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

Abstract

Ferritin is a nanocage protein made of 24 subunits. Its major role is to manage intracellular concentrations of free Fe(ii) and Fe(iii) ions, which is pivotal for iron homeostasis across all domains of life. This function of the protein is regulated by a conserved di-iron catalytic center and has been the subject of extensive studies over the past 50 years. Yet, it has not been fully understood how Fe(ii) is oxidized in the di-iron catalytic center and it is not known why eukaryotic and microbial ferritins oxidize Fe(ii) with different kinetics. In an attempt to obtain a new insight into the mechanism of Fe(ii) oxidation and understand the origin of the observed differences in the catalysis of Fe(ii) oxidation among ferritins we studied and compared the mechanism of Fe(ii) oxidation in the eukaryotic human H-type ferritin (HuHF) and the archaeal ferritin from Pyrococcus furiosus (PfFtn). The results show that the spectroscopic characteristics of the intermediate of Fe(ii) oxidation and the Fe(iii)-products are the same in these two ferritins supporting the proposal of unity in the mechanism of Fe(ii) oxidation among eukaryotic and microbial ferritins. Moreover, we observed that a site in the di-iron catalytic center controls the distribution of Fe(ii) among subunits of HuHF and PfFtn differently. This observation explains the reported differences between HuHF and PfFtn in the kinetics of Fe(ii) oxidation and the amount of O consumed per Fe(ii) oxidized. These results provide a fresh understanding of the mechanism of Fe(ii) oxidation by ferritins.

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

“…1B), and conformational changes in amino acid residues of sites C and B have been observed 19. 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.…”

“…Before the addition of dioxygen to ferritin containing 48 Fe(II) per ferritin 24‐mer, the percentages of 57 Fe(II) in sites A, B, and C were 41%, 40%, and 19%, respectively (Table 1). According to the distribution model we explained previously 10 it could be concluded that in 32% of the subunits (~ 7 subunits per 24‐mer) sites A, B, and C were occupied with Fe(II) (A II B II C II subunits). After 0.7 s all the Fe(II) in sites A and B was converted to the peroxodiferric species but the Fe(II) in site C was not oxidized (Table 1).…”

“…1B) with potentially variable occupancy patterns 1, 2, 10, 13, at least two parallel pathways for catalysis of Fe(II) oxidation exist (Fig. 2A) 1, 13: a pathway for subunits in which only sites A and B are occupied with Fe(II) ions (A II B II C 0 subunits; pathway 1 in Fig.…”

“…2A). Mössbauer spectroscopy showed that in PfFtn upon the addition of 48 Fe(II) per ferritin 24‐mer ~ 80–82% of Fe(II) ions fill sites A and B, and about 18–19% of Fe(II) ions fill site C. Based on the affinity of each site for Fe(II) and an statistical model it was shown that ~ 56% of subunits is in the A II B II C 0 form and 32% is in the A II B II C II form 10. In the A II B II C 0 subunits Fe(II) is oxidized via the peroxodiferric intermediate to form the Fe(III) products and hydrogen peroxide, while in the A II B II C II subunits two Fe(II) in the ferroxidase center and an Fe(II) in site C are somehow oxidized together to form Fe(III) products and reduce a molecule of oxygen to two water molecules 13.…”

“…1D) and a high-valent Fe(IV) species has never been reported. The exact molecular structure of the peroxodiferric intermediate in ferritin is under debate [1,10]. While it was originally proposed that the dioxygen in the peroxodiferric species has a l-1,2 bonding mode [5,11], recent data obtained by X-ray crystallography [12] and M€ ossbauer spectroscopy [10] suggest a l-g 1 -g 2 bonding mode.…”