Resonance Raman studies on the flavohemoglobin of the protist Giardia intestinalis: evidence of a type I/II-peroxidase-like heme environment and roles of the active site distal residues

Original Paper

Abstract

Flavohemoglobins are microbial enzymes that counter nitrosative stress, but the details of their underlying enzymatic activities and structure–function relationships are not completely understood. These enzymes have been identified in Gram-negative bacteria, certain fungi, and the parasitic protist Giardia intestinalis (gFlHb) which, despite lacking the ability to make heme, encodes several hemeproteins. To gain knowledge about the biophysical properties of the active site of gFlHb, we used resonance Raman spectroscopy to probe the wild-type protein and variants at globin domain positions E11, E7, and B10 on the distal, ligand-binding side of the heme. The heme of gFlHb has a peroxidase-like environment resembling that of the well-characterized E. coli flavohemoglobin HMP. We provide evidence that gFlHb has two Fe–His stretching modes, a feature that also occurs in type I/II-peroxidases in which a proximal histidine with strong imidazolate character and a nearby carboxylic acid residue can exist as a tautomeric pair depending on the position of a shared proton. Characterization of the distal variants Tyr30Phe, Gln54Leu, and Leu59Ala shows that TyrB10 and GlnE7 but not LeuE11 are implicated in stabilisation of bound exogenous ligands such as CO and O2. Our work revealed that several biophysical properties of the heme active site of gFlHb are highly conserved compared to HMP and suggest that they are conserved across the flavohemoglobin family.

Keywords

Flavohemoglobin Giardia Resonance Raman spectroscopy Nitric oxide 

Abbreviations

gFlHb

Giardia intestinalis flavohemoglobin

HMP

Flavohemoglobin of Escherichia coli

FHP

Flavohemoglobin of Ralstonia metallidurans (formally Alcaligenes eutrophus)

FeIII heme

Ferric heme

FeII heme

Ferrous heme

FeIICO complex

Carbon monoxide-bound complex of ferrous heme

NO

Nitric oxide

CO

Carbon monoxide

O2

Molecular oxygen

Supplementary material

775_2017_1487_MOESM1_ESM.pdf (1.3 mb)
Supplementary material 1 (PDF 1381 kb)

References

  1. 1.
    Forrester MT, Foster MW (2012) Free Radic Biol Med 52:1620–1633CrossRefPubMedGoogle Scholar
  2. 2.
    Bonamore A, Boffi A (2008) IUBMB Life 60:19–28CrossRefPubMedGoogle Scholar
  3. 3.
    Ilari A, Bonamore A, Farina A, Johnson KA, Boffi A (2002) J Biol Chem 277:23725–23732CrossRefPubMedGoogle Scholar
  4. 4.
    El Hammi E, Warkentin E, Demmer U, Marzouki NM, Ermler U, Baciou L (2012) FEBS J 279:4565–4575CrossRefPubMedGoogle Scholar
  5. 5.
    Ermler U, Siddiqui RA, Cramm R, Friedrich B (1995) EMBO J 14:6067–6077PubMedPubMedCentralGoogle Scholar
  6. 6.
    Mukai M, Mills CE, Poole RK, Yeh SR (2001) J Biol Chem 276:7272–7277CrossRefPubMedGoogle Scholar
  7. 7.
    Egawa T, Yeh SR (2005) J Inorg Biochem 99:72–96CrossRefPubMedGoogle Scholar
  8. 8.
    Gardner AM, Martin LA, Gardner PR, Dou Y, Olson JS (2000) J Biol Chem 275:12581–12589CrossRefPubMedGoogle Scholar
  9. 9.
    Ferreiro DN, Boechi L, Estrin DA, Marti MA (2013) J Inorg Biochem 119:75–84CrossRefPubMedGoogle Scholar
  10. 10.
    Ollesch G, Kaunzinger A, Juchelka D, Schubert-Zsilavecz M, Ermler U (1999) Eur J Biochemistry FEBS 262:396–405CrossRefGoogle Scholar
  11. 11.
    Bonamore A, Farina A, Gattoni M, Schinina ME, Bellelli A, Boffi A (2003) Biochemistry 42:5792–5801CrossRefPubMedGoogle Scholar
  12. 12.
    Mastronicola D, Testa F, Forte E, Bordi E, Pucillo LP, Sarti P, Giuffre A (2010) Biochem Biophys Res Commun 399:654–658CrossRefPubMedGoogle Scholar
  13. 13.
    Rafferty S, Luu B, March RE, Yee J (2010) Biochem Biophys Res Commun 399:347–351CrossRefPubMedGoogle Scholar
  14. 14.
    Andersson JO, Sjogren AM, Davis LA, Embley TM, Roger AJ (2003) Curr Biol 13:94–104CrossRefPubMedGoogle Scholar
  15. 15.
    Rafferty SP, Dayer G (2015) Exp Parasitol 159:13–23CrossRefPubMedGoogle Scholar
  16. 16.
    Einarsson E, Ma’ayeh S, Svard SG (2016) Curr Opin Microbiol 34:47–52CrossRefPubMedGoogle Scholar
  17. 17.
    Di Genova BM, Tonelli RR (2016) Frontiers in microbiology 7:256CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lundberg JO, Weitzberg E (2013) Gut 62:616–629CrossRefPubMedGoogle Scholar
  19. 19.
    Spiro TG, Soldatov AV, Balakrishnan G (2013) Coord Chem Rev 257:511–527CrossRefPubMedGoogle Scholar
  20. 20.
    D’Angelo P, Lucarelli D, della Longa S, Benfatto M, Hazemann JL, Feis A, Smulevich G, Ilari A, Bonamore A, Boffi A (2004) Biophys J 86:3882–3892CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bonamore A, Chiancone E, Boffi A (2001) Biochim Biophys Acta 1549:174–178CrossRefPubMedGoogle Scholar
  22. 22.
    Chartier FJ, Couture M (2004) Biophys J 87:1939–1950CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lu C, Egawa T, Wainwright LM, Poole RK, Yeh SR (2007) J Biol Chem 282:13627–13636CrossRefPubMedGoogle Scholar
  24. 24.
    Shepherd M, Barynin V, Lu C, Bernhardt PV, Wu G, Yeh SR, Egawa T, Sedelnikova SE, Rice DW, Wilson JL, Poole RK (2010) J Biol Chem 285:12747–12754CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Shepherd M, Bernhardt PV, Poole RK (2011) Nitric Oxide 25:229–233CrossRefPubMedGoogle Scholar
  26. 26.
    Smulevich G, Feis A, Howes BD, Ivancich A (2010) In: Kadish KM, Smith KM, Guilard R (eds) Handbook of porphyrin science. World Scientific, HackensackGoogle Scholar
  27. 27.
    Frey AD, Kallio PT (2003) FEMS Microbiol Rev 27:525–545CrossRefPubMedGoogle Scholar
  28. 28.
    Spiro TG (1985) In: Anfinsen CB, Edsall JT, Richards FM (eds) Advances in protein chemistry. Academic Press Inc., Cambridge, pp 111–159Google Scholar
  29. 29.
    Van Wart HE, Zimmer J (1985) J Biol Chem 260:8372–8377PubMedGoogle Scholar
  30. 30.
    Mylrajan M, Valli K, Wariishi H, Gold MH, Loehr TM (1990) Biochemistry 29:9617–9623CrossRefPubMedGoogle Scholar

Copyright information

© SBIC 2017

Authors and Affiliations

  1. 1.Environmental and Life Sciences Graduate ProgramTrent UniversityPeterboroughCanada
  2. 2.Biology DepartmentTrent UniversityPeterboroughCanada
  3. 3.Department of Biochemistry, Microbiology and BioinformaticsUniversité Laval, IBIS and PROTEOQuébecCanada

Personalised recommendations