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

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 O2 consumed per Fe(II) oxidized. These results provide a fresh understanding of the mechanism of Fe(II) oxidation by ferritins.

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Molecular BioSystems
www.rsc.org/molecularbiosystems Fe(II) distribution among three sites is different in HuHF and PfFtn. The first step in catalysis of 1 Fe(II) oxidation is binding of the Fe(II) ions to the metal ion binding sites in each subunit. As discussed in 2 the introduction three Fe(II) binding sites exist in different eukaryotic and microbial ferritins 1,6 , i.e. sites 3 A and B of the ferroxidase center and site C close to this center. We have showed previously that Fe(II) 4 distributes among these sites 4 . However, we could not determine the Fe(II) occupation of each site to 5 define the amount of different types of Fe(II)-occupied subunits under single-turnover conditions, i.e. 6 addition of circa 2 Fe(II) per ferritin subunit. This knowledge is essential for understanding the 7 mechanism of Fe(II) oxidation. To determine the Fe(II) occupation of each site before addition of 8 dioxygen we used Mössbauer spectroscopy and combined the results with knowledge of the binding 9 affinity of each site for Fe(II), which we had determined in a previous study using detailed isothermal 10 titration calorimetry experiments under anaerobic conditions 4 Table 1). Two Fe(II) ions 11 per ferritin subunit were added to apo-HuHF or apo-PfFtn under anaerobic conditions. Simulation of the 12

(Supplementary
Mössbauer spectra required a model of three distinct Fe(II) doublets ( Figure 2A and Supplementary 13 Figures 2-3). We attribute these doublets to the three individual sites, i.e. sites A, B, and C (Table 1), in 14 agreement with the observation of three sites with different coordination environments using X-ray 15 crystallography in various ferritins 1 including PfFtn 25 and HuHF 6 . These observations are inconsistent 16 with the possibility that only one or two sites might exist. Furthermore, the hypothesis that two of the 17 Fe(II) doublets might be assigned to a single site with alternative coordination ligands can also be ruled 18 out based on our Mössbauer data. The sum of the amount of any combination of two different doublets 19 exceeds the total number of site A, or B, or C present in a ferritin 24-mer. For example the second and the 20 third doublets in PfFtn together account for circa 60% of the Fe(II)-added. This means circa 29 Fe(II) per 21 ferritin 24-mer. Because there are only 24 sites A, or B, or C per ferritin 24-mer available, the second and 22 the third doublets in PfFtn cannot be assigned to the same site with alternative coordination ligands. The 23 Mössbauer parameters of the first doublet in HuHF and in PfFtn are very close (Table 1). Because the 24 Mössbauer parameters of Fe(II) in the absence of dioxygen are mainly affected by its amino acid 1 coordinating residues, the coordination environments of the Fe(II) associated with the doublet in HuHF 2 and PfFtn should be the same. The available structural data 1 shows exactly the same coordination 3 environment for site A in PfFtn and HuHF ( Figure 2B), but not for sites B and C. Consequently, we 4 attribute the first Fe(II) doublet to the Fe(II) in site A of the ferroxidase center. In PfFtn the second 5 (purple trace in figure 2A) and the third (orange trance in figure 2A) Fe(II) doublets have 40% and 19% 6 abundance respectively (Table 1). In PfFtn as we reported previously 4 the affinity of site B for Fe(II), i.e. 7 (5.5 ± 1.0) × 10 4 M -1 , is 50-fold higher than that of site C, i.e. (1.0 ± 0.3) × 10 3 M -1 (Supplementary Table  8 1). Therefore, in PfFtn the doublet with 40% abundance is attributed to site B and the doublet with 19% 9 abundance is attributed to site C. In HuHF the abundances of the second (purple trace in figure 2A) and 10 the third (orange trace in figure 2A) Fe(II) doublets are within experimental error the same (  Table 1). Thus, site A should first be occupied with Fe(II). Occupation of site A will be 22 followed by Fe(II) binding to sites B and C, possibly in a cooperative fashion. Therefore, among the 23 above seven Fe(II)-occupation scenarios four predominate ( Figure 3): (A II B II C 0 ) subunits with Fe(II)-1 occupied sites A and B but empty site C; (A II B II C II ) subunits with Fe(II)-occupied sites A, B, and C; 2 (A II B 0 C II ) subunits with Fe(II)-occupied sites A and C but empty site B; and (A II B 0 C 0 ) subunits with 3 Fe(II)-occupied site A only. To estimate the percentage of each subunit type per ferritin 24-mer using the 4 results of Mössbauer spectroscopy we define three variables: 5 in which X is the sum of the percentages of all subunit types divided by 100, Y is the sum of the 9 percentages of subunits with sites A and B occupied divided by 100, and Z is the percentages of subunits 10 with site B empty divided by 100. As we discussed above, site A is first occupied with Fe(II) and 11 subsequently sites B and C are filled. Thus, 'X' or 'Y' are a factor of the amount of Fe(II) added per 12 subunit and the percentage of Fe(II) assigned to site A or B respectively. Accordingly we may write: 13 In which "n" is the amount of Fe(II) added per ferritin 24-mer for a single turnover experiment. In our 16 experiments "n" was 50 Fe(II) per ferritin 24-mer. % Fe(II) in site A or B is the percentage of Fe(II) 17 doublet assigned to site A or B based on the results of Mössbauer spectroscopy for samples before 18 addition of dioxygen (Table 1). X and Y are calculated using equations 4 and 5, and subsequently the 19 percentage of four different Fe(II)-occupied subunit types (Figure 3) was found using the following 1 equations (see supplementary materials for details): 2 in which % Fe(II) in site B or C is obtained from the results of Mössbauer spectroscopy for samples 7 before addition of dioxygen. Using equations 6-9 we found that the percentage of (A II B II C 0 ) subunits in 8 PfFtn and HuHF is circa 52% and 42% respectively and that of (A II B II C II ) subunits in PfFtn and HuHF is 9 32% and 14% respectively ( Figure 3). The percentages of (A II B 0 C II ) and (A II B 0 C 0 ) subunits in PfFtn is 10 circa 1% each, while in HuHF they are circa 13% and 12% respectively ( Figure 3). Because in some 11 subunits, i.e. (A II B II C II ) subunits, three sits are occupied upon addition of circa 2 Fe(II) per subunit and 12 because the percentage of (A II B II C II ) subunits is more than that of (A II B 0 C 0 ) subunits, in total only circa 13 80-90% of the subunits is observed to be occupied. Moreover, it should be noted that although we could 14 not specifically assign the second and the third Fe(II) doublets in HuHF to sites B and C, because their 15 amounts are within experimental error the same the results obtained using our statistical model are valid 16 for HuHF. Our observations regarding the distribution of Fe(II) are consistent with a possible positive 17 cooperativity among subunits and among three binding sites, i.e. binding of Fe(II) to site A in one subunit 18 induces binding of Fe(II) to site A in a nearby subunit and to sites B and C. Indeed kinetics of Fe(II) 19 oxidation have shown positive cooperativity in eukaryotic and microbial ferritins due to a yet to be 20 identified mechanism 13, 27 . 21 1

The same peroxodiferric intermediate is formed in HuHF and PfFtn. An intermediate with visible 2
absorbance between 500-800 nm and centered at a different wavelength in different ferritins 1,7,19,[28][29][30] has 3 been reported during catalysis of Fe(II) oxidation. For example the progress curves of this intermediate in 4 HuHF (650 nm) and PfFtn (620 nm) are shown in figure 4A and 4B respectively. We applied freeze 5 quench EPR and Mössbauer spectroscopy to obtain molecular insight into the origin of this intermediate  and PfFtn (green and purple traces in figure 4C) was constrained to 1:1 abundance ( to the μ-1,2-peroxodiferric binding mode span over a wide range, but for the majority of cases, at least 23 one of the reported values for the ΔE Q is above 1.4 (mm/s) (Table 3). On the other hand for the cases in which the peroxo species is assigned to η 2 -O 2 binding mode a ΔE Q of less than 0.8 (mm/s) is reported. 1 Similar to the η 2 -O 2 binding mode of the peroxo, in PfFtn and HuHF one of the ΔE Q of the peroxodiferric 2 intermediate is less than 0.8 (mm/s) ( Table 3). Because EPR spectroscopy showed that two Fe(III) in the 3 ferroxidase center are antiferromagnetically coupled, we propose that the peroxodiferric intermediate in 4 HuHF and PfFtn has a μ-η1: η2 core structure. Further investigations with e.g. resonance Raman or 5 EXAFS spectroscopy may be used to corroborate this proposal. It should be noted that the Mössbauer 6 parameters we found in HuHF are different from those previously reported 42 . Previous Mössbauer studies 7 with HuHF 42 were performed at pH ≤ 6.5, a pH value at which Fe(II) binding to the site A of the 8 ferroxidase center is known to be disrupted 26 . Fe(II) binding under anaerobic conditions to sites A, B, and 9 C in HuHF has been observed by isothermal titration calorimetry 4 or X-ray crystallography 10 at pH ≥ 7. the Fe(II) doublets before addition of dioxygen ( Table 1) and those of the Fe(II)/Fe(III) doublets 0.7 s 20 after addition of dioxygen ( Table 2). As discussed above the results of Mössbauer spectroscopy before 21 addition of dioxygen revealed the amounts of different forms of Fe(II)-occupied subunits for PfFtn and 22 HuHF (Figure 3). In PfFtn and HuHF 0.7 s after addition of dioxygen the amount of Fe(III) observed as 23 the peroxodiferric intermediate was circa 84% and 58% (Table 2), which represent circa 84% of subunits 1 in PfFtn and 58% of subunits in HuHF. Comparison of these values with the percentages of (A II B II C 0 ) 2 and (A II B II C II ) subunits in figure 5 shows that they are within experimental error the same as the sum of 3 the percentages of (A II B II C 0 ) and (A II B II C II ) subunits in PfFtn (84%) and in HuHF (56%) respectively. 4 These data suggest to us that both in PfFtn and in HuHF the Fe(II) ions in sites A and B of the (A II B II C 0 ) 5 and (A II B II C II ) subunits were oxidized concurrently within 0.7 s to form the peroxodiferric intermediate, 6 but the Fe(II) ions in site C of the (A II B II C II ) subunits or sites A and C of the (A II B 0 C 0 ) and (A II B 0 C II ) 7 subunits were not oxidized rapidly ( Figure 5). Consistently, in PfFtn one Fe(II) doublet (16%) was 8 observed (Table 2) whose amount was within experimental error close to the amount of the Fe(II) doublet 9 attributed to site C (19%) under anaerobic conditions (Table 1). However, the Mössbauer parameters of 10 the Fe(II) doublet attributed to site C before (Table 1) and after (Table 2) addition of dioxygen were 11 different. The reason for this difference is not known but may suggest a change in the coordination 12 environment of site C in PfFtn upon Fe(II) oxidation in the ferroxidase center. In HuHF 0.7 s after 13 addition of dioxygen two Fe(II) doublets were observed ( Table 2). The Mössbauer parameters of the first 14 Fe(II) doublet (12%) ( Table 2) are the same as the Fe(II) doublet attributed to site A before addition of 15 dioxygen ( Table 1) (Table 2) should be the Fe(II) in site C, since this Fe(II) has not entered the 21 ferroxidase center and cannot be oxidized rapidly together with the Fe(II) in site A of the ferroxidase 22 center. In summary, the data for PfFtn and HuHF together demonstrate that only in (A II B II C 0 ) and 23 (A II B II C II ) subunits two Fe(II) are oxidized simultaneously in the ferroxidase center. In subunits in which 24 site B is not occupied, Fe(II) in site A cannot be oxidized ( Figure 5). We speculate that site B might be 1 the initial dioxygen binding site. This suggestion is in line with a previous site directed mutagenesis study 2 of HuHF in which differences between sites A and B of the ferroxidase center were observed 45 . 3 Replacement of a glutamate residue of each site resulted in a different response to Fe(II) oxidation. Based 4 on this observation it has been proposed that differences exist between sites A and B, and that site B is 5 possibly the initial dioxygen binding site 45 . 6 7 Site B tunes the kinetics of Fe(II) oxidation. Progress curves of Fe(III) formation, which are typically 8 measured between 300-350 nm, have been recorded for various ferritins using stopped-flow spectroscopy 9 6,7,17,21 . Even though previous Mössbauer data showed that when the peroxodiferric intermediate has its 10 maximum absorbance not all the Fe(II) ions are oxidized 18,20,29 , the progress curves have been interpreted 11 as formation of the peroxodiferric in each subunit as a sudden increase in the absorbance followed by  (Table 2). Under single-turnover conditions, a two-exponential equation (Equation 10) was 20 required to obtain a fit to the data using global fit analysis: 21 in which M and N are the pre-exponential amplitude factor (the absorbance of each exponential phase), 1 T 1 and T 2 are time constants, and M ∞ is the absorbance at infinite time. The values of M, N, T 1 , T 2 , and 2 M ∞ for PfFtn and HuHF are given in table 4. In PfFtn and HuHF the ratio of the M to M ∞ was circa 80% 3 and 50% respectively. This suggests that in PfFtn circa 80% and in HuHF circa 50% of the Fe(II) added 4 was rapidly oxidized in the first phase. This is consistent with the observation of circa 84% and circa 58% 5 Fe(III) as the peroxodiferric intermediate in PfFtn and in HuHF respectively (Table 2). Thus, the fast 6 phase should present the rapid formation of the peroxodiferric intermediate in the (A II B II C 0 ) and 7 (A II B II C II ) subunits and not the Fe(III) products. Moreover, the ratio of N to M ∞ in PfFtn and HuHF was 8 circa 20% and 50% respectively. These ratios represent the percentages of Fe(II) not oxidized in the first 9 phase but oxidized in the second slow phase plus a possible small change in the absorbance due to 10 conversion of the peroxodiferric intermediate to the Fe(III) products. They are close to the percentages of 11 Fe(II) observed by Mössbauer spectroscopy in PfFtn (16%) and in HuHF (37%) 0.7 s after addition of 12 dioxygen (Table 2). Therefore, the Fe(II) that was not oxidized rapidly in the first phase was oxidized at a 13 slower rate in the second phase. These data demonstrate that the kinetics of Fe(II) oxidation are defined Mössbauer parameters of these doublets were different from those of the peroxodiferric intermediate. The 22 first doublet in PfFtn and HuHF accounts for circa 42% of the Fe(III) ( Table 5), which is the same as the 23 amount of Fe(II) in site A before addition of dioxygen (Table 1). Therefore, this doublet is assigned to 1 Fe(III) in site A. The second doublet accounts for 58% Fe(III) ( Table 5), which is the same as the sum of 2 the Fe(II) in sites B and C before addition of dioxygen ( Table 1). The Mössbauer parameters of the Fe(III) 3 products in ferritin are similar to those reported for oxo or hydroxo bridged di-iron complexes 46 . This is 4 consistent with the results of EPR spectroscopy. Only circa 2-5 % of the total Fe(II) added showed up as Fe(III) stayed in site C and was observed as mononuclear Fe(III) and some moved to the internal cavity to 12 form the Fe(III)-mineral core. Further detailed low temperature high-field Mössbauer measurements are 13 required to study the nature of the mineral core in each ferritin. 14 15

Discussion. 16
Because oxidation of Fe(II) by ferritin is vital for the iron homeostasis machinery of all life forms, for 17 more than half a century, this reaction has been studied intensively using ferritins from different 18 organisms. Although the quaternary structure of ferritins is highly conserved, differences exist in the 19 amino acid residues essential for the functioning of protein. A notable variation among ferritins is in one 20 of the amino acids in the coordination environment of site B of the ferroxidase center ( Figure 2B). As a 21 consequence, studies of the kinetics of Fe(II) oxidation with various ferritins have resulted in the 22 suggestion of core differences and sometimes mutually inconsistent proposals regarding the mechanism 23 of Fe(II) oxidation in eukaryotic, bacterial, and archaeal ferritins. Some of these differences are listed 1 below and have been discussed in more detail previously 1 : (i) measurement of the amount of dioxygen 2 consumed for oxidation of two Fe(II) per ferritin subunit led to the report of differences in eukaryotic and Fe(III) products, which spontaneously move to the core 17, 18,29,42,43,49,50  (circa 60%) and some Fe(III) monomer (circa 30%), while the data obtained for HuHF 52 have been 10 interprted as formation of Fe(III) dimer as the main product (circa 70%) and some Fe(III) mineral core 11 (circa 30%). (vi) In bacteria a variant of ferritin, named bacterioferritin, is found, which has a very similar 12 structure to that of ferritin except that it has a heme group between pairs of subunits 53 with a role in iron 13 release 54 . While studies with E.coli bacterioferritin have led to the conclusion that in bacterioferritins the 14 Fe(III) mineralization process is different from that in eukaryotic and microbial ferritins and proceeds via 15 a diiron cofactor site 15, 55 , studies with a bacterioferritin isolated from Desulfovirio vulgaris 16 Hildenborough (DvHBfr) have led to the proposal of an Fe(III) mineralization mechanism that is similar 17 to the proposed Fe(III) mineralization process for vertebrate H-type ferritin 56 , the ferroxidase center is a 18 substrate site and not a stable cofactor center. Based on the data and interpretations discussed above the 19 diversity view has emerged: the mechanism of Fe(II) oxidation and storage is different among eukaryotic 20 and microbial ferritins 15,22 . For example, , in eukaryotic ferritin two Fe(II) are simultanously oxidized in 21 each ferroxidase center and in bacterial ferritin three Fe(II) are simultanously oxidized in the ferroxidase 22

center. 23
In contrast to this diversity view our recent studies using HuHF and PfFtn have led to the emergence of 1 the unifying view of a single mechanism of Fe(II) oxidation and storage by ferritins and bacterioferritins 1 . PfFtn and HuHF was the relative amount of each Fe(II)-occupied subunit type. This difference is 2 interpreted to originate from the difference in the affinity of site B in these ferritins for Fe(II) ion. 3 In the next step we analysed the Fe(II)/Fe(III) intermediates during catalysis of Fe(II) oxidation. 4 The Mössbauer parameters that we found for the peroxodiferric intermediate were compared to those 5 reported for different peroxodiferric species in other proteins and model compounds. We observed that 6 the values of the quadrupole splitting (ΔE Q ) in HuHF and PfFtn (Table 3) are not close to those assigned 7 to the μ-1,2-peroxodiferric binding mode in most of the di-iron cofactor enzymes and model compounds. those reported for the η 2 -O 2 binding mode of dioxygen to Fe(III) in model compounds (Table 3)  Raman data should be used in combination with Mössbauer data. Because of the available inconsistencies 20 in the reported Mössbauer data for BfMF (Table 3)  Comparison of the Mössbauer data before addition of dioxygen and 0.7 s after addition of dioxygen 1 revealed that only in the (A II B II C 0 ) and (A II B II C II ) subunits the Fe(II) in sites A and B could be oxidized 2 rapidly to form the peroxodiferric intermediate ( Figure 8). Thus, the rapid increase in the absorbance at 3 310 nm in HuHF and PfFtn (Figure 4) is indeed due to formation of the peroxodiferric intermediate and 4 not Fe(III) products. The slower phase of the progress curves of Fe(III) formation at 310 nm (Figure 4), 5 which occurs after 0.7 s, represents the slow oxidation of Fe(II) in site C of the (A II B II C II ) subunits and 6 that of Fe(II) in (A II B 0 C 0 ) and (A II B 0 C II ) subunits. In PfFtn less than 16% of the total Fe(II) added is 7 oxidized slowly and in HuHF circa 37% of the total Fe(II) added is oxidized slowly ( Table 2). The 8 difference in the kinetics of Fe(II) oxidation between HuHF and PfFtn originates from the amount of 9 (A II B 0 C II ), (A II B 0 C 0 ), and (A II B II C II ) subunits. In PfFtn the percentages of (A II B 0 C II ) and (A II B 0 C 0 ) 10 subunits were negligible and almost all of the Fe(II) in site C was next to fully occupied ferroxidase 11 centers, i.e. (A II B II C II ) subunits. The Fe(II) in site C of (A II B II C II ) subunits is proposed to be oxidized 12 presumably by the peroxodiferric intermediate 13 , and in this mechanism the conserved tyrosine provides a 13 fourth electron for complete reduction of molecular oxygen to water 13 . In contrast in HuHF the percentage 14 of (A II B II C II ) subunits was less than half of that in PfFtn and instead the percentage of (A II B 0 C II ) and In summary, we demonstrated that in PfFtn and HuHF a difference in the occupation of site B 4 with Fe(II) exists, but the same peroxodiferric intermediate forms upon addition of dioxygen, which 5 decays to a major Fe(III)-dimer product. While the exact molecular structure of the peroxodiferric 6 intermediate remains to be determined, the data support the proposal of unity in the biochemistry of 7 ferritins, and they provide a possible explanation for the observed differences among ferritins in the 8 reaction rates, the amount of Fe(II) oxidized per molecular oxygen, and the formation of different Fe(III) 9 products besides the major Fe(III)-dimer. We propose that because of the variation in an amino acid 10 residue of site B, variation in the affinity of this site for Fe(II) among ferritins exists. As a consequence 11 the amount of (A II B II C II ), (A II B II C 0 ), (A II B 0 C II ), and (A II B 0 C 0 ) subunits formed upon addition of Fe(II) will 12 vary. In ferritins with higher percentages of (A II B II C II ) and (A II B II C 0 ) subunits, more Fe(II) will be 13 oxidized at a fast rate via the peroxodiferric intermediate because Fe(II) in site B is required for catalysis. 14 This will result in different reaction rates as we here observed for HuHF and PfFtn. A higher percentage 15 of (A II B II C II ) subunits means more Fe(II) will be oxidized in site C together with the Fe(II) in sites A and 16 B to form two water molecules and as a result the amount of Fe(II) oxidized per dioxygen consumed will 17 be different in PfFtn and HuHF as we reported previously 13 . Moreover, differences in the relative number 18 of (A II B II C II ), (A II B II C 0 ), (A II B 0 C II ), and (A II B 0 C 0 ) subunits among ferritins can lead to formation of minor 19 Fe(III) products such as Fe(III)-monomer, Fe(III)-trimer, and Fe(III) mineral core, next to the main 20 Fe(III)-dimer product in the ferroxidase center. The validity of this proposal to other microbial and 21 eukaryotic ferritins remains to be evaluated. It is conceivable that the variation observed in the kinetics of 22 This work was supported in part by an EMBO Long Term Fellowship (2015-157) to KHE. We thank 3 Marc Strampraad for his assistance with the freeze-quench setup. During the preparation of this work, Dr. 4 Simon de Vries whose work on development of rapid freeze-quench techniques has inspired our present 5 research and who has been a long-time friend, passed away unexpectedly. We dedicate this work to his 6 memory. 7 8

Competing financial interest 9
The authors declare no competing financial interest. and HuHF before addition of dioxygen. In human H-type ferritin (HuHF) a small amount of Fe(III) (less 4 than 9%) is observed which was due to oxidation of Fe(II) before addition to ferritin. The simulation 5 results are not biased by this low amount of 'dirty' Fe(III). Both in HuHF and PfFtn three distinct Fe(II) 6 doublets are observed which are assigned to Fe(II) in sites A, B, and C. Measurements were performed at 7 80 K. (B) Coordination environment of site A is highly conserved and a residue in the coordination 8 environment of site B, which is also nearby site C, varies among ferritins. The structure shows the amino 9 acid residues in the coordination environment of the ferroxidase center of PfFtn. The amino acids that are 10 conserved among ferritins are numbered in black. An amino acid residue in the coordination environment 11 of site B, which varies among ferritins, is numbered in red. Site C is not shown for clarity. and C filled (A II B II C II subunits), subunits with sites A and C filled (A II B 0 C II subunits), and subunits with 5 site A only filled (A II B 0 C 0 subunits). The percentage of each subunit type varies between HuHF and 6 PfFtn. The major difference between PfFtn and HuHF is in the percentages of (A II B 0 C II ), (A II B 0 C 0 ), and 7 (A II B II C II ) subunits. The percentage of (A II B 0 C II ) and (A II B 0 C 0 ) subunits in PfFtn is negligible. Fe(II) per ferritin subunit and that of HuHF was recorded 60 s after addition of circa 2 Fe(II) per subunit. 4 Measurements were performed at 80 K. Fe(II) in sites A and B of the (A II B II C II ) and (A II B II C 0 ) subunits is oxidized rapidly via the peroxodiferric 7 intermediate, which presumably has a μ-η1: η2 structure. In these subunits the Fe(II) in site C is possibly 8 oxidized via the peroxodiferric intermediate in the ferroxidase center as proposed previously 13 . In 9 (A II B 0 C II ) and (A II B 0 C 0 ) subunits, whose site B is empty, Fe(II) is first rearranged to fill sites A and B. 10 The kinetic of this rearrangement process is the rate limiting step in oxidation of Fe(II) in (A II B 0 C II ) and 11 (A II B 0 C 0 ) subunits. The model shows a single turnover in the ferroxidase center after addition of Fe(II) to 12 apo-ferritin, i.e. ferritin with no Fe(III) bound, in the presence of molecular oxygen. For subsequent 13 turnovers Fe(III) present in the ferroxidase center is displaced by incoming Fe(II). 14 Tables  (1) 3.27 (1) C In HuHF at t=0 s less than 9% of the 57 Fe(II) was observed as Fe(III) (gray line in figure 2a), which we attribute to dirty Fe(III) possibly due to the presence of Fe(III) in Fe(II) solution before addition to HuHF (Supplementary Figure 1). Circa 2 Fe(II) per ferritin subunit were added to PfFtn (45 μM 24-mer) or HuHF (55 μM 24-mer). Measurements were performed under exactly the same conditions. In HuHF Fe(II) is equally distributed among sites B and C and the exact assignment of the second and the third Fe(II) doublet to sites B and C was not possible at this stage.  (1)  3 Fe(II) 16 (2) 1.20 (1) 2.77(1) C Measurements were performed under exactly the same conditions. Circa 2 Fe(II) per ferritin subunit were added. In HuHF a minor Fe(III) doublet (< 6 %) was observed ( Figure 3). The Mössbauer parameters of this doublet were different from those of dirty Fe(III) observed in sample before addition of dioxygen: δ (mm/s) = 0.38 (1) and ΔE Q (mm/s)=1.52 (1). This minor Fe(III) species might be the mononuclear Fe(III) observed by EPR spectroscopy (Supplementary  Table 2), whose origin is unknown. * The Fe(III) doublets that form the peroxodiferric intermediate. ] 3-. The bonding modes proposed for dioxygen in RNR and complexes 1, 2, 3, 4, and 6 are based on detailed spectroscopic studies. The bonding mode proposed for complex 5 is a suggestion due to the considerable difference between the Mössbauer parameters of this complex and those reported for complexes with μ-1,2-peroxo bonding mode. a For BfMF inconsistent Mössbauer parameters have been obtained from simulation of exactly the same Mössbauer spectra. Based on these inconsistent data a μ-1,2-peroxo binding mode has been proposed. b Signs unknown. * Postulated. HuHF 0.027 ± 0.001 0.031 ± 0.002 0.27 ± 0.01 12.5 ± 0.1 0.058 ± 0.001 PfFtn 0.045 ± 0.001 0.012 ± 0.001 0.03 ± 0.001 0.7 ± 0.02 0.058 ± 0.001 The kinetic parameters were obtained from a global analysis of the progress curves of Fe(III) formation in figure 6 using equation 10. The M, N, and M ∞ are dimensionless.  (1) *B, C, and mineral core Measurements were performed under exactly the same conditions. *Circa 42% of the second Fe(III) doublet in HuHF and PfFtn should be the Fe(III) in site B, because EPR spectroscopy shows negligible amount of Fe(III) monomer. From the remaining amount of the second Fe(III) doublet (circa 16%) some is possibly in site C and is observed as mononuclear Fe(III), and some forms the Fe(III) mineral core.