Spectroscopic Evidence for and Characterization of a Trinuclear Ferroxidase Center in Bacterial Ferritin from Desulfovibrio vulgaris Hildenborough

Abstract

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 Å in diameter. The main function of ferritin is to oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe2+ (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino-acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant were loaded with sub-stoichiometric amounts of Fe2+ and products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe2+Fe3+ species and a mononuclear Fe2+ species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) site B and C are bridged by a μ-OH group forming a diiron sub-center within the trinuclear center, and (3) this sub-center can afford both a mixed valence Fe2+Fe3+ state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

A Conserved Tyrosine in Ferritin Is a Molecular Capacitor

“…These results suggested to us that H 2 O 2 is the product of the ferroxidase reaction in HuHF and PfFtn, consistent with previous reports for HuHF20–21 and PfFtn 15b. We conclude that simultaneous oxidation of three Fe II ions in each ferroxidase center and the production of water only (reaction (2), Figure 4), as proposed for bacterial ferritin,2b, 11, 12 is not valid for PfFtn, although its ferroxidase center has a coordination environment identical to that of bacterial ferritin (Figure S1). One explanation is that as the amount of Fe II increases, H 2 O 2 produced during the ferroxidase reaction reacts with other components in the reaction mixture to produce oxygen, and, as a result, the stoichiometry decreases.…”

“…In the model for eukaryotic H and M ferritin, two Fe II ions are simultaneously oxidized in the ferroxidase center to form a blue intermediate, which subsequently decays to the Fe III products and hydrogen peroxide (Figure 1 C). 2a, 8b,10 For Escherichia coli ferritin A (EcFtnA)11 and Desulfovibrio vulgaris ferritin (DvFtn),12 a different model proposes that three Fe II ions (two in the ferroxidase center and one in the gateway site, commonly referred to as the Fe C site in bacterial and archaeal ferritin5a, 6,1 3) are simultaneously oxidized by one molecular oxygen,2b,11–1 2 and that water is produced (Figure 1 C). In this model the origin of the fourth electron necessary for the reduction of molecular oxygen to water is not specified.…”

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.…”

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

“…The amino acid sequences of five ferritins (Helicobacter pylori, HpF; Archaeoglobus fulgidus, AfFtn; Pyrococcus furiosus, PfFtn; Desulfovibrio vulgaris , DvFtn and Escherichia coli, EcFtnA) all show highly conserved C-site residues, suggesting an important function of this site (35–41,50). Indeed, the C-site of EcFtnA is not a passive player in the ferroxidase activity of the protein but rather modulates the stoichiometric and kinetic properties of the protein.…”