Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms.

Membranes from Spirillum itersonii reduce ferric iron to ferrous iron with reduced nicotinamide adenine dinucleotide or succinate as a source of reductant. Iron reduction was measured spectrophotometrically at 562 nm using ferrozine, which chelates ferrous iron specifically. Reduced nicotinamide adenine dinucleotide or succinate was also effective as a source of iron. The effects of respiratory inhibitors suggested that reduction of iron occurs at one or more sites on the respiratory chain before cytochrome c. Reduction of iron and synthesis of protoheme with the physiological reductants were also observed with crude extracts of other bacteria, including Rhodopseudomonas spheroides, Rhodopseudomonas capsulata, Paracoccus denitrificans, and Escherichia coli. The effect of oxygen upon reduction of iron and formation of protoheme was examined with membranes from S. itersonii, using succinate as a source of reductant. Both systems were inhibited by oxygen, but this effect was completely reversed by addition of antimycin A. We conclude that reduced components of the respiratory chain serve as reductants for ferric iron, but with oxygen present they are oxidized preferentially by the successive members of the chain. This could be a mechanism for regulating synthesis of heme and cytochrome by oxygen.     

Metabolic characterization of lactic acid bacterium Lactococcus garvieae sk11, capable of reducing ferric iron, nitrate, and fumarate

A lactic acid bacterium capable of anaerobic respiration was isolated from soil with ferric iron-containing glucose basal medium and identified as L. garvieae by using 16S rDNA sequence homology. The isolate reduced ferric iron, nitrate, and fumarate to ferrous iron, nitrite, and succinate, respectively, under anaerobic N2 atmosphere. Growth of the isolate was increased about 30-39% in glucose basal medium containing nitrate and fumarate, but not in the medium containing ferric iron. Specifically, metabolic reduction of nitrate and fumarate is thought to be controlled by the specific genes fnr, encoding FNR-like protein, and nir, regulating fumarate-nitrate reductase. Reduction activity of ferric iron by the isolate was estimated physiologically, enzymologically, and electrochemically. The results obtained led us to propose that the isolate metabolized nitrate and fumarate as an electron acceptor and has specific enzymes capable of reducing ferric iron in coupling with anaerobic respiration.     

Changing the heme ligation in flavocytochrome b 2: substitution of histidine-66 by cysteine

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.     

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.     

Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M _r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M _r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with ⁵⁵Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with ⁹⁹Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae.

We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.     

sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus

Staphylococcus aureus has membrane-associated sn-glycerol-3-phosphate dehydrogenase activity that is strongly activated by detergents. The enzyme can be measured spectrophotometrically in intact cells in assay systems containing lauryldimethylamine oxide (Ammonyx LO). The dehydrogenase activity was located exclusively in the membrane fraction of cells grown with glycerol under aerobic conditions or under anaerobic conditions with the addition of nitrate; there was no evidence of multiple forms. Development of sn-glycerol-3-phosphate dehydrogenase activity was studied with suspensions of cells grown previously under semianaerobic conditions with glucose and nitrate. The wild-type strain rapidly formed the enzyme when incubated with glycerol under aerobic conditions or under semianaerobic conditions in the presence of nitrate. Under similar conditions, suspensions of hem mutant H-14 required the addition of hemin. Induction of the enzyme was strongly repressed by glucose with both organisms. A procedure was established to obtain cells of mutant H-14 with sn-glycerol-3-phosphate dehydrogenase and nitrate reductase activities, but which could not link the systems unless supplemented with hemin. The coupled activity could also be reconstructed in vitro by the addition of hemin to the depleted membranes.     

Enzymatic reduction of iron oxide by fungi

The occurrence of the iron-reducing phenomenon among some common fungi was studied. Results indicated that (i) the reduction of ferric iron to the ferrous state by fungi seems to be restricted to nitrate reductase-inducible strains such as Actinomucor repens, Alternaria tenuis, Fusarium oxysporum, and F. solani and (ii) the amount of dissolved ferrous iron may be reduced progressively by increasing the amount of nitrate added to the medium. Compared with a complex medium (Sabouraud medium), less iron became reduced if NO₃^- was the only nitrogen source (Czapek Dox medium). These data strongly support the view that ferric iron is acting as an hydrogen acceptor in respiration, competing with nitrate for electrons that are mediated by the enzyme nitrate reductase. The significance of this property from an ecological viewpoint is discussed.     

The reactions of heme- and verdoheme-heme oxygenase-1 complexes with FMN-depleted NADPH-cytochrome P450 reductase. Electrons required for verdoheme oxidation can be transferred through a pathway not involving FMN

Electrons utilized in the heme oxygenase (HO) reaction are provided by NADPH-cytochrome P450 reductase (CPR). To investigate the electron transfer pathway from CPR to HO, we examined the reactions of heme and verdoheme, the second intermediate in the heme degradation, complexed with rat HO-1 (rHO-1) using a rat FMN-depleted CPR; the FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/Y178A, against 2 m KBr. Degradation of heme in complex with rHO-1 did not occur with FMN-depleted CPR, notwithstanding that the FMN-depleted CPR was able to associate with the heme-rHO-1 complex with a binding affinity comparable with that of the wild-type CPR. Thus, the first electron to reduce the ferric iron of heme complexed with rHO-1 must be transferred from FMN. In contrast, verdoheme was converted to the ferric biliverdin-iron chelate with FMN-depleted CPR, and this conversion was inhibited by ferricyanide, indicating that electrons are certainly required for conversion of verdoheme to a ferric biliverdin-iron chelate and that they can be supplied from the FMN-depleted CPR through a pathway not involving FMN, probably via FAD. This conclusion was supported by the observation that verdoheme dimethyl esters were accumulated in the reaction of the ferriprotoporphyrin IX dimethyl ester-rHO-1 complex with the wild-type CPR. Ferric biliverdin-iron chelate, generated with the FMN-depleted CPR, was converted to biliverdin by the addition of the wild-type CPR or desferrioxamine. Thus, the final electron for reducing ferric biliverdin-iron chelate to release ferrous iron and biliverdin is apparently provided by the FMN of CPR.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity

Tyrosine hydroxylase (TyrH) is a pterin-dependent enzyme that catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine. The oxidation state of the active site iron atom plays a central role in the regulation of the enzyme. The kinetics of reduction of ferric TyrH by several reductants were determined by anaerobic stopped-flow spectroscopy. Anaerobic rapid freeze-quench EPR confirmed that the change in the near-UV absorbance of TyrH upon adding reductant corresponded to iron reduction. Tetrahydrobiopterin reduces wild-type TyrH following a simple second-order mechanism with a rate constant of 2.8 +/- 0.1 mM(-)(1) s(-)(1). 6-Methyltetrahydropterin reduces the ferric enzyme with a second-order rate constant of 6.1 +/- 0.1 mM(-)(1) s(-)(1) and exhibits saturation kinetics. No EPR signal for a radical intermediate was detected. Ascorbate, glutathione, and 1,4-benzoquinone all reduce ferric TyrH, but much more slowly than tetrahydrobiopterin, suggesting that the pterin is a physiological reductant. E332A TyrH, which has an elevated K(m) for tetrahydropterin in the catalytic reaction, is reduced by tetrahydropterins with the same kinetic parameters as those of the wild-type enzyme, suggesting that BH(4) does not bind in the catalytic conformation during the reduction. Oxidation of ferrous TyrH by molecular oxygen can be described as a single-step second-order reaction, with a rate constant of 210 mM(-)(1) s(-)(1). S40E TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of the wild-type enzyme, suggesting that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.     

Toxicity and bioaccumulation of iron in soil microalgae

Microalgae are extensively used in the remediation of heavy metals like iron. However, factors like toxicity, bioavailability and iron speciation play a major role in its removal by microalgae. Thus, in this study, toxicity of three different iron salts (FeSO₄, FeCl₃ and Fe(NO₃)₃) was evaluated towards three soil microalgal isolates, Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11. Interestingly, all the three iron salts gave different EC50 concentrations; however, ferric nitrate was found to be significantly more toxic followed by ferrous sulphate and ferric chloride. The EC₅₀ analysis revealed that Chlorella sp. was significantly resistant to iron compared to other microalgae. However, almost 900 μg g^?1 iron was accumulated by Chlamydomonas sp. grown with 12 mg L^?1 ferric nitrate as an iron source when compared to other algae and iron salts. The time-course bioaccumulation confirmed that all the three microalgae adsorb the ferric salts such as ferric nitrate and ferric chloride more rapidly than ferrous salt, whereas intracellular accumulation was found to be rapid for ferrous salts. However, the amount of iron accumulated or adsorbed by algae, irrespective of species, from ferrous sulphate medium is comparatively lower than ferric chloride and ferric nitrate medium. The Fourier transform infrared spectroscopy (FTIR) analysis shows that the oxygen atom and P?=?O group of polysaccharides present in the cell wall of algae played a major role in the bioaccumulation of iron ions by algae.     

Reduction of ferric citrate catalyzed by NADH:nitrate reductase

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.     

Influence of nicotianamine and iron supply on formation and elongation of adventitious roots in hypocotyl cuttings of the tomato mutant 'chloronerva' (Lycopersicon esculentum)

The influence of nicotianamine (NA) on formation and elongation of adventitious roots in hypocotyls of de-rooted NA-less mutant seedlings of Lycopersicon esculentum Mill, was examined in relation to the iron supply [ferric N-N'-ethylenediaminedi-(2-hydroxyphenylacetate) (FEDDHA), ferric ethylenediaminetetracetate (FeEDTA), ferric N-(2-hydroxyethyl)-ethylenediaminetriacetate (FeHEDTA, Fe-citrate and FeCl₃] in the nutrient solution. The initiation of root primordia in hypocotyl cuttings was independent of NA and occurred with about the same frequency in both, mutant and wild-type. In the mutant the development of primordia to adventitious roots was blocked at all iron sources used, except FeEDTA. Addition of NA (5x 10⁻⁶ to 2 × 10⁻⁵ M ) to the rooting medium resulted in a fast growth of adventitious roots in mutant cuttings with all iron sources tested. Rooting of wild-type cuttings was independent from NA application and iron sources. We suppose that NA is involved in the intracellular transport of iron. Its function is possibly linked with chelation of ferrous iron in the cell.     

Studies on the binding of iron by rabbit intestinal microvillus membranes.

1. 59Fe binding by microvillus membranes purified from rabbit intestine was studied by means of a microfiltration procedure. 2. Binding activity from ferrous ascorbate chelates was 100-fold greater than from ferric chelates of citrate and nitrilotriacetate. Dual-label experiments indicated dissociation of iron complexes before binding to the membranes. 3. Binding was inhibited at low incubation temperatures and was optimal at neutral pH. 4. Binding activity was reduced in ileal preparations when compared with membranes prepared from proximal intestine. 5. Initial binding velocity followed saturation kinetics over the range 45-450 microM-iron: it was weakly inhibited in the presence of excess Co2+ and V3+. 6. The data provide additional evidence for high-affinity iron-binding sites on the intestinal microvillus membrane and indicate properties that may reflect the functional significance of the binding step in the absorption pathway for iron.     

Role of the chlC gene in formation of the formate-nitrate reductase pathway in Escherichia coli.

Five temperature-sensitive chlC mutants were isolated from Escherichia coli by the technique of localized mutagenesis. All of the mutants produced severely reduced levels of both nitrate reductase and formate dehydrogenase when grown at 43 degrees C. In three of the mutants, the nitrate reductase activity produced at the permissive temperature was shown to be thermolabile compared with the activity produced by the parent wild-type strain, both in membrane preparations and in preparations released from the membrane by deoxycholate. In each case, formate dehydrogenase activity was similar to the wild-type activity in its stability to heat. It is concluded that the chlC gene codes for at least one of the polypeptide chains of nitrate reductase and that the chlC mutations affect indirectly the formation of formate dehydrogenase.     

Different effects of 2-n-heptyl-4-hydroxyquinoline-N-oxide on oxygen and nitrate respiration in Klebsiella aerogenes

The inhibition of the oxidase and respiratory nitrate reductase activity in membrane preparations from Klebsiella aerogenes by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) has been investigated. Addition of HQNO only slightly affected the aerobic steady-state reduction of cytochrome b₅₅₉ with NADH, but caused a significantly lower nitrate reducing steady-state of this cytochrome. The changes in the redox states of the cytochromes during a slow transition from anaerobic to aerobic conditions in the presence and absence of HQNO showed that the inhibition site of HQNO is located before cytochrome d. Inhibition patterns obtained upon titration of the NADH oxidase and NADH nitrate reductase activity with HQNO indicated one site of inhibitor interaction in the NADH nitrate reductase pathway and suggested a multilocated inhibition of the NADH oxidase pathway. Difference spectra with ascorbate-dichlorophenolindophenol as electron donor indicated the presence of a cytochrome b₅₆₃ component which was not oxidized by nitrate, but was rapidly oxidized by oxygen. The latter oxidation was prevented by HQNO. A scheme for the electron transport to oxygen and nitrate is presented. In the pathway to oxygen, HQNO inhibits both at the electron-accepting side of cytochrome b₅₅₉ and at the electron-donating side of cytochrome b₅₆₃, whereas in the pathway to nitrate, inhibition occurs only at the electron-accepting side of cytochrome b₅₅₉.