Pauline M. Harrison

Structure, function, and evolution of ferritins

The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation. The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues. Hence they probably arose from a common H-type ancestor. The recent discovery in E. coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism. Mammalian L ferritins lack ferroxidase centers and form a distinct group. Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework. The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains.

Identification of the ferroxidase centre in ferritin

Ferroxidase activity in human H‐chain ferritin has been studied with the aid of site‐directed mutagenesis. A site discovered by X‐ray crystallography has now been identified as the ferroxidase centre. This centre is present only in H‐chains and is located within the four‐helix bundle of the chain fold.

Crystal Structure of Manganese Catalase from Lactobacillus plantarum

BACKGROUND Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex. RESULTS The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase. CONCLUSIONS A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.

Ferric Oxyhydroxide Core of Ferritin

Ferritin is an iron-protein complex which plays an important part in the storage of iron. Here an atomic structure is proposed for the iron containing core and its synthesis is discussed.

Influence of site-directed modifications on the formation of iron cores in ferritin.

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.

The structure of apoferritin: Molecular size, shape and symmetry from X-ray data†

Apoferritin is the protein component of the iron-storage protein ferritin, and forms a shell round the iron oxide core. Iron-free apoferritin crystallizes in a cubic form isomorphous with one form of ferritin and having four molecules in a unit cell of side 184 A and apparent space group F 432. From the X-ray data apoferritin molecules have a molecular weight of 480,000 and a form approximating on the average at a resolution of 26 A to a spherical shell having an external radius of 61 ± 3 A and internal to external radius ratio about 0·6. The shell is constructed from subunits and chemical results suggest that there are about twenty of these. Recent X-ray photographs of wet apoferritin crystals are found to contain both sharp reflections, extending to spacings of 1/1·4 A −1 , and diffuse streaks indicative of a structure having “ordered disorder”. The X-ray patterns are interpreted as being due to molecules which are situated regularly on a face-centred lattice, but having different orientations at random, so that the apparent point-group 4:3:2† is produced statistically from molecules with lower symmetry. The diffraction patterns suggest that the molecules have pseudo-icosahedral symmetry, but not exact icosahedral nor tetrahedral symmetry. The molecules probably consist of twenty subunits situated at the vertices of a pentagonal dodecahedron with point group 5:2, 2:2:2 or 2. An examination of the diffraction pattern of orthorhombic ferritin shows that the symmetry is almost certainly 5:2.

On the structure of hemosiderin and its relationship to ferritin

The atomic structures and morphology of the mineral components of ferritin and hemosiderin prepared from a single horse spleen are compared by X-ray diffraction, Mossbauer spectroscopy, and electron microscopy. The atomic structures could not be distinguished by X-ray diffraction and Mossbauer spectroscopy, although the average particle size in hemosiderin appears to be somewhat smaller than that in ferritin, as judged by X-ray diffraction and electron microscopy. The results are compatible with the conclusion of earlier studies that hemosiderin may consist largely of ferritin ironcores, which have aggregated on removal of their protein coats by proteolytic digestion. However, formation of such particles independently of ferritin is possible.

Reconstituted and native iron-cores of bacterioferritin and ferritin

The structural and magnetic properties of the iron-cores of reconstituted horse spleen ferritin and Azotobacter vinelandii bacterioferritin have been investigated by high-resolution transmission electron microscopy, electron diffraction and Mossbauer spectroscopy. The structural properties of native horse spleen ferritin, native Az. vinelandii, and native and reconstituted Pseudomonas aeruginosa bacterioferritins have also been determined. Reconstitution in the absence of inorganic phosphate at pH 7.0 showed sigmoidal behaviour in each protein but was approximately 30% faster in initial rate for the Az. vinelandii protein when compared with horse spleen apoferritin. The presence of Zn2+ reduced the initial rate of Fe(II) oxidation in Az. vinelandii to 22% of the control rate. The iron-cores of the reconstituted bacterioferritins adopt defect ferrihydrite structures and are more highly ordered than their native counterparts, which are both amorphous. However, the blocking temperature for reconstituted Az. vinelandii (22.2 K) is almost identical to that for the native protein (20 K). Particle size measurements indicate that the reconstituted Az. vinelandii cores are smaller in median diameter than the native cores and this reduction in particle volume (V) offsets the increased magnetocrystalline contribution to the magnetic anisotropy constant (K) in such a way that the magnetic anisotropy barrier (KV), and hence the blocking temperature, is similar for both proteins. Reconstituted horse spleen ferritin exhibits a similar blocking temperature (38 K) to that determined for the native protein, although it is structurally more disordered. The possibility of introducing structural and compositional modifications in both horse ferritin and bacterioferritins by in-vitro reconstitution suggests that these proteins do not function primarily as a crystallochemical-specific interface for core development in vivo.

The structures of ferritin and apoferritin: Some preliminary X-ray data

Ferritin is a protein containing about 20 % by weight of iron in the form of a micelle of ferric hydroxide: this is surrounded by a shell of apoferritin. The iron can be removed and the apoferritin crystallised as colourless octahedra similar to the brown octahedral crystals formed by ferritin. An X-ray study has been made of horse spleen apoferritin and of two crystalline forms of ferritin A and B from the same source. Apoferritin and ferritin B crystals are isotropic, while those of ferritin A are anisotropic. All three crystals have identical lattice repeats; wet crystals of apoferritin and ferritin B are face-centred cubic with a = 184 A, while ferritin A is only pseudo-cubic, being truly face-centred on only one face. The lattice of ferritin A can therefore be described in terms of an orthorhombic unit cell with a = 130 A, b = 130 A, c = 184 A. None of the space-groups are determined unambiguously from the X-ray data, but depend on the exact configurations of the molecules. The space group of apoferritin is probably F432, the unit cell containing four molecules. This being the case each molecule should have point group symmetry 432, and hence be composed of 24n identical sub-units, where n is an integer. A possible molecular structure is suggested, that of 24 sub-units situated at the vertices of a snub-cube. In ferritin B the protein structure is probably identical with that of apoferritin, but point groups 432, 43m or m3m are allowable for the iron hydroxide micelle. If, as suggested by electron microscopy, the micelle consists of four particles at the vertices of a tetrahedron, or eight particles at the vertices of a cube, then each of these particles is probably composed of three sub-units. The possibility that the observed symmetries of apoferritin and ferritin B are produced statistically is also discussed briefly. The X-ray data for ferritin A are consistent with the molecules containing a single two-fold axis, the space group of the orthorhombic unit cell being P21212, P21221 or P22121. Thus either there are two species of ferritin in which the micelles are oriented differently with respect to the protein, or the lattice of ferritin A does not use the full symmetry of the molecule. A molecular diameter of 111–112 A is derived for apoferritin and ferritin A, the air-dried crystals having a face-centred cubic arrangement with a respectively 158 A and 156 A. A molecular weight of 747,000 is obtained for ferritin A. If certain assumptions are made, a molecular weight close to that obtained from sedimentation-diffusion studies can be calculated for apoferritin.

Three-dimensional structure of the enzyme dimanganese catalase from Thermus Thermophilus at 1 Å resolution

The crystal structures of two forms of the enzyme dimanganese catalase from Thermus Thermophilus (native and inhibited by chloride) were studied by X-ray diffraction analysis at 1.05 and 0.98 Å resolution, respectively. The atomic models of the molecules were refined to the R factors 9.8 and 10%, respectively. The three-dimensional molecular structures are characterized in detail. The analysis of electron-density distributions in the active centers of the native and inhibited enzyme forms revealed that the most flexible side chains of the amino acid residues Lys162 and Glu36 exist in two interrelated conformations. This allowed us to obtain the structural data necessary for understanding the mechanism of enzymatic activity of the dimanganese catalase.