How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >1011 -fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a "shutter" that cytoplasmic factors might open or close to regulate iron release in vivo.Ferritins are vesicle-like assemblies of 24 polypeptide (4-helix bundle) subunits that concentrate iron in cells by directing the formation of a ferric mineral in the hollow protein interior (8 nm diameter) (1-3). Effective cellular iron concentrations Ͼ1011 times the solubility of the ferric ion are achieved by ferritins, which are found in microorganisms, plants, and animals. The complexity and the sophistication of the genetic regulation of the ferritins, involving both DNA and mRNA (4 -7), emphasize the central role of iron and ferritin in life. Rates of Fe(II) oxidation, translocation of Fe(II) and Fe(III) (1.0 -2.0 nm), and mineralization are all controlled by the protein (1, 2). Fe(II) release from ferritin following reduction of the mineral is slow and poorly understood (8, 9) but is important for the biosynthesis of iron-proteins, such as those required in respiration, photosynthesis, nitrogen fixation, and cell division, (1, 2) and as dietary iron (10). How and where the iron exits from ferritin in vivo is not known.We now show that localized unfolding in the assembled protein, at sites of cooperative subunit interactions, can increase the rate of exit of iron from ferritin. When conserved leucine 134 was replaced by proline (L134P), the protein assembled, oxidized Fe(II), and mineralized Fe(III), but the time for complete dissolution of mineral (480 iron) in vitro was greatly decreased (5 min compared with 150 min for the parent protein). X-ray diffraction studies of crystals of H-L134P ferritin showed a flexible region localized near the termini of two subunit helices (C, D), which form the interfaces of subunit trimers and a channel. The results indicate that iron can exit from ferritin at the trimer subunit junction. A possible mechanism for regulated iron release in vivo could be localized disorder in the assembled protein, enhanced by cytoplasmic changes with effects analogous to the effect of H-L134P. EXPERIMENTAL PROCEDURES Expression and Purification of Recombinant Ferritin Proteins-The coding sequence for H ferritin, H-L134P, K82Q, and H-L134P, R86Qwere obtained by the mutagenesis of PJD5F12L134P sequence (12) with a Chameleon TM double-stranded, site-directed mutagenesis kit (Stratagene). The oligonucleotides, 5F12P134L (5Ј-CACCTGTTCCTC-CAGGTATTCAGTCTCC-3Ј), 5F12K82Q (5Ј-CGCTCTGGTTTCTGGA-CATCCTGCAG-3Ј), and 5F12R86Q (5Ј-CCCCATTCATCCTGCTCTG-GTTTCTTGACATCC-3Ј), were used as the...

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.

The crystal structure of human ornithine transcarbamoylase complexed with the bisubstrate analog Nphosphonacetyl-L-ornithine has been solved at 1.85-Å resolution by molecular replacement. Deleterious mutations produce clinical hyperammonia that, if untreated, results in neurological symptoms or death (ornithine transcarbamylase deficiency). The holoenzyme is trimeric, and as in other transcarbamoylases, each subunit contains an N-terminal domain that binds carbamoyl phosphate and a C-terminal domain that binds L-ornithine. The active site is located in the cleft between domains and contains additional residues from an adjacent subunit. Binding of N-phosphonacetyl-L-ornithine promotes domain closure. The resolution of the structure enables the role of active site residues in the catalytic mechanism to be critically examined. The side chain of Cys-303 is positioned so as to be able to interact with the ␦-amino group of L-ornithine which attacks the carbonyl carbon of carbamoyl phosphate in the enzymecatalyzed reaction. This sulfhydryl group forms a charge relay system with Asp-263 and the ␣-amino group of L-ornithine, instead of with His-302 and Glu-310, as previously proposed. In common with other ureotelic ornithine transcarbamoylases, the human enzyme lacks a loop of ϳ20 residues between helix H10 and ␤-strand B10 which is present in prokaryotic ornithine transcarbamoylases but has a C-terminal extension of 10 residues that interacts with the body of the protein but is exposed. The sequence of this C-terminal extension is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, suggesting a possible mode of interaction with the inner mitochondrial membrane.

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