JBIC Journal of Biological Inorganic Chemistry

, Volume 17, Issue 8, pp 1231–1239 | Cite as

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities

  • Han Fang
  • Jonathan D. Caranto
  • Rosalinda Mendoza
  • Alexander B. Taylor
  • P. John Hart
  • Donald M. KurtzJr.Email author
Original Paper


Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O2R). While most FDPs show similar structural, spectroscopic, and redox properties, O2R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O2R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and −A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O2R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O2 were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O2R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O2R and NOR mechanisms of FDPs.


Non-heme iron Nitric oxide Dioxygen X-ray crystallography Enzyme kinetics Site-directed mutagenesis 



Flavo-diiron protein


FMN-diferric FDP


FMNH2-diferrous FDP


Flavin mononucleotide


FMN hydroquinone


Nitric oxide reductase


Dioxygen reductase


NADH:rubredoxin oxidoreductase




Rubredoxin:oxygen oxidoreductase


Protein Data Bank


Thermotoga maritima


3-(N-morpholino)propanesulfonic acid



This work was supported by grants from National Institute of Health (RO1 GM040388 to D.M.K. Jr.) and the Robert A. Welch Foundation (AQ-1399 to P. J. H.). Support for the X-ray Crystallography Core Laboratory by the University of Texas Health Sciences Center at San Antonio Executive Research Committee and the Cancer Therapy & Research Center is gratefully acknowledged. We thank Professor Heather Shipley for access to and assistance with the inductively coupled plasma mass spectrometer.

Supplementary material

775_2012_938_MOESM1_ESM.pdf (6.7 mb)
Supplementary material 1 (PDF 6905 kb)


  1. 1.
    Kurtz DM Jr (2007) Dalton Trans 37:4115–4121Google Scholar
  2. 2.
    Saraiva LM, Vicente JB, Teixeira M (2004) Adv Microb Physiol 49:77–129PubMedCrossRefGoogle Scholar
  3. 3.
    Smutna T, Goncalves VL, Saraiva LM, Tachezy J, Teixeira M, Hrdy I (2009) Eukaryot Cell 8:47–55PubMedCrossRefGoogle Scholar
  4. 4.
    Hayashi T, Caranto JD, Wampler DA, Kurtz DM Jr, Moenne-Loccoz P (2010) Biochemistry 49:7040–7049PubMedCrossRefGoogle Scholar
  5. 5.
    Seedorf H, Dreisbach A, Hedderich R, Shima S, Thauer RK (2004) Arch Microbiol 182:126–137PubMedCrossRefGoogle Scholar
  6. 6.
    Vicente JB, Carrondo MA, Teixeira M, Frazao C (2008) Methods Enzymol 437:3–19PubMedCrossRefGoogle Scholar
  7. 7.
    Seedorf H, Hagemeier CH, Shima S, Thauer RK, Warkentin E, Ermler U (2007) FEBS J 274:1588–1599PubMedCrossRefGoogle Scholar
  8. 8.
    Di Matteo A, Scandurra FM, Testa F, Forte E, Sarti P, Brunori M, Giuffre A (2008) J Biol Chem 283:4061–4068PubMedCrossRefGoogle Scholar
  9. 9.
    Vu V, Makris TM, Lipscomb JD, Que L (2011) J Am Chem Soc 133:6938–6941PubMedCrossRefGoogle Scholar
  10. 10.
    Frazão C, Silva G, Gomes CM, Matias P, Coelho R, Sieker L, Macedo S, Liu MY, Oliveira S, Teixeira M, Xavier AV, Rodrigues-Pousada C, Carrondo MA, Le Gall J (2000) Nature Struct Biol 7:1041–1045PubMedCrossRefGoogle Scholar
  11. 11.
    Gardner AM, Helmick RA, Gardner PR (2002) J Biol Chem 277:8172–8177PubMedCrossRefGoogle Scholar
  12. 12.
    Kern M, Volz J, Simon J (2011) Environ Microbiol 13:2478–2494PubMedCrossRefGoogle Scholar
  13. 13.
    Baptista JM, Justino MC, Melo AM, Teixeira M, Saraiva LM (2012) J Bacteriol 194:3611–3617PubMedCrossRefGoogle Scholar
  14. 14.
    Hillmann F, Riebe O, Fischer RJ, Mot A, Caranto JD, Kurtz DM Jr, Bahl H (2009) FEBS Lett 583:241–245PubMedCrossRefGoogle Scholar
  15. 15.
    Li H, Jubelirer S, Costas AMG, Frigaard NU, Bryant DA (2009) Arch Microbiol 191:853–867PubMedCrossRefGoogle Scholar
  16. 16.
    Karr EA (2010) J Bacteriol 192:5914–5922PubMedCrossRefGoogle Scholar
  17. 17.
    Le Fourn C, Brasseur G, Brochier-Armanet C, Pieulle L, Brioukhanov A, Ollivier B, Dolla A (2011) Environ Microbiol 13:2132–2145PubMedCrossRefGoogle Scholar
  18. 18.
    Allahverdiyeva Y, Ermakova M, Eisenhut M, Zhang PP, Richaud P, Hagemann M, Cournac L, Aro EM (2011) J Biol Chem 286:24007–24014PubMedCrossRefGoogle Scholar
  19. 19.
    Wildschut JD, Lang RM, Voordouw JK, Voordouw G (2006) J Bacteriol 188:6253–6260PubMedCrossRefGoogle Scholar
  20. 20.
    Rodrigues R, Vicente JB, Felix R, Oliveira S, Teixeira M, Rodrigues-Pousada C (2006) J Bacteriol 188:2745–2751PubMedCrossRefGoogle Scholar
  21. 21.
    Silaghi-Dumitrescu R, Coulter ED, Das A, Ljungdahl LG, Jameson GN, Huynh BH, Kurtz DM Jr (2003) Biochemistry 42:2806–2815PubMedCrossRefGoogle Scholar
  22. 22.
    Silaghi-Dumitrescu R, Ng KY, Viswanathan R, Kurtz DM Jr (2005) Biochemistry 44:3572–3579PubMedCrossRefGoogle Scholar
  23. 23.
    Vicente JB, Testa F, Mastronicola D, Forte E, Sarti P, Teixeira M, Giuffre A (2009) Arch Biochem Biophys 488:9–13PubMedCrossRefGoogle Scholar
  24. 24.
    Silaghi-Dumitrescu R, Kurtz DM Jr, Ljungdahl LG, Lanzilotta WN (2005) Biochemistry 44:6492–6501PubMedCrossRefGoogle Scholar
  25. 25.
    Le Fourn C, Fardeau ML, Ollivier B, Lojou E, Dolla A (2008) Environ Microbiol 10:1877–1887PubMedCrossRefGoogle Scholar
  26. 26.
    Blomberg LM, Blomberg MRA, Siegbahn PEM (2007) J Biol Inorg Chem 12:79–89PubMedCrossRefGoogle Scholar
  27. 27.
    Victor BL, Baptista AM, Soares CM (2009) J Biol Inorg Chem 14:853–862PubMedCrossRefGoogle Scholar
  28. 28.
    Stookey LL (1970) Anal Chem 42:779–781CrossRefGoogle Scholar
  29. 29.
    Otwinowski Z, Minor W (1997) Methods Enzymol 276:307–326CrossRefGoogle Scholar
  30. 30.
    Mccoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) J Appl Crystallogr 40:658–674PubMedCrossRefGoogle Scholar
  31. 31.
    Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) Acta Crystallogr D 66:213–221PubMedCrossRefGoogle Scholar
  32. 32.
    Emsley P, Cowtan K (2004) Acta Crystallogr D 60:2126–2132PubMedCrossRefGoogle Scholar
  33. 33.
    Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Crystallogr 26:283–291Google Scholar
  34. 34.
    Iyer RB, Silaghi-Dumitrescu R, Kurtz DM Jr, Lanzilotta WN (2005) J Biol Inorg Chem 10:407–416PubMedCrossRefGoogle Scholar
  35. 35.
    Vicente JB, Scandurra FM, Forte E, Brunori M, Sarti P, Teixeira M, Giuffre A (2008) Methods Enzymol 437:47–62PubMedCrossRefGoogle Scholar
  36. 36.
    Hayashi T, Caranto JD, Matsumura H, Kurtz DM Jr, Moënne-Loccoz PJ (2012) J Am Chem Soc 134:6878–6884PubMedCrossRefGoogle Scholar

Copyright information

© SBIC 2012

Authors and Affiliations

  • Han Fang
    • 1
  • Jonathan D. Caranto
    • 1
  • Rosalinda Mendoza
    • 1
  • Alexander B. Taylor
    • 2
  • P. John Hart
    • 2
    • 3
  • Donald M. KurtzJr.
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of Texas at San AntonioSan AntonioUSA
  2. 2.Department of BiochemistryUniversity of Texas Health Science CenterSan AntonioUSA
  3. 3.Department of Veterans Affairs, Geriatric Research, Education, and Clinical CenterSouth Texas Veterans Health Care SystemSan AntonioUSA

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