Abstract
The iron(III) mineral cores of bacterioferritins (BFRs), as isolated, contain a significant component of phosphate, with an iron-to-phosphate ratio approaching 1:1 in some cases. In order to better understand the in vivo core-formation process, the effect of phosphate on in vitro core formation in Escherichia coli BFR was investigated. Iron cores reconstituted in the presence of phosphate were found to have iron-to-phosphate ratios similar to those of native cores, and possessed electron paramagnetic resonance properties characteristic of the phosphate-rich core. Phosphate did not affect the stoichiometry of the initial iron(II) oxidation reaction that takes place at the intrasubunit dinuclear iron-binding sites (phase 2 of core formation), but did increase the rate of oxidation. Phosphate had a more significant effect on subsequent core formation (the phase 3 reaction), increasing the rate up to five-fold at pH 6.5 and 25 °C. The dependence of the phase 3 rate on phosphate was complex, being greatest at low phosphate and gradually decreasing until the point of saturation at ~2 mM phosphate (for iron(II) concentrations <200 μM). Phosphate caused a significant decrease in the absorption properties of both phase 2 and phase 3 products, and the phosphate dependence of the latter mirrored the observed rate dependence, suggesting that distinct iron(III)-phosphate species are formed at different phosphate concentrations. The effect of phosphate on absorption properties enabled the observation of previously undetected events in the phase 2 to phase 3 transition period.
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Abbreviations
- BFR:
-
Bacterioferritin
- EcBFR:
-
Escherichia coli BFR
- PaBFR:
-
Pseudomonas aeruginosa BFR
- AvBFR:
-
Azotobacter vinelandii BFR
- ITPG:
-
Isopropyl β-d-thiogalactopyranoside
- MES:
-
2-(N-morpholino)-ethanesulfonic acid
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Acknowledgements
This work was supported by a grant from the BBSRC to G.R.M. and N.L.B. N.L.B. thanks the Royal Society for supporting his work on metals and metal cofactors in biology and H.A. thanks her family for financial support. The authors thank Dr. Myles Cheesman and Prof. Andrew Thomson for valuable assistance with EPR measurements.
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Aitken-Rogers, H., Singleton, C., Lewin, A. et al. Effect of phosphate on bacterioferritin-catalysed iron(II) oxidation. J Biol Inorg Chem 9, 161–170 (2004). https://doi.org/10.1007/s00775-003-0504-1
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DOI: https://doi.org/10.1007/s00775-003-0504-1