Effect of deoxyribonucleic acid on the production of reduced oxygen by bleomycin and iron

The binding of bleomycin to DNA in the presence and absence of ferric iron was measured by fluorescence spectroscopy. In millimolar concentrations of tris(hydroxymethyl)aminomethane, pH 7.5, approximately 80% of the bleomycin binds to DNA. Ferric iron seems to have no significant effect on the binding of DNA to bleomycin. The induction of oxygen uptake by ferrous iron and bleomycin was monitored in the presence and absence of DNA. DNA has no effect on the rate of oxygen uptake. Therefore, the iron binding site and the DNA binding site appear to be independent of each other. Under conditions where 80% of the bleomycin is bound to DNA, the ferrous iron-bleomycin-induced reduction of oxygen follows Michaelis-Menten kinetics. Ferrous iron autoxidation produces ethylene from methional. The addition of bleomycin greatly increases ethylene production. DNA, under conditions where 80% of the bleomycin is bound to DNA, inhibits ethylene production. Since ethylene is a measure of hydroxyl radical production, we conclude that DNA is able to compete with methional for the hydroxyl radical. We postulate a mechanism for DNA double-strand breaks in which the bleomycin selectively binds to DNA and recurrently produces the hydroxyl radical at that site. The localized generation of many hydroxyl radicals as provided by the proposed oxidation-reduction cycle mechanism may cause multiple strand breaks taking place on both strands of the DNA duplex leading to double-strand breaks. Since catalase, but not superoxide dismutase, is able to inhibit ferrous iron-bleomycin-induced products of the hydroxyl radical, hydrogen peroxide, but not the superoxide radical, is the immediate precursor of the hydroxyl radical.     

Superoxide dismutase and catalase protect cultured hepatocytes from the cytotoxicity of acetaminophen

Superoxide dismutase, catalase and mannitol prevent the killing of cultured hepatocytes by acetaminophen in the presence of an inhibitor of glutathione reductase, BCNU. Under these conditions, the cytotoxicity of acetaminophen depends upon its metabolism, since beta-naphthoflavone, an inhibitor of mixed function oxidation, prevents the cell killing. In hepatocytes made resistant to acetaminophen by pretreatment with the ferric iron chelator, deferoxamine, addition of ferric or ferrous iron restores the sensitivity to acetaminophen. In such a situation, both superoxide dismutase and catalase prevent the killing by acetaminophen in the presence of ferric iron. By contrast, catalase, but not superoxide dismutase, prevents the cell killing dependent upon addition of ferrous iron. These results document the participation of both superoxide anion and hydrogen peroxide in the killing of cultured hepatocytes by acetaminophen and suggest that hydroxyl radicals generated by an iron catalyzed Haber-Weiss reaction mediate the cell injury.     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

Free radical mechanism of oxidation of uroporphyrinogen in the presence of ferrous iron

Human porphyria cutanea tarda is an unusual consequence of common hepatic disorders such as alcoholic liver disease. Hepatic iron plays a key role in the expression of the metabolic lesions, i.e., defective hepatic decarboxylation of porphyrinogens, catalyzed by uroporphyrinogen decarboxylase. This prompted the present study to determine the in vitro effects of iron on the uroporphyrinogen substrate in the absence and presence of atmospheric oxygen. We observed that (i) unless oxygen is the limiting reactant, autoxidation of ferrous iron and iron-catalyzed oxidation of uroporphyrinogen occurred soon after initiating the reaction at pH 7.4 and 30 degrees C in buffers which are non- or poor chelators of iron; (ii) the rates of uroporphyrinogen oxidation were proportional to the initial concentration of ferrous ion; (iii) about 70% of the oxidations of uroporphyrinogen were accountable due to a free-radical chain reaction pathway involving superoxide radical and hence inhibitable by superoxide dismutase; (iv) uroporphyrinogen could be further oxidized to completion by the hydroxyl radical since the reaction was partially inhibited by both mannitol and catalase which prevent hydroxyl radical production; (v) the oxidizing effects of ferric ion on uroporphyrinogen were none or negligible as compared to those of ferrous ion. Ferric was reduced to ferrous ion in the presence of dithiothreitol. When the ferrous ion thus formed was reoxidized in the presence of atmospheric oxygen, minor but definite oxidations of both uroporphyrinogen and dithiothreitol were observed. The oxidations of Fe2+ and uroporphyrinogen could be blocked by 1,10-phenanthroline, a ferrous iron chelator. The data suggest that ferrous is the reactive form of iron that may contribute to pathogenic development of the disease by irreversibly oxidizing the porphyrinogen substrates to nonmetabolizable porphyrins, which accumulate in porphyric liver.     

Inhibition of the Fenton reaction by the protein caeruloplasmin and other copper complexes. Assessment of ferroxidase and radical scavenging activities

The copper-containing protein caeruloplasmin is an important biological extracellular protein. By catalysing the oxidation of ferrous ions to the ferric state (ferroxidase activity) it can inhibit lipid peroxidation and the Fenton reaction. This activity is readily destroyed by heat-denaturation. When a ferric-EDTA complex is added to hydrogen peroxide, OH X radicals are formed in a reaction inhibitable by superoxide dismutase (SOD). This reaction is also inhibited by caeruloplasmin both before and after heat-denaturation, suggesting a non-catalytic scavenging role for the protein. A combination of ferroxidase and radical scavenging activities in fluids containing iron complexes and hydrogen peroxide, but no SOD or catalase, would make caeruloplasmin an important extracellular antioxidant.     

Ferric iron and superoxide ions are required for the killing of cultured hepatocytes by hydrogen peroxide. Evidence for the participation of hydroxyl radicals formed by an iron-catalyzed Haber-Weiss reaction

Cultured hepatocytes pretreated with the ferric iron chelator deferoxamine were resistant to the toxicity of H2O2 generated by either glucose oxidase or by the metabolism of menadione (2-methyl-1,4-naphthoquinone). Ferric, ferrous, or cupric ions restored the sensitivity of the cells to H2O2. Deferoxamine added to hepatocytes previously treated with this chelator prevented the restoration of cell killing by only ferric iron. The free radical scavengers mannitol, thiourea, benzoate, and 4-methylmercapto-2-oxobutyrate protected either native cells exposed to H2O2 or pretreated hepatocytes exposed to H2O2 and given ferric or ferrous iron. Superoxide dismutase prevented the killing of native hepatocytes by either glucose oxidase or menadione. With deferoxamine-pretreated hepatocytes, superoxide dismutase prevented the cell killing dependent upon the addition of ferric but not ferrous iron. Catalase prevented the killing by menadione of deferoxamine-pretreated hepatocytes given either ferric or ferrous iron. Deferoxamine pretreatment did not prevent the toxicity of t-butyl hydroperoxide but did, however, prevent that of cumene hydroperoxide. It is concluded that both ferric iron and superoxide ions are required for the killing of cultured hepatocytes by H2O2. The toxicity of H2O2 is also dependent upon its reaction with ferrous iron to form hydroxyl radicals by the Fenton reaction. The ferrous iron needed for this reaction is formed by the reduction of cellular ferric iron by superoxide ions. Such a sequence corresponds to the so-called iron-catalyzed Haber-Weiss reaction, and the present report documents its participation in the killing of intact hepatocytes by H2O2. Cumene hydroperoxide but not t-butyl hydroperoxide closely models the toxicity of hydrogen peroxide.     

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.     

Nonenzymatic reduction of ferric leghemoglobin

Ferric leghemoglobin isolated from soybean root nodules was reduced nonenzymatically to ferrous leghemoglobin in vitro at pH 5.2 using either 1.0 mM NADH or NADPH as the reductant. In the pH range of 5.2 to 7.0, the highest rates of reduction occurred below pH 6.5 with a maximum rate observed at pH 5.2. Rates of nonenzymatic ferric leghemoglobin reduction above pH 6.5 or at reduced-pyridine nucleotide concentrations below 0.4 mM were insignificant. Oxygen was required for the nonenzymatic reduction. Inhibition of ferric leghemoglobin reduction by superoxide dismutase and catalase indicated that superoxide and hydrogen peroxide may be intermediates in the reaction.     

Cytochrome P450 and steroid hydroxylase activity in mouse olfactory and vomeronasal mucosa

Neopterin and 7,8-dihydroneopterin, two compounds which are secreted by activated macrophages, have been shown to interfere with radicals generated by cellular and certain chemical systems. Reduced pterins were reported to scavenge whereas aromatic pterins promoted or reduced radical mediated reactions or had no effect. However, recently it was found that high concentrations of 7, 8-dihydroneopterin enhanced luminol dependent chemiluminescence and T-cell apoptosis, suggesting an enhancement of free radical formation. In this study hydroxylation of salicylic acid was used for detection of hydroxyl radicals. It is shown that in solutions of 7,8-dihydroneopterin hydroxyl radicals were formed in the absence of any radical source. The presence of EDTA chelated iron enhanced hydroxyl radical formation. Whereas the addition of iron accelerated the hydroxylation reaction, 7,8-dihydroneopterin was responsible for the amount of hydroxylation products. In the presence of superoxide dismutase or catalase, as well as by helium purging, hydroxylation was inhibited. Our data suggest that in solutions of 7, 8-dihydroneopterin superoxide radicals are generated which are converted to hydroxyl radicals by Fenton or Haber-Weiss type reactions. While superoxide might be generated during autoxidation of ferrous iron, dihydroneopterin seems to be involved in regeneration of ferrous iron from the ferric form.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase

An enzyme system from Escherichia coli activates an inactive form of ribonucleotide reductase by transforming a tyrosine residue of the enzyme into a cationic free radical. The process requires NAD(P)H, a flavin, dithiothreitol, and oxygen and at least three proteins. After purification to near homogeneity two of the proteins were identified as superoxide dismutase and NAD(P)H:flavin oxidoreductase (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). The nature of the third protein, provisionally named Fraction b, is unknown. The flavin reductase is believed to reduce the ferric iron center of the ribonucleotide reductase as a prerequisite for radical generation. Here we demonstrate that the flavin reductase under aerobic conditions generates superoxide anions which inactivate ribonucleotide reductase. Superoxide dismutase protects the enzyme or a sensitive intermediate formed during the generation of the tyrosyl radical from the harmful effects of superoxide. Hydrogen peroxide, formed by superoxide dismutase, is also harmful. In this case, catalase present in Fraction b might protect the system. Fraction b has, however, an additional unknown function in the overall process of radical generation.     

Role of iron in the interaction of red blood cells with methylglyoxal. Modification of L-arginine by methylglyoxal is catalyzed by iron redox cycling.

Diabetes mellitus is characterized by increased methylglyoxal (MG) production. The aim of the present study was to investigate the role of iron in the cellular and molecular effects of MG. A red blood cell (RBC) model and L-arginine were used to study the effects of MG in the absence and presence of iron. Intracellular free radical formation and calcium concentration were measured using dichlorofluorescein and Fura-2-AM, respectively. Effects of MG were compared to the effect of ferrous iron. Reaction of L-arginine with MG was investigated by electron spin resonance (ESR) spectroscopy and by a spectrophotometric method. MG caused an iron dependent oxidative stress in RBCs and an elevation of the intracellular calcium concentration due to formation of reactive oxygen species. Results of co-incubation of MG with ferrous iron in the RBC model suggested an interaction of MG and iron; one interaction was a reduction of ferric iron by MG. A role of iron in the MG-L-arginine reaction was also verified by ESR spectroscopy and by spectrophotometry. Ferric iron increased free radical formation as detected by ESR in the MG-L-arginine reaction; however, ferrous iron decreased it. The reaction of MG with L-arginine yielded a brown product as detected spectrophotometrically and this reaction was catalyzed at a lower rate with ferric iron but at a higher rate with ferrous iron. These data suggest that MG causes oxidative stress in cells, which is due at least in part to ferric iron reduction by MG and to the modification of amino acids e.g. L-arginine by MG, which is catalyzed by iron redox cycling.     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

The reaction of ferric- and ferrous salts with bleomycin.

1. A comparative study shows that ferrous ions give a much better yield of Fe(III)-bleomycin than ferric ions, when iron salt is added to bleomycin in a buffer solution (pH 7.2). 2. The amount of Fe(III)-bleomycin formed after addition of ferric ions was markedly increased in the presence of ferric ion binding compounds (BSA, citrate) or reducing agents (ascorbate, cysteine).     

Ferritin and superoxide-dependent lipid peroxidation

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.     

DNA breakage induced by 1,2,4-benzenetriol: relative contributions of oxygen-derived active species and transition metal ions

We report here the relative roles of metals and selected reactive oxygen species in DNA damage by the genotoxic benzene metabolite 1,2,4-benzenetriol, and the interactions of antioxidants in affording protection. 1,2,4-Benzenetriol induces scission in supercoiled phage DNA in neutral aqueous solution with an effective dose (ED(50)) of 6.7 microM for 50% cleavage of 2.05 microg/ml supercoiled PM2 DNA. In decreasing order of effectiveness: catalase (20 U/ml), formate (25 mM), superoxide dismutase (20 U/ml), and mannitol (50 mM) protected, from 85 to 28%. Evidently, H(2)O(2) is the dominant active species, with O(2)(*)(-) and *OH playing subordinate roles. Desferrioxamine or EDTA inhibited DNA breakage by 81-85%, despite accelerating 1,2,4-benzenetriol autoxidation. Consistent with this suggestion of a crucial role for metals, addition of cupric, cuprous, ferric, or ferrous ions enhanced DNA breakage, with copper being more active than iron. Combinations of scavengers protected more effectively than any single scavenger alone, with implications for antioxidants acting in concert in living cells. Synergistic combinations were superoxide dismutase with *OH scavengers, superoxide dismutase with desferrioxamine, and catalase with desferrioxamine. Antagonistic (preemptive) combinations were catalase with superoxide dismutase, desferrioxamine with *OH scavengers, and catalase with *OH scavengers. The most striking aspect of synergism was the extent to which metal chelation (desferrioxamine) acted synergistically with either catalase or superoxide dismutase to provide virtually complete protection. Concluding, 1,2,4-benzenetriol-induced DNA damage occurs mainly by site-specific, Fenton-type mechanisms, involving synergism between several reactive intermediates. Multiple antioxidant actions are needed for effective protection.     

A catalyst function for MPTP in superoxide formation

We demonstrate that 1-methyl-4-phenyl-1,2-dihydropyridine (MPDP) can be generated, in an alternate pathway, from the catalyst action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon the iron redox equilibrium reaction. Superoxide and ferric iron are instantaneously produced after addition of MPTP to a solution of ferrous iron. This reaction is oxygen and pH dependent. Superoxide, through a iron dependent Haber-Weiss reaction with peroxide, can generate the cytotoxic hydroxyl radical. A small portion of the superoxide reacts with MPTP to produce the reactive species X. which, in the presence of Fe+3 can also generate MPDP.     

The iron superoxide dismutase from the filamentous cyanobacterium Nostoc PCC 7120. Localization, overexpression, and biochemical characterization

The nitrogen-fixing filamentous cyanobacterium Nostoc PCC 7120 (formerly named Anabaena PCC 7120) possesses two genes for superoxide dismutase, a unique membrane-associated manganese superoxide dismutase (MnSOD) and a soluble iron superoxide dismutase (FeSOD). A phylogenetic analysis of FeSODs shows that cyanobacterial enzymes form a well separated cluster with filamentous species found in one subcluster and unicellular species in the other. Activity staining, inhibition patterns, and immunogold labeling show that FeSOD is localized in the cytosol of vegetative cells and heterocysts (nitrogenase containing specialized cells formed during nitrogen-limiting conditions). The recombinant Nostoc FeSOD is a homodimeric, acidic enzyme exhibiting the characteristic iron peak at 350 nm in its ferric state, an almost 100% occupancy of iron per subunit, a specific activity using the ferricytochrome assay of (2040 +/- 90) units mg(-1) at pH 7.8, and a dissociation constant Kd of the azide-FeSOD complex of 2.1 mM. Using stopped flow spectroscopy it was shown that the decay of superoxide in the presence of various FeSOD concentrations is first-order in enzyme concentration allowing the calculation of the catalytic rate constants, which increase with decreasing pH: 5.3 x 10(9) M(-1) s(-1) (pH 7) to 4.8 x 10(6) M(-1) s(-1) (pH 10). FeSOD and MnSOD complement each other to keep the superoxide level low in Nostoc PCC 7120, which is discussed with respect to the fact that Nostoc PCC 7120 exhibits oxygenic photosynthesis and oxygen-dependent respiration within a single prokaryotic cell and also has the ability to form differentiated cells under nitrogen-limiting conditions.     

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.