Abstract
The recombinant Chlorobium tepidum ferritin (rCtFtn) is able to oxidize iron using ferroxidase activity but its ferroxidase activity is intermediate between the H-chain human ferritin and the L-chain human ferritin. The rCtFtn has an unusual C-terminal region composed of 12 histidine residues, as well as aspartate and glutamate residues. These residues act as potential metal ion ligands, and the rCtFtn homology model predicts that this region projects inside the protein cage. The rCtFtn also lacks a conserved Tyr residue in position 19. In order to know if those differences are responsible for the altered ferroxidase properties of rCtFtn, we introduced by site-directed mutagenesis a stop codon at position 166 and a Tyr residue replaced Ala19 in the gene of rCtFtn (rCtFtn 166). The rCtFtn166 keeps the canonical sequence considered important for the activity of this family of proteins. Therefore, we expected that rCtFtn 166 would possess similar properties to those described for this protein family. The rCtFtn 166 is able to bind, oxidize and store iron; and its activity is inhibit by Zn(II) as was described for other ferritins. However, the rCtFtn 166 possesses a decrease ferroxidase activity and protein stability compared with the wild type rCtFtn. The analysis of the Ala19Tyr rCtFtn shows that this change does not affect the kinetic of iron oxidation. Therefore, these results indicate that the C-terminal regions have an important role in the activity of the ferroxidase center and the stability of rCtFtn.
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Abbreviations
- Bfr:
-
Bacterioferritin
- FC:
-
Ferroxidase center
- GdnHCl:
-
Guanidine hydrochloride
- IPTG:
-
Isopropyl-β-d-thiogalactoside
- MOPS:
-
3-(N-morpholino)propane sulfonic acid
- PMSF:
-
Phenylmethane sulfonyl fluoride
- TEM:
-
Transmission electron microscopy
- WT:
-
Wild type
- YF :
-
Maximum fluorescence intensity changes emission
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Acknowledgments
This work was supported by Conicyt: Proyecto Inserción a la Academia 79090038 and Proyecto Anillo ACT 1107.
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Brito, C., Matias, C., González-Nilo, F.D. et al. The C-Terminal Regions Have an Important Role in the Activity of the Ferroxidase Center and the Stability of Chlorobium tepidum Ferritin. Protein J 33, 211–220 (2014). https://doi.org/10.1007/s10930-014-9552-3
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DOI: https://doi.org/10.1007/s10930-014-9552-3