JBIC Journal of Biological Inorganic Chemistry

, Volume 22, Issue 7, pp 1099–1108 | Cite as

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


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.


Flavohemoglobin Giardia Resonance Raman spectroscopy Nitric oxide 



Giardia intestinalis flavohemoglobin


Flavohemoglobin of Escherichia coli


Flavohemoglobin of Ralstonia metallidurans (formally Alcaligenes eutrophus)

FeIII heme

Ferric heme

FeII heme

Ferrous heme

FeIICO complex

Carbon monoxide-bound complex of ferrous heme


Nitric oxide


Carbon monoxide


Molecular oxygen



This work was supported by NSERC Discovery Grants 250073 and 216849 to M. Couture and to S.P. Rafferty, respectively. B.L. is grateful for the support of an NSERC Postgraduate Scholarship.

Supplementary material

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


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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

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