Mössbauer spectroscopy of Escherichia coli and its iron-storage protein.

57Fe Mössbauer spectra of whole frozen Escherichia coli cells and of an iron storage protein isolated from iron-rich cells of E. coli have been measured over a range of temperatures down to 0.08 K. The spectra of E. coli cells with high iron content and of the iron storage protein were found to be very similar. Above 4 K these spectra consist of a quadrupole split doublet characteristic of Fe3+. Below 3.5 K, the spectra display magnetic hyperfine splitting which is temperature dependent, and point to the existence of an ordered magnetic phase associated with a saturation magnetic hyperfine field of 43 tesla in both samples. The results indicate that the bulk of iron in the iron-rich cells is in the form of aggregates similar in nature to the iron cores in the isolated protein, although the latter account for not more than 1% of the total iron in the cells. The Mössbauer spectra of the isolated protein are different from those observed in ferritin, the iron-storage protein of plants and higher animals, showing that the iron cores in these two proteins are different.

Properties of a new crystal form of the complex of concanavalin A with methyl alpha-D-glucopyranoside.

The complex of concanavalin A with methyl alpha-D-glucopyranoside crystallizes as regular rhombic dodecahedra containing 35% protein by weight. The crystal is of space group I23 with a = 167.8 A (1 A = 0.1 nm) and contains one concanavalin A dimer per asymmetric unit. It diffracts to a resolution of 1.9 A and is suitable for crystallographic investigation of the structure of the saccharide-binding site.

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms.

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

13C NMR analysis of methionine sulfoxide in protein.

The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls.

Circumstantial evidence for cytochrome b1 involvement in the functioning of lac-permease in respiring Escherichia coli.

The structure of the haem-binding site of cytochrome b1 and particularly the fact that the two protein ligands of the haem are methionines could explain a correlation found between loss of lac-permease activity and replacement of methionine by norleucine in the protein of aerobically respiring E. coli. If cytochrome b1 is essential for lac-permease mediated transport in whole bacteria as this correlation suggests, translocation of substrate by this permease must be coupled to electron transport. Such a dependence would invalidate the chemiosmotic interpretation of lactose transport in E. coli in its present form and would be in variance with the coupling-by-energy theories of lactose transport that exempted translocation from dependence on energy yielding processes.

Preliminary results for the primary structure of bacterioferritin of Escherichia coli.

Bacterioferritins are type-b cytochromes which resemble ferritin. Amino acid analysis combined with chemical modification and partial sequence analysis characterize bacterioferritin of Escherichia coli in terms of its primary structure. It is a protein composed of one kind of polypeptide chain that commences with methionine and terminates with glutamic acid. The length of the polypeptide chain is, tentatively, 146 residues. Besides the N-terminal methionine residue there are three more methionine residues, which yield four CNBr peptides, which have been aligned. The identity of the following positions in the sequence has been ascertained: residues 1-25, 30-37, 83-88, 127-132 and 143-146. No homology with ferritin was found.

The molecular composition of the volutin granule of yeast.

The volutin granule was isolated from yeast by disruption of freeze-dried cells in an organic solvent and density-gradient-gradient centrifugation. The granule is composed of two types of macromolecule, a linear-chain polyphosphate and four basic proteins, of molecular weights ranging from 10 000 to 20 000. In the dissolved granule these macromolecules are in a complex that is uniform by hydrodynamic criteria (s20,w = 22.3 S). The polyphosphate separated from this complex gives a single 31P n.m.r. resonance and in the analytical ultracentrifuge behaves as a monodisperse solute of molecular weight 245 000 +/- 1000. In the 31P n.m.r. spectrum of yeast used for its isolation, this polyphosphate accounts for 14% of total cell polyphosphate.

Properties of a crystal of the complex of methyl D-arabinofuranoside with concanavalin A.

The complex of methyl alpha-D-arabinofuranoside with concanavalin A crystallizes in the orthorhombic space group P2(1)22(1) with cell dimensions a = 97.5, b = 87.0 and c = 61.5 A. The asymmetric unit contains one dimer and the unit cell consists of two tetrahedral clusters of point-group symmetry 222. The crystals diffract to 2.0 A resolution.

Location of haem in bacterioferritin of E. coli.

. A low-resolution partial structure of bacterioferritin was solved using a combination of molecular replacement and rigid-body refinement methods. Modification of bacterioferritin crystals by soaking in tetrachloroplatinate results in a phase transition from tetragonal symmetry (space group P4(2)2(1)2) to a pseudo-cubic one (approximate space group I432). Helical parts of human H ferritin structure stripped of side chains beyond the C(beta) atoms were used as the model. An electron-density map of the refined model revealed a region of extended density which by its shape and position in a pocket between helices was identified as haem. Inclusion of haem in the refinement showed that it can occupy only one of two symmetry-related sites near a twofold axis of the molecule.

Structure of a unique twofold symmetric haem-binding site.

Bacterioferritin of Escherichia coli, also known as cytochrome b1, is a hollow, nearly spherical shell made up of 24 identical protein subunits and 12 haems. We have solved this structure in a tetragonal crystal form at 2.9 A resolution. We find that each haem is bound in a pocket formed by the interface between a pair of symmetry-related subunits. The quasi-twofold axis of the haem is closely aligned with the local twofold axis relating these subunits. The axial ligands of the haem are sulphurs of two equivalent methionyl residues (Met 52) from the symmetry-related subunits. A cluster of four water molecules is trapped in the gap between the upper edge of the haem and two extended protein loops which close off the haem from the outer aqueous environment. This is the first structure of a bis-methionine ligated haem-binding site and the first case of a twofold symmetric haem-binding site.

A crystallographic study of haem binding to ferritin.

Ferritin, the iron-storage protein, binds porphyrins, metalloporphyrins and the fluorescent dyes ANS (8-anilino-1-naphthalenesulfonic acid) and TNS (2-p-toluidinyl-6-naphthalenesulfonic acid), similarly to apo-myoglobin. Octahedral crystals of horse-spleen apo-ferritin (HSF; 174 amino acids) complexes prepared by the addition of haem, hematoporphyrin or Sn-protoporphyrin IX to a solution of apo-ferritin crystallize in space group F432 with cell parameter a = 184.0 A. X-ray crystallographic analysis of single crystals prepared from a mixture containing haem or Sn-protoporphyrin IX shows that the haem-binding sites in these crystals are occupied by protoporphyrin IX, which is free of metal, rather than by the original metalloporphyrin. The present paper describes the structure of horse-spleen apo-ferritin cocrystallized with Sn-protoporphyrin IX. The 6797 reflections up to 2.6 A resolution used in the refinement were obtained from a data set recorded on a Nicolet/Xentronics area detector with Cu Kalpha radiation from a Rigaku RU 200 rotating anode. The final structure comprises 1613 non-H atoms, two Cd atoms and 170 solvent molecules. Four residues are described as disordered. The root-mean-square deviations from ideal bond lengths and angles are 0.013 A and 2.88 degrees, respectively. Protoporphyrins are observed in special positions on the twofold axes of the ferritin molecule with a stoichiometry of 0.4 per subunit.

Dynamics of heme iron in crystals of metmyoglobin and deoxymyoglobin.

The 57Fe gamma-ray resonance absorption spectra have been measured in crystals of metmyoglobin and deoxymyoglobin over a wide range of temperatures. Above a critical temperature common to both proteins (220 K), the dynamics of heme iron display a dramatic change, in that two kinds of thermal fluctuations come into play--a fast fluctuation associated with a steep decrease of the total fluctuation of characteristic time 10(-8) sec, associated with bounded diffusive motion. By using both discrete jump and continuous diffusion models, the latter based on the Brownian motion of an overdamped harmonic oscillator, the essential parameters of the iron motion (mean square displacement and jump frequency or diffusion constant) can be derived as a function of temperature. Thus, for deoxy Mb at 288 K, the mean square displacement for the fast fluctuation is about 6 X 10(-2) A2 and for the diffusive motion is 1.6 X 10(-2) A2; the diffusion constant is 4 X 10(-10) cm2/sec. The diffusive process is associated with an activation energy of about 0.75 kcal/mol. Although the same general kinds of phenomena are observed in crystals of MetMb and deoxy Mb, significant differences in behavior are found, which suggest that the main dynamical phenomenon observed reflects internal large-scale motions of the protein.

The composition and the structure of bacterioferritin of Escherichia coli.

Bacterioferritin isolated from Escherichia coli is of two kinds: a protein containing a polynuclear iron compound, the bacterioferritin proper and a protein free of the polynuclear iron compound, the apo-bacterioferritin. Bacterioferritin of both kinds is characterized by absorption maxima at 417,530 and 560 nm, contributed by protohaem IX. Single crystals of bacterioferritin of the space group I432 suggest that the molecule is made up of 24 identical subunits related by a cubic point symmetry. The molecular weight of the protein subunit, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is 15000. In the electron microscope the bacterioferritin molecule appears to be a sphere of 9.5 nm (95 A) diameter composed of a negatively staining outer shell and an inner electron-dense core of 6 nm (60 A) diameter.