@article{theil_takagi_small_he_tipton_danger_2000, title={The ferritin iron entry and exit problem}, volume={297}, ISSN={["1873-3255"]}, DOI={10.1016/S0020-1693(99)00375-8}, abstractNote={The entry and exit of Fe(II) ions in ferritin are endpoints in the process which concentrates iron as a solid (hydrated ferric oxide) to be used by living cells. Ferritin is a response to the trillion fold mismatch between the solubility of iron in neutral, aqueous, aerated solutions and the requirements for protein biosynthesis. The supramolecular structure of 24 polypeptides (subunits) joined by non-covalent bonds in a highly symmetrical (4, 3, 2), large (12 nm diameter) protein with a cavity (8 nm diameter) is found in plants, animals and microorganisms. Of the five types of iron sites which can be defined, iron entry (site 1) is likely at the junction of three subunits. A ferroxidase site (site 2), present in H-type ferritins, binds Fe(II) which reacts with dioxygen to form an initial, μ-1,2 diferric peroxo complex. The peroxo complex decays into hydrogen peroxide and the multiple diferric oxo complexes that are mineral precursors (sites 3a, 3b, 3x) in transit across the protein to the cavity; the ferroxidase site is sensitive to both natural and engineered variations in Fe ligands and ‘second shell amino’ acids such as tyrosine 30 and leucine 134. Studies of ferritin with subunits which lack the ferroxidase site (L-type) show that the mineral anchor sites of clustered R-COOH (site 4) are sensitive to RCH3 and RSH replacements. Iron exit (site 5), studied by adding NADH/FMN as a trigger, is at or near the entry site. Iron exit can be enhanced by localized unfolding of the polypeptide at the junction of three subunits, suggesting a regulation-sensitive biological signal for iron exit. The entry and exit of iron to and from the mineral, unique to ferritin has steps which parallel those in channel ion transport, and ion transport to and from biomineral such as tooth and bone. Iron entry involves oxidation at non-heme iron catalytic centers, similar to ribonucleotide reductase and methane monoxygenase, but diverging in the role of iron as a substrate rather than a cofactor. Sorting out the evolutionary and mechanistic relationships of ferritin and other proteins which use metal ions, as well as fully characterizing all five types of the functional iron sites are challenges which will keep biological inorganic chemists occupied for some time to come.}, number={1-2}, journal={INORGANICA CHIMICA ACTA}, author={Theil, EC and Takagi, H and Small, GW and He, L and Tipton, AR and Danger, D}, year={2000}, month={Jan}, pages={242–251} }
 @article{pereira_tavares_lloyd_danger_edmondson_theil_huynh_1997, title={Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization}, volume={36}, ISSN={["0006-2960"]}, DOI={10.1021/bi970348f}, abstractNote={Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.}, number={25}, journal={BIOCHEMISTRY}, author={Pereira, AS and Tavares, P and Lloyd, SG and Danger, D and Edmondson, DE and Theil, EC and Huynh, BH}, year={1997}, month={Jun}, pages={7917–7927} }
 @article{fetter_cohen_danger_sanders_theil_1997, title={The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation}, volume={2}, DOI={10.1007/s007750050180}, abstractNote={Ferritins uniquely direct the vectorial transfer of hydrated Fe(II)/Fe(III) ions to a condensed ferric phase in the central cavity of the soluble protein. Secondary, tertiary and quaternary structure are conserved in ferritin, but only five amino acid residues are conserved among all known ferritins. The sensitivity of ferroxidation rates to small differences in primary sequence between ferritin subunits that are cell-specifically expressed or to the conservative replacement of the conserved tyrosine 30 residue was demonstrated by examining recombinant (frog) H-type (red blood cell predominant) and M-type subunit (liver predominant) proteins which are both fast ferritins; the proteins form two differently colored Fe(III)-protein complexes absorbing at 550 nm or 650 nm, respectively. The complexes are convenient reporters of Fe(III)-protein interaction because they are transient in contrast to the Fe(III)-oxy complexes measured in the past at 310–420 nm, which are stable because of contributions from the mineral itself. The A650-nm species formed 18-fold faster in the M-subunit protein than did the 550-nm species in H-subunit ferritin, even though all the ferroxidase residues are the same; the Vmax was fivefold faster but the Hill coefficents were identical (1.6), suggesting similar mechanisms. In H-subunit ferritin, substitution of phenylalanine for conserved tyrosine 30 (located in the core of the subunit four-helix bundle) slowed ferroxidation tenfold, whereas changing surface tyrosine 25 or tyrosine 28 had no effect. The Fe(III)-tyrosinate was fortunately not changed by the mutation, based on the resonance Raman spectrum, and remained a suitable reporter for Fe(III)-protein interactions. Thus, the A550/650 nm can also report on post-oxidation events such as transport through the protein. The impact of Y30F on rates of formation of Fe(III)-protein complexes in ferritin, combined with Mössbauer spectroscopic studies that showed the parallel formation of multiple Fe(III) postoxidation species (three dinuclear oxy and one trinuclear oxy species) (A. S. Periera et al., Biochemistry 36 : 7917–7927, 1997) and the loss of several of the multimeric Fe(III) post-oxidation species in a Y30F alteration of human recombinant H-ferritin (E. R. Bauminger et al., Biochem J. 296 : 709–719, 1993), indicate that at least one of the pathways for Fe oxidation/transfer in ferritin is through the center of the four-helix bundle and is influenced by structural features dependent on tyrosine 30.}, number={5}, journal={Journal of Biological Inorganic Chemistry}, author={Fetter, J. and Cohen, J. and Danger, D. P. and Sanders, Loehr J. and Theil, E. C.}, year={1997}, pages={652–661} }