Reaction of O2 with a diiron protein generates a mixed-valent Fe2+/Fe3+ center and peroxide.

The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron-O2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron-O2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2 O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+ , was exposed to O2 or H2 O2 . We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2 O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O2 . Initially formed Fe3+ can further react with H2 O2 (producing protein bound radicals) but relaxes within seconds to an H2 O2 -unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2 O2 rather than sequester iron.

New insights into controlling radical migration pathways in heme enzymes gained from the study of a dye-decolorising peroxidase.

In heme enzymes, such as members of the dye-decolorising peroxidase (DyP) family, the formation of the highly oxidising catalytic Fe(iv)-oxo intermediates following reaction with hydrogen peroxide can lead to free radical migration (hole hopping) from the heme to form cationic tyrosine and/or tryptophan radicals. These species are highly oxidising (∼1 V vs. NHE) and under certain circumstances can catalyse the oxidation of organic substrates. Factors that govern which specific tyrosine or tryptophan the free radical migrates to in heme enzymes are not well understood, although in the case of tyrosyl radical formation the nearby proximity of a proton acceptor is a recognised facilitating factor. By using an A-type member of the DyP family (DtpAa) as an exemplar, we combine protein engineering, X-ray crystallography, hole-hopping calculations, EPR spectroscopy and kinetic modelling to provide compelling new insights into the control of radical migration pathways following reaction of the heme with hydrogen peroxide. We demonstrate that the presence of a tryptophan/tyrosine dyad motif displaying a T-shaped orientation of aromatic rings on the proximal side of the heme dominates the radical migration landscape in wild-type DtpAa and continues to do so following the rational engineering into DtpAa of a previously identified radical migration pathway in an A-type homolog on the distal side of the heme. Only on disrupting the proximal dyad, through removal of an oxygen atom, does the radical migration pathway then switch to the engineered distal pathway to form the desired tyrosyl radical. Implications for protein design and biocatalysis are discussed.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.

Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn.

Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres.