@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{ha_shi_small_theil_allewell_1999, title={Crystal structure of bullfrog M ferritin at 2.8 angstrom resolution: analysis of subunit interactions and the binuclear metal center}, volume={4}, ISSN={["1432-1327"]}, DOI={10.1007/s007750050310}, abstractNote={Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.}, number={3}, journal={JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, author={Ha, Y and Shi, DS and Small, GW and Theil, EC and Allewell, NM}, year={1999}, month={Jun}, pages={243–256} }