Functionality of the Three-Site Ferroxidase Center of Escherichia coli Bacterial Ferritin (EcFtnA)

Abstract

At least three ferritins are found in the bacterium Escherichia coli, the heme-containing bacterioferritin (EcBFR) and two non-heme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A- and B-sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C-site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA has been further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-visible, fluorescence and EPR spectroscopic measurements on the wild-type protein and site-directed variants of the A-, B- and C-sites. The data reveal that, while H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(II)/O2 ~ 3:1, most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(II)/H2O2 stoichiometry, thus suppressing hydroxyl radical formation. While the A- and B-sites are essential for rapid iron oxidation, the C-site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, our data do not support a universal mechanism for iron oxidation in all ferritins whereby the C-site acts as transit site, as recently proposed.

Spectroscopic evidence for the presence of a high‐valent Fe(IV) species in the ferroxidase reaction of an archaeal ferritin

“…Moreover, consistent with this speculation is our previous Mössbauer study of PfFtn, in which we found that the Mössbauer parameters of Fe(II) in site C before the addition of dioxygen are different than those after the addition of dioxygen (Table 1) 10. The importance of site C for efficient catalysis of Fe(II) oxidation has been reported for other ferritins namely Escherichia coli ferritin A 25, 26 and Desulfovibrio vulgaris Hildenborough ferritin 27.…”

Spectroscopic evidence for the role of a site of the di-iron catalytic center of ferritins in tuning the kinetics of Fe(ii) oxidation

“…An intermediate with visible absorbance between 500 and 800 nm and centered at a different wavelength in different ferritins 1,7,19,[28][29][30] has been reported during the catalysis of Fe(II) oxidation. For example the progress curves of this intermediate in HuHF (650 nm) and PfFtn (620 nm) are shown in Fig.…”

Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution

“…However, all cells do use inorganic bio-mineralization to maintain near constant concentrations of essential trace metals via high affinity chelators and storage proteins for times of excess. One prominent example are the ferritins, a ubiquitous class of proteins found in all domains of life that play a crucial role in iron homeostasis212131415161718. Ferritins form shells composed of 24 monomers each, creating an inner cavity in order to store iron in a hydrated amorphous form of iron oxide similar to the mineral ferrihydrite.…”