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.     

A bacterioferritin from the strict anaerobe Desulfovibrio desulfuricans ATCC 27774

A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as the terminal electron acceptor, which is the first example of a bacterioferritin from a strict anaerobic organism. This new bacterioferritin was isolated mainly as a 24-mer of 20 kDa identical subunits, containing 0.5 noncovalently bound heme and 2 iron atoms per monomer. Although its N-terminal sequence is significantly homologous with ferritins from other microorganisms and the ligands to the di-iron ferroxidase center are conserved, it is one of the most divergent bacterioferritins so far characterized. Also, in contrast to all other known bacterioferritins, its heme is not of the B type; its chromatographic behavior is identical to that of iron uroporphyrin. Thus, D. desulfuricans bacterioferritin appears to be the second example of a protein unexpectedly containing this heme cofactor, or a closely related porphyrin, after its finding in Desulfovibrio gigas rubredoxin:oxygen oxidoreductase ?Timkovich, R., Burkhalter, R. S., Xavier, A. V., Chen, L., and Le Gall, J. (1994) Bioorg. Chem. 22, 284-293. The oxidized form of the protein has a visible spectrum characteristic of low-spin ferric hemes, exhibiting a weak absorption band at 715 nm, indicative of bis-methionine heme axial coordination; upon reduction, the alpha-band appears at 550 nm and a splitting of the Soret band occurs, with two maxima at 410 and 425 nm. The heme center has a reduction potential of 140 +/- 10 mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca. -200 mV).     

Bacterial ferritin contains 24 haem groups

Pseudomonas aeruginosa bacterioferritin, also known as cytochrome b1 or cytochrome b557, has been isolated with 9 haems per 24 subunits. Various forms of the protein have been prepared including the completely haem-free protein and the fully haem-loaded protein with 24 haems per 24 subunits. The presence of the core does not significantly affect haem addition or removal. The absorbance ratio of the non-haem-iron-loaded protein, 278 nm:417 nm (oxidised), can be used to estimate the haem loading.     

Isolation and characterization of a molybdenum iron-sulfur protein from Desulfovibrio africanus.

A molybdenum-containing iron-sulfur protein has been isolated from the sulfate reducer $\text{Desulfovibrio}\text{africanus}$. The protein appears to be a complex protein of high molecular weight (112,000) composed of 10 subunits (mol. wt. 11,500) and containing a high amount of molybdenum (5–6 atoms/mole) with approx. 20 atoms each of iron and labile sulfide. The spectrum shows peaks at around 615, 410 and 325 nm with a protein peak at 280 nm. Its millimolar extinction coefficients at 615, 410 and 280 nm are 48.4, 64.4 and 141 respectively. The protein contains 106 amino-acid residues per subunit of mol. wt. 11,262 and the number of cysteine residues is 2 per subunit. The N-terminal sequence which has been determined up to 26 residues is characterized by its high degree of hydrophobicity.     

Isolation of a ferritin from Bacteroides fragilis

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.     

A facile method of isolation for the iron molybdenum cofactor of nitrogenase and bacterial ferritin from extracts of azotobacter vinelandii

An alternative method has been developed for the isolation of both the iron molybdenum cofactor of nitrogenase (FeMoco), a small molecular weight FeMoS cluster which is the putative nitrogen- reducing site of the enzyme, and bacterioferritin, an iron storage protein similar to other ferritins, but containing heme prosthetic groups. Previously the isolation of these two species, the characterization of which is of significant current interest, has been dependent on the purification of the nitrogenase enzyme from Azotobacter vinelandii. Out new procedure eliminates the use of the anaerobic column chromatography necessary to obtain pure nitrogenase components, involving instead the heat and RNAase/ DNAase treatment of crude extracts of ruptured cells followed by sedimentation (150000 × g for 18 h) of both the 'nitrogenase complex' and bacterioferritin. The redissolved pellet from this centrifugation yields the pure crystalline bacterioferritin on addition of Mg²⁺. and cooling, the iron content of the protein being higher by this method than in previous reports. Likewise, denaturation by acid/base treatment of this protein mixture yields a precipitate which can be extracted with either N-methylformamide or N,N-dimethylformamide containing dithionite ion to yield solutions of FeMoco, as evidenced by UW 45 reconstitution and EPR spectral criteria. Unfortunately, preparations of FeMoco obtained by this method have a variable, but consistently low, Fe/Mo ratio and additional visible spectral features, indicating that they are significantly less pure than that those generated from purified nitrogenase. The aqueous supernatant from the denaturation also yields bacterioferritin, but with a lower iron content than that from the direct crystallization method.     

Purification and characterization of three proteins from a halophilic sulfate-reducing bacterium,Desulfovibrio salexigens

Summary Hydrogenase, desulfoviridin and molybdenum proteins have been isolated from a halophilic sulfate-reducing bacteria,Desulfovibrio salexigens strain British Guiana. At least 50% of the hydrogenase was found to be located in the periplasm. The hydrogenase has a typical absorption spectrum, a 400/280 nm ratio of 0.28, a molecular weight by sedimentation equilibrium of 81 000 and is composed of two subunits. It has one nickel, one selenium and 12 iron atoms per molecule. The sulfite reductase has a typical desulfoviridin absorption spectrum, a molecular weight of 191 000 and iron and zinc associated with it. The molybdenum-iron protein is gray-green in color and exhibits an absorbtion spectrum with peaks around 612, 410, 275 nm and a shoulder at 319 nm. It is composed of subunits of approximately 13 250 and has an approximate molecular weight of 110 000. Three molybdenum and 20 iron atoms are found associated with it.An extensive study of these three proteins will allow a better understanding of the function of these enzymes and also of their possible role in microbially caused corrosion.     

M?ssbauer spectroscopic studies of iron in Pseudomonas aeruginosa.

The present M?ssbauer spectroscopic studies of isolated bacterioferritin and whole cells of Pseudomonas aeruginosa have shown that the iron core of bacterioferritin is not altered on isolation. These studies have also shown that the bacterioferritin core is typically 85% oxidized within the cell and may contain a significant proportion of its iron as small clusters during the early stage of the stationary phase of cell growth.     

Haem binding to ferritin and possible mechanisms of physiological iron uptake and release by ferritin.

Haem binding to horse spleen ferritin and Pseudomonas aeruginosa bacterioferritin has been studied by spectroscopic methods. A maximum of 16 haems per ferritin molecule, and 24 haems per bacterioferritin molecule, has been shown to bind. The influence of the bound haem on the rate of reductive iron release has been investigated. With a range of reductants and in the absence of haem the rate of release varied with the reductant, but in the presence of haem the rate was both independent of the reductant and faster than with any of the reductants alone. This indicates the rate-limiting step for iron release in the absence of haem was electron-transfer across the protein shell. Based on the results obtained with the in vitro assay system and from a consideration of data currently in the literature, plausible schemes for ferritin and bacterioferritin iron uptake and release are described.     

Characterization of the soluble hydrogenase from Desulfovibrio africanus

The soluble hydrogenase from Desulfovibrio africanus has been isolated and characterized. The enzyme consists of two subunits of 65 kDa and 27 kDa. Its absorption spectrum is typical of an iron-sulfur protein. The protein contains 12 iron atoms, 10 labile sulfur atoms and 0.9 nickel atom per molecule. D. africanus hydrogenase is rapidly activated under reducing conditions and exhibits a specific activity of 570 mumoles H2 evolved/min/mg. The EPR spectrum of the oxidized enzyme shows no Ni(III) signals. Upon reduction under hydrogen, the protein sample exhibits signals due to nickel with g values at 2.21, 2.17 and 2.01 correlating with the active state of the enzyme.     

The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin-2 genes: involvement of rubredoxin in iron metabolism

The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.     

Isolation and properties of the complex nonheme-iron-containing cytochrome b557 (bacterioferritin) from Pseudomonas aeruginosa

A nonheme-iron-containing cytochrome b557, also known as bacterioferritin, was isolated from Pseudomonas aeruginosa. Gel electrophoresis showed that the protein subunits had a molecular weight of 18 kD and it is suggested that the intact molecule contained 24 subunits. The isolated protein contained 8.7% by weight Fe and 8% by weight phosphate. Most of the Fe was contained in the nonheme-iron core and could be readily removed by dialysis against 0.12 M thioglycollic acid. The resulting apobacterioferritin contained approximately one protoporphyrin IX group for every five subunits and, in addition, an unidentified fluorophor. The nonheme-iron core was found to be amorphous with a mean core size of 60-65 A.     

Amino acid sequence of the bacterioferritin (cytochrome b1) of Escherichia coli-K12

The complete amino acid sequence of bacterioferritin (cytochrome b1) from Escherichia coli-K12 has been derived from the nucleotide sequence of the cloned gene. It comprises 158 amino acid residues giving an Mr of 18,495. The identity of the gene product was confirmed by an 87 residue N-terminal sequence obtained from the purified protein, but it differs significantly from much of the previously published partial amino acid sequence (1). Secondary structure prediction indicates a high alpha-helical content consistent with a 4-helix-bundle conformation. The fully assembled bacterioferritin molecule comprising 24 identical subunits and 12 haem moieties is a tetracosamer with an Mr of approximately 452,000.     

The nature of the di-iron site in the bacterioferritin from Desulfovibrio desulfuricans

The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 A resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.     

Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate

Abstract The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti- M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weigh ( M _r) M. leprae protein (MLP) with a subunit M _r of 22000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis . It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed.     

Studies on the utilization of ferritin iron in the ferrochelatase reaction of isolated rat liver mitochondria.

The utilization of ferritin as a source of iron for the ferrochelatase reaction has been studied in isolated rat liver mitochondria. 1. It was found that isolated rat liver mitochondria utilized ferritin as a source of iron for the ferrochelatase reaction in the presence of succinate plus FMN (or FAD). 2. Under optimal experimental conditions, i.e., approx. 50 micromol/1 FMN, 37 degrees C, pH 7.4 and 0.5 mmol/l Fe(III) (as ferritin iron), the release process, as shown by the formation of deuteroheme, amounted to approx. 0.5 nmol iron/min per mg protein. 3. The release process could not be elicited by ultrasonically treated mitochondria, lysosomes, microsomes or cytosol, i.e., the release of iron from ferritin was due to mitochondria and was a function of the in situ orientation of the mitochondrial inner membrane. 4. The release of iron from ferritin by the mitochrondria might be of relevance not only for the in situ synthesis of heme in the hepatocyte, but also with respect to the mechanism(s) by means of which iron is mobilized for transport to the erythroid tissue.     

Bacterioferritin from Mycobacterium smegmatis contains zinc in its di-nuclear site

Bacterioferritins, also known as cytochrome b (1), are oligomeric iron-storage proteins consisting of 24 identical amino acid chains, which form spherical particles consisting of 24 subunits and exhibiting 432 point-group symmetry. They contain one haem b molecule at the interface between two subunits and a di-nuclear metal binding center. The X-ray structure of bacterioferritin from Mycobacterium smegmatis (Ms-Bfr) was determined to a resolution of 2.7 A in the monoclinic space group C2. The asymmetric unit of the crystals contains 12 protein molecules: five dimers and two half-dimers located along the crystallographic twofold axis. Unexpectedly, the di-nuclear metal binding center contains zinc ions instead of the typically observed iron ions in other bacterioferritins.     

Isolation and partial characterization of a hemolytic and toxic protein from the nematocyst venom of the Portuguese Man-of-War, Physalia physalis

Nematocysts isolated from the stinging tentacles of the Atlantic Portuguese Man-of-War (Physalia physalis) possess a potent venom composed of several proteins. A hemolytic protein lethal to mice has been isolated from this nematocyst venom. This protein, physalitoxin, appears to be responsible for both the venom's hemolytic and lethal activities. The hemolysin has a molecular weight of approx. 240 000, a sedimentation coefficient of 7.8 S, and is rod-like in shape with a calculated axial ratio of about 1 : 10. It appears to be composed of three subunits of unequal size, each of which is glycosylated. Two of these subunits seem to have pKi values near 8.2 and the third near 5.5. Physalitoxin comprises about 28% of the total nematocyst venom protein. It is 10.6% carbohydrate by weight and represents the major glycoprotein of the venom. Physalitoxin is inactivated by concanavalin A and this inactivation can be blocked with alpha-methyl-mannoside. The inactivation by concanavalin A is temperature-dependent about 12 degrees C and the hemolytic activity of untreated venom is temperature-dependent below 12 degrees C. Physalitoxin is the first hemolytic toxin from a cnidarian to be purified directly from isolated nematocysts.