Iron Oxidase Research Articles

The mechanism of copper leaching by intact cells of Thiobacillus ferrooxidans.

Under aerobic conditions, washed intact cells of Thiobacillus ferrooxidans AP19-3 solubilized copper enzymatically from a copper concentrate containing Cu(20.48%), Fe(31.61%), 8(38.22%), Pb(3.84%), and Zn(4.22%). The optimum pH of copper solubilization from copper concentrate was at pH 3.0. In contrast, under anaerobic conditions or under aerobic conditions in the presence of sodium cyanide, inhibitor of iron oxidase, the amount of copper solubilized enzymatically from the ore markedly decreased, indicating that iron oxidase is involved in the copper solubilization. Under the conditions under which iron oxidase of the cells cannot operate, Fe2+ and Cu+ ions were produced enzymatically, suggesting that a hydrogen sulfide: ferric ion oxidoreductase (SFORase) was involved in this copper solubilization from the ore. A short treatment of the strain with 0.5% phenol completely destroyed the SFORase. However, this treatment did not affect the iron oxidase of the cells. A concomitant loss of the activity of copper solubilization from the ore was observed in 0.5% phenol-treated cells. The results strongly suggest that both iron oxidase and SFORase are crucial in copper ore leaching by T. ferrooxidans AP19-3.

Spectroscopic Studies of Isopenicillin N Synthase: A Mononuclear Nonheme Fe2+ Oxidase with Metal Coordination Sites for Small Molecules and Substrate

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the β-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. Mossbauer spectra of the 57Fe-enriched enzyme exhibit parameters (δ = 1.30 mm/s, Δ EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter δ (δ = 1.10 mm/s, ΔEQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme·ACV·NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme· ACV·NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme·ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.

Synthesis of an Iron-Oxidizing System during Growth of Thiobacillus ferrooxidans on Sulfur-Basal Salts Medium

It was found that the de novo synthesis of not only sulfur:ferric ion oxidoreductase (ferric ion-reducing system) but also iron oxidase was absolutely required when Thiobacillus ferrooxidans AP19-3 was grown on sulfur-salts medium. The results strongly suggest that iron oxidase is involved in sulfur oxidation. This bacterium could not grow on sulfur-salts medium under anaerobic conditions with Fe as a terminal electron acceptor, suggesting that energy conservation by electron transfer between elemental sulfur and Fe is not available for this bacterium.

Mechanism of Tetravalent Manganese Reduction with Elemental Sulfur byThiobacillus ferrooxidans

Washed intact cells of iron-grown T. ferrooxidans AP19-3 could reduce manganese dioxide (MnO2) enzymatically with elemental sulfur (S°) and the mechanism of Mn4+ reduction was studied. The optimum pH for Mn4+ reduction was 2.5. The amount of Mn4+ reduced by the strain was proportional to the amount of S° added to the reaction mixture. An enzyme that directly catalyzes the reduction of Mn4+ to Mn2+ with S° was not found in the cell-free extract of this strain. T. ferrooxidans AP19-3 was found to possess sulfur: ferric ion oxidoreductase (SFORase), which catalyzes the reduction of Fe3+ with S° to give Fe2+ and sulfite. The reduction of Mn4+ was inhibited by a specific inhibitor of SFORase or diamide, strongly suggesting that SFORase is involved in the Mn4+ reduction in this strain. The involvement of SFORase in Mn4+ reduction was further supported by the following results. Both the amounts of Fe2 + and sulfite produced by SFORase were markedly reduced by MnO2. The amount of Mn4+ reduced increased 5.4-fold with 1 mm ferric ion and 10-fold with 2.5 mm cyanide or an inhibitor of iron oxidase, respectively. The sulfite and Fe2+ were chemically oxidized by MnO2 instantly. From the results, it is concluded that the Mn4+ reduction with S° in this strain occurs through two steps. In the first step S° is enzymatically oxidized by SFORase to give Fe2+ and sulfite, and in the second step these two reduced compounds are chemically reduced by MnO2 to give Mn2 +

Mechanism of tetravalent manganese reduction with elemental sulfur by Thiobacillus ferrooxidans.

Washed intact cells of iron-grown T. ferrooxidans AP19-3 could reduce manganese dioxide (MnO2) enzymatically with elemental sulfur (S0) and the mechanism of Mn4+ reduction was studied. The optimum pH for Mn4+ reduction was 2.5. The amount of Mn4+ reduced by the strain was proportional to the amount of S0 added to the reaction mixture. An enzyme that directly catalyzes the reduction of Mn4+ to Mn2+ with S0 was not found in the cell-free extract of this strain. T. ferrooxidans AP19-3 was found to possess sulfur: ferric ion oxidoreductase (SFORase), which catalyzes the reduction of Fe3+ with S0 to give Fe2+ and sulfite. The reduction of Mn4+ was inhibited by a specific inhibitor of SFORase or diamide, strongly suggesting that SFORase is involved in the Mn4+ reduction in this strain. The involvement of SFORase in Mn4+ reduction was further supported by the following results. Both the amounts of Fe2+ and sulfite produced by SFORase were markedly reduced by MnO2. The amount of Mn4+ reduced increased 5.4-fold with 1 mM ferric ion and 10-fold with 2.5 mM cyanide or an inhibitor of iron oxidase, respectively. The sulfite and Fe2+ were chemically oxidized by MnO2 instantly. From the results, it is concluded that the Mn4+ reduction with S0 in this strain occurs through two steps. In the first step S0 is enzymatically oxidized by SFORase to give Fe2+ and sulfite, and in the second step these two reduced compounds are chemically reduced by MnO2 to give Mn2+

Dependence of intestinal iron absorption on the valency state of iron.

1. In rats iron was absorbed after administration into the gut lumen as ferric iron bound to serum albumin, to nitrilotriacetic acid, and to 8-OH-quinoline sulfonic acid, or as isolated diferri-transferrin. 2. Iron absorption from 59Fe-labelled transferrin was inhibited by the addition of rat plasma. 3. The inhibitory component in the rat plasma turned out to be ceruloplasmin (ferrous iron oxidase, EC 1.16.2.1). 4. The absorption of iron from these ferric iron complexes was also inhibited by addition to the incubation medium of ferrozine, a strong anionic Fe(II)-ligand. 5. Uptake and absorptive utilization of transferrin-bound ferric iron was decreased after a prewash of the gut lumen and could be restored by the addition of ascorbate to the incubation medium. 6. The conclusion was drawn from these results that luminal reduction precedes ferric iron absorption and that this is a prerequisite for the uptake into the mucosa.

Production of ferrous ions as intermediates during aerobic sulfur oxidation in Thiobacillus ferrooxidans.

When Thiobacillus ferrooxidans AP19-3 that had been treated to remove iron from the cell surface was incubated aerobically with both elemental sulfur and 6>-phenanthroline, Fe3+ still present was reduced by the ferric-ion reducing (FIR) system of the cells. Ferrous ion chelated with o-phenanthroline was not be oxidized by iron oxidase, and leaked out into the reaction mixture. In the absence of o-phenanthroline, however, Fe2+ was not detected, suggesting that it was rapidly reoxidized by iron oxidase. Sulfur oxidation by this strain under aerobic conditions was strongly inhibited by o-phenanthroline, so Fe2+ was probably produced as an intermediate during the oxidation of elemental sulfur. Sulfur-grown cells that were cultured successively in a sulfur-salt medium always had high levels of iron-oxidizing activity. The strain could use Fe2+ produced earlier by the FIR system for growth. We concluded that when T. ferrooxidans AP19-3 grows on a sulfur-salt medium under aerobic conditions, the strain used a su...

Role of a Ferric Ion-Reducing System in Sulfur Oxidation of Thiobacillus ferrooxidans

The properties of a ferric ion-reducing system which catalyzes the reduction of ferric ion with elemental sulfur was investigated with a pure strain of Thiobacillus ferrooxidans. In anaerobic conditions, washed intact cells of the strain reduced 6 mol of Fe with 1 mol of elemental sulfur to give 6 mol of Fe, 1 mol of sulfate, and a small amount of sulfite. In aerobic conditions, the 6 mol of Fe produced was immediately reoxidized by the iron oxidase of the cell, with a consumption of 1.5 mol of oxygen. As a result, Fe production was never observed under aerobic conditions. However, in the presence of 5 mM cyanide, which completely inhibits the iron oxidase of the cell, an amount of Fe production comparable to that formed under anaerobic conditions was observed under aerobic conditions. The ferric ion-reducing system had a pH optimum between 2.0 and 3.8, and the activity was completely destroyed by 10 min of incubation at 60 degrees C. A short treatment of the strain with 0.5% phenol completely destroyed the ferric ion-reducing system of the cell. However, this treatment did not affect the iron oxidase of the cell. Since a concomitant complete loss of the activity of sulfur oxidation by molecular oxygen was observed in 0.5% phenol-treated cells, it was concluded that the ferric ion-reducing system plays an important role in the sulfur oxidation activity of this strain, and a new sulfur-oxidizing route is proposed for T. ferrooxidans.

Structural and functional aspects of copper transporting proteins

The biogenic nature and esssentiality of copper in biological systems is well known. Due to both its oxidations–reduction activity and the intriguing coordination of chemistry, copper is most suited to play a vital role in cellular biochemistry. Hydrated copper ions themselves are highly reactive and would be in a position to interfere in many a biochemical pathway. For example, the Fenton Type generation of ·OH radicals would destroy the cellular architecture in an uncontrolled manner. Unwanted and non-specific binding of ionic copper to functional and/or structurally important biopolymers must be considered hazardous for living system. In electron transport and oxygen reduction the specific reaction of the bound copper is dictated by the macromolecular protein portion [1,2]. While some progress has been made on the structure-function correlation of some of these copper proteins little is known how the metal is implanted into these functionally important copper proteins. In order to avoid the uncontrolled reactivity of copper and to ascertain the proper insertion of this metal into a specific enzyme the need of an efficient transportation is obvious. At present only three copper-transporting proteins including the cupreins, caeruloplasmin and copper-thionein are fairly well characterized. The binding site of the cuprein copper has been elucidated by X-ray crystallography. The other two proteins have been chemically and spectroscopically studied, in the serum protein caeruloplasmin three different types of copper coordination are known while the copper chromophore in copperthionein appears to be homogeneous. Special emphasis will be placed on this thiolate rich copper protein. This type of copper binding protein appears to be very old origin. It is found in microorganisms as well as in animal cells. Its amino acid composition is even more simple compared to the amino acid residues found in the iron sulphur proteins. A striking phenomenon is the extraordinarily high cysteine content ranging up to 30%. In freshly isolated Cuthionein the copper is the cuprous state. Apart from the copper transportation in either protein some of these proteins display catalytic functions. For example, some of the copper in caerulophenoloxidase and is reported to function like a terminal cytochrome c oxidase. Furthermore, the function of a more or less specific iron oxidase has been suggested. The cupreins are discussed with great enthusiasm to catalyse the spontaneous superoxide dismutation. In the case of the copper-thioneins their role apparently remains to lie exclusively in metal transport.

Ferrous iron oxidation by Ferrobacillus ferrooxidans. Purification and properties of Fe++-cytochrome c reductase.

The electron transport chain and Fe++-cytochrome c reductase of Ferrobacillus ferrooxidans were studied to elucidate the mechanism of iron oxidation by this autotrophic bacterium.The iron oxidation involved the cytochrome c and a type cytochrome of F. ferrooxidans in the electron transport system. The initial enzyme of the oxidation system was found to be Fe++-cytochrome c reductase. The iron oxidase system was labile to freezing or sonication; either treatment disrupted some link between the cellular cytochromes c and a. Fe++-cytochrome c reductase and cytochrome oxidase retained their individual activities after either treatment.Fe++-cytochrome c reductase was purified 60-fold to 70-fold. The enzyme was judged to be approximately 90% pure by disc electrophoresis, sedimentation, and DEAE-cellulose chromatography. A suitable assay system with a veronal–acetate buffer was developed for the determination of enzyme activity. The effects of inhibitors and potential activators were studied. No specific inhibitor or cofactor was found, although the enzyme was inhibited by various ionic compounds.Fe++-cytochrome c reductase was dissociated into two subunits, one protein and the other ribonucleic acid (RNA). Neither of the subunits had enzymatic activity and efforts to reconstitute the holoenzyme from the two subunits were unsuccessful. The molecular weights of the holoenzyme, protein subunit, and RNA subunit were determined as 100,000–110,000, 27,000–30,000, and 315,000–330,000, respectively. The protein subunit contained one non-heme iron atom per protein molecule. It was concluded that RNA is an essential component of the enzyme and the failure to recover the activity from subunits is due to the aggregation of RNA after dissociation.

Electron Transport Systems of the Chemoautotroph Ferrobacillus ferrooxidans: II. PURIFICATION AND PROPERTIES OF A HEAT-LABILE IRON-CYTOCHROME c REDUCTASE

Abstract A heat-labile iron-cytochrome c reductase was isolated from Ferrobacillus ferrooxidans by sonic oscillation of the cells at pH 5.5 in the presence of protamine sulfate and purified 20-fold with 10% recovery by successive fractionation of the high speed (144,000 x g) supernatant solution with ammonium sulfate and Sephadex G-200. The enzyme displayed a pH optimum of 5.7 to 6.2 and typical substrate saturation curves with a Km of 2.75 x 10-5 m for cytochrome c and 1.6 x 10-3 m for FeSO4·7H2O. The protein nature of the catalyst was established by its protein content and susceptibility to the proteolytic action of Pronase and to heat. Exposure for 1 min at 100° was sufficient to destroy the activity completely. The most highly purified fraction of iron-cytochrome c reductase contained cytochrome c and apparently nonfunctional cytochrome b. Enzymatic activity, proportional to protein concentration and linear with time, was inhibited by reduced glutathione, p-hydroxymercuribenzoate, and cysteine, but not by atebrin, Amytal, or antimycin A. The inhibition by p-hydroxymercuribenzoate could be entirely reversed by equivalent concentrations of reduced glutathione or cysteine. Reduced cytochrome c, ferric ions, and several other metal ions also inhibited the reductase activity. The close association of the enzyme with the iron oxidase activity of F. ferrooxidans was partially shown by the generally parallel distribution of the two activities in the various fractions resulting from Sephadex treatment and from differential centrifugation. Various attempts to separate the iron oxidase and cytochrome c reductase activities from one another in the less purified fractions were unsuccessful leading to destruction of either both activities or the iron oxidase activity. The most highly purified iron-cytochrome c reductase fraction (F5) possessed no iron oxidase activity and contained a factor that stimulated less purified fractions several-fold in both their cytochrome c oxidase and iron oxidase activities but not in their iron-cytochrome c reductase activity.

ENERGY SUPPLY FOR THE CHEMOAUTOTROPH FERROBACILLUS FERROOXIDANS.

Dugan, Patrick R. (Syracuse University, Syracuse, N.Y.), and Donald G. Lundgren. Energy supply for the chemoautotroph Ferrobacillus ferrooxidans. J. Bacteriol. 89:825-834. 1965.-A working model is proposed to explain dissimilatory ferrous iron oxidation by Ferrobacillus ferrooxidans, that is, oxidation linked to an energy source. The model is supported by experimental evidence reported here as well as in the literature. Polarographic assays of the culture medium demonstrated an iron "complex" involving oxygen. The initial "complex" would be oxygenated, but not oxidized because no electron transport has taken place. The "complex" is formed in solution or on the cell surface and is somehow reacted with iron oxidase (or oxygenase), resulting in the release of an electron. Either sulfate or a flavoprotein is suggested as involved in the initial electron-transfer link between iron and the cell. The electron is transported in the cell through a typical electron-transport system involving coenzyme Q(6), cytochrome c, and cytochrome a; oxygen is the final electron acceptor. Electron micrographs of intact and sectioned cells are included to show structural detail in support of the model.