnson JL, Rajagopalan KV, Enemark JH (2002) J Am Chem Soc 124:6109–6118

    Article  CAS  PubMed  Google Scholar 

  18. Astashkin AV, Mader ML, Pacheco A, Enemark JH, Raitsimring AM (2000) J Am Chem Soc 122:5294–5302

    Article  CAS  Google Scholar 

  19. Astashkin AV, Hood BL, Feng C, Hille R, Mendel RR, Raitsimring AM, Enemark JH (2005) Biochemistry 44:13274–13281

    Article  CAS  PubMed  Google Scholar 

  20. Raitsimring AM, Kappler U, Feng C, Astashkin AV, Enemark JH (2005) Inorg Chem 44:7283–7285

    Article  CAS  PubMed  Google Scholar 

  21. Kappler U, Bennett B, Rethmeier J, Schwarz G, Deutzmann R, McEwan AG, Dahl C (2000) J Biol Chem 275:13202–13212

    Article  CAS  PubMed  Google Scholar 

  22. Kappler U, Bailey S, Feng CJ, Honeychurch MJ, Hanson GR, Bernhardt PV, Tollin G, Enemark JH (2006) Biochemistry 45:9696–9705

    Article  CAS  PubMed  Google Scholar 

  23. Raitsimring AM, Astashkin AV, Feng C, Wilson HL, Rajagopalan KV, Enemark JH (2008) Inorg Chim Acta 361:941–946

    Article  CAS  Google Scholar 

  24. Gutteridge S, Lamy MT, Bray RC (1980) Biochem J 191:285–288

    CAS  PubMed  Google Scholar 

  25. Pacheco A, Basu P, Borbat P, Raitsimring AM, Enemark JH (1996) Inorg Chem 35:7001–7008

    Article  CAS  PubMed  Google Scholar 

  26. George GN, Garrett RM, Graf T, Prince RC, Rajagopalan KV (1998) J Am Chem Soc 120:4522–4523

    Article  CAS  Google Scholar 

  27. Astashkin AV, Johnson-Winters K, Klein EL, Feng C, Wilson HL, Rajagopalan KV, Raitsimring AM, Enemark JH (2008) J Am Chem Soc 130:8471–8480

    Article  CAS  PubMed  Google Scholar 

  28. Doonan C, Wilson HL, Rajagopalan KV, Garrett RM, Bennett B, Prince RC, George GN (2007) J Am Chem Soc 129:9421–9428

    Article  CAS  PubMed  Google Scholar 

  29. Kappler U, McEwan AG (2002) FEBS Lett 529:208–214

    Article  CAS  PubMed  Google Scholar 

  30. Astashkin AV, Johnson-Winters K, Klein EL, Byrne RS, Hille R, Raitsimring AM, Enemark JH (2007) J Am Chem Soc 129:14800–14810

    Article  CAS  PubMed  Google Scholar 

  31. Astashkin AV, Enemark JH, Raitsimring AM (2006) Concepts Magn Reson Part B Magn Reson Eng 29:125–136

    Article  Google Scholar 

  32. Dikanov SA, Tsvetkov YD (1992) Electron spin echo envelope modulation. CRC Press, Boca Raton

    Google Scholar 

  33. Van Doorslaer S, Schweiger A (1997) Chem Phys Lett 281:297–305

    Article  Google Scholar 

  34. Astashkin AV, Raitsimring AM (2000) J Magn Reson 143:280–291

    Article  CAS  PubMed  Google Scholar 

  35. Astashkin AV, Raitsimring AM (2001) J Magn Reson 148:379–387

    Article  CAS  PubMed  Google Scholar 

  36. Astashkin AV, Feng C, Raitsimring AM, Enemark JH (2005) J Am Chem Soc 127:502–503

    Article  CAS  PubMed  Google Scholar 

  37. Astashkin AV, Klein EL, Ganyushin D, Johnson-Winters K, Neese F, Kappler U, Enemark JH (2009) Phys Chem Chem Phys 11:6733–6742

    Article  CAS  PubMed  Google Scholar 

  38. Astashkin AV, Neese F, Raitsimring AM, Cooney JJA, Bultman E, Enemark JH (2005) J Am Chem Soc 127:16713–16722

    Article  CAS  PubMed  Google Scholar 

  39. Astashkin AV, Kawamori A, Kodera Y, Kuroiwa S, Akabori K (1995) J Chem Phys 102:5583–5592

    Article  CAS  Google Scholar 

  40. Doan PE, Hoffman BM (1997) Chem Phys Lett 269:208–214

    Article  CAS  Google Scholar 

  41. Enemark JH, Astashkin AV, Raitsimring AM (2008) Biochem Soc Trans Transition Met Biochem 36:1129–1133

    Article  CAS  Google Scholar 

  42. Kappler U, Bailey S (2004) Acta Crystallogr D Biol Crystallogr 60:2070–2072

    Article  PubMed  Google Scholar 

  43. Feng C, Kappler U, Tollin G, Enemark JH (2003) J Am Chem Soc 125:14696–14697

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge support of this research by the NIH (GM-37773 to J.H.E.) and by grants from the NSF (DBI-0139459, DBI-9604939, BIR-9224431) and the NIH (S10RR020959) for development of the pulsed EPR facility. K. J.-W. thanks the NIH for a Ruth L. Kirschstein National Service Award. We thank Eric L. Klein for preparing the sample of 33S-labeled sulfite. T.D.R. is the recipient of an International Postgraduate Research Scholarship (IPRS) and a UQ Graduate School Research Travel Grant from the University of Queensland. Support of the Australian Research Council (ARC Grant DP0878525 to U.K.) is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, 4072, Australia

    Trevor D. Rapson, Paul V. Bernhardt & Ulrike Kappler

  2. Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721-0041, USA

    Andrei V. Astashkin, Kayunta Johnson-Winters, Arnold M. Raitsimring & John H. Enemark

Authors
  1. Trevor D. Rapson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrei V. Astashkin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kayunta Johnson-Winters
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paul V. Bernhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ulrike Kappler
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arnold M. Raitsimring
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John H. Enemark
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ulrike Kappler or John H. Enemark.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 276 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rapson, T.D., Astashkin, A.V., Johnson-Winters, K. et al. Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella . J Biol Inorg Chem 15, 505–514 (2010). https://doi.org/10.1007/s00775-009-0619-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-009-0619-0

Keywords

Search

Navigation