Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)₃Cit₂Mal₂, Fe(III)₃Cit₃Mal₁, Fe(III)Cit₂). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled ⁵⁵Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

Investigation of iron pools in cucumber roots by Mössbauer spectroscopy: direct evidence for the Strategy I iron uptake mechanism

Distinct chemical species of iron were investigated by Mössbauer spectroscopy during iron uptake into cucumber roots grown in unbuffered nutrient solution with or without ⁵⁷Fe-citrate. Mössbauer spectra of iron deficient roots supplied with 10–500 μM ⁵⁷Fe-citrate for 30–180 min and 24 h and iron-sufficient ones, were recorded. The roots were analysed for Fe concentration and Fe reductase activity. The Mössbauer parameters in the case of iron-sufficient roots revealed high-spin iron(III) components suggesting the presence of Fe^III-carboxylate complexes, hydrous ferric oxides and sulfate–hydroxide containing species. No Fe^II was detected in these roots. However, iron-deficient roots supplied with 0.5 mM ⁵⁷Fe^III-citrate for 30 min contained significant amount of Fe^II in a hexaaqua complex form. This is a direct evidence for the Strategy I iron uptake mechanism. Correlation was found between the decrease in Fe reductase activity and the ratio of Fe^II–Fe^III components as the time of iron supply was increased. The data may refer to a higher iron reduction rate as compared to its uptake/reoxidation in the cytoplasm in accordance with the increased reduction rate in iron deficient Strategy I plants.     

Yellow stripe1. Expanded roles for the maize iron-phytosiderophore transporter

Graminaceous monocots, including most of the world's staple grains (i.e. rice, corn, and wheat) use a chelation strategy (Strategy II) for primary acquisition of iron from the soil. Strategy II plants secrete phytosiderophores (PS), compounds of the mugineic acid family that form stable Fe(III) chelates in soil. Uptake of iron-PS chelates, which occurs through specific transporters at the root surface, thus represents the primary route of iron entry into Strategy II plants. The gene Yellow stripe1 (Ys1) encodes the Fe(III)-PS transporter of maize (Zea mays). Here the physiological functions performed by maize YS1 were further defined by examining the pattern of Ys1 mRNA and protein accumulation and by defining YS1 transport specificity in detail. YS1 is able to translocate iron that is bound either by PS or by the related compound, nicotianamine; thus, the role of YS1 may be to transport either of these complexes. Ys1 expression at both the mRNA and protein levels responds rapidly to changes in iron availability but is not strongly affected by limitation of copper or zinc. Our data provide no support for the idea that YS1 is a transporter of zinc-PS, based on YS1 biochemical activity and Ys1 mRNA expression patterns in response to zinc deficiency. YS1 is capable of transporting copper-PS, but expression data suggest that the copper-PS uptake has limited significance in primary uptake of copper.     

Arsenic accumulation by aquatic macrophyte coontail (Ceratophyllum demersum L.) exposed to arsenite,and the effect of iron on the uptake of arsenite and arsenate

In this study, the aquatic macrophyte Ceratophyllum demersum L. (coontail or hornwort) was tested for its efficiency of arsenic (As) uptake under laboratory conditions. Our results revealed that the solution pH had a significant effect on As accumulation by C. demersum (p < 0.001). The accumulation was highest at pH 5 and decreased as pH values increased. Plants that were exposed to various concentrations of arsenite (As(III)) for 24 and 48 h, exhibited tolerance and toxic responses, respectively. As accumulation by C. demersum depended on the concentrations of As(III) and the duration of exposure (p < 0.001). At 40 μM after 24 h, plants accumulated 227.5 μg As g⁻¹ dw and showed no visible symptoms of toxicity. However, after 48 h, As level reached 302.4 μg g⁻¹ dw and biomass production decreased significantly. Toxic effects were evident by plant necrosis and negative biomass production, leading to a decrease in the amount of accumulated As. Also, the addition of iron (Fe) into the nutrient solutions (0.18 mM) had contrasting effects on the uptake of 2 As species – the uptake of As(III) was enhanced by the presence of Fe, but the uptake of arsenate (As(V)) was considerably inhibited.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)₃], with 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m_S = ± 1/2 (g_eff ~ 9) or m_S = ± 3/2 (g_eff ~ 4.3) states. Mössbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H₃₇Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the “second-line” therapeutic drugs.     

Iron uptake and storage in the HAB dinoflagellate <Emphasis Type="Italic">Lingulodinium polyedrum</Emphasis>

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive–oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to ⁵⁷Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.     

Role of membrane surface potential and other factors in the uptake of non-transferrin-bound iron by reticulocytes

Reticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non-transferrin-bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo-cytes incubated with ⁵⁹Fe-labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K⁺, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono-, di-, tri-, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley-Liss, Inc.     

New insights into iron acquisition by cyanobacteria: an essential role for ExbB-ExbD complex in inorganic iron uptake

Cyanobacteria are globally important primary producers that have an exceptionally large iron requirement for photosynthesis. In many aquatic ecosystems, the levels of dissolved iron are so low and some of the chemical species so unreactive that growth of cyanobacteria is impaired. Pathways of iron uptake through cyanobacterial membranes are now being elucidated, but the molecular details are still largely unknown. Here we report that the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 contains three exbB-exbD gene clusters that are obligatorily required for growth and are involved in iron acquisition. The three exbB-exbDs are redundant, but single and double mutants have reduced rates of iron uptake compared with wild-type cells, and the triple mutant appeared to be lethal. Short-term measurements in chemically well-defined medium show that iron uptake by Synechocystis depends on inorganic iron (Fe′) concentration and ExbB-ExbD complexes are essentially required for the Fe′ transport process. Although transport of iron bound to a model siderophore, ferrioxamine B, is also reduced in the exbB-exbD mutants, the rate of uptake at similar total [Fe] is about 800-fold slower than Fe′, suggesting that hydroxamate siderophore iron uptake may be less ecologically relevant than free iron. These results provide the first evidence that ExbB-ExbD is involved in inorganic iron uptake and is an essential part of the iron acquisition pathway in cyanobacteria. The involvement of an ExbB-ExbD system for inorganic iron uptake may allow cyanobacteria to more tightly maintain iron homeostasis, particularly in variable environments where iron concentrations range from limiting to sufficient.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His⁴⁴, Met⁹⁰, His⁹⁷, and His¹²⁷, and CuB, a second degenerate octahedral geometry with the addition of Glu⁴⁶. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.     

Metal chelating properties of pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas spp. and the biological activities of the formed complexes

We evaluated the ability of pyridine-2,6-bis(thiocarboxylic acid) (pdtc) to form complexes with 19 metals and 3 metalloids. Pdtc formed complexes with 14 of the metals. Two of these metal:pdtc complexes, Co:(pdtc)₂ and Cu:pdtc, showed the ability to cycle between redox states, bringing to 4 the number of known redox-active pdtc complexes. A precipitant formed when pdtc was added to solutions of As, Cd, Hg, Mn, Pb, and Se. Additionally, 14 of 16 microbial strains tested were protected from Hg toxicity when pdtc was present. Pdtc also mediated protection from the toxic effects of Cd and Te, but for fewer strains. Pdtc by itself does not facilitate iron uptake, but increases the overall level of iron uptake of Pseudomonas stutzeri strain KC and P. putida DSM301. Both these pseudomonads could reduce amorphous Fe(III) oxyhydroxide in culture. In vitro reactions showed that copper and pdtc were required for this activity. This reaction may derive its reducing power from the hydrolysis of the thiocarboxyl groups of pdtc.     

Iron assimilation in Chlamydomonas reinhardtii involves ferric reduction and is similar to Strategy I in higher plants

The mechanism of adaptation to Fe-deficiency stress was investigated in the unicellular green alga, Chlamydomonas reinhardtii. Upon removal of nutritional Fe, the activity of a cell surface Fe(III)-chelate reductase was increased by at least 15-fold within 24 h. This increase was negatively corelated with the Fe concentration in the growth media. Incubation of cells in the presence of the Fe²⁺-specific chelator, bathophenanthrolinedisulphonic acid, led to an increased Fe³⁺ reductase activity, even when sufficient Fe was present. Growth of cells in Cu-free media for 48 h led to no statistically significant increase in Fe³⁺ reductase activity. The Fe(III)-chelate reductase activity in Fe-starved cells was saturable with an apparent Km of 31 M and was inhibited by uncouplers of the transmembrane proton gradient but not by SH-specific reagents.Fe uptake was only observed in Fe-deficient cells. Uptake was specific for Fe in that at 100-fold excess of a number of metal ions in the transport assay did not inhibit uptake activity. However, a 100-fold excess of Cu resulted in a 87% inhibition of Fe uptake. The Vmax for Fe³⁺ reduction activity was 250-fold greater than for Fe uptake; although the Km values for both processes differed by only 10-fold. Thus, the rate limiting step in Fe assimilation was transport and not reduction. These results indicate that Fe assimilation in C. reinhardtii involves a reductive step and thus resembles the mechanism of Fe uptake in Strategy I higher plants.Keywords: Ferric chelate reduction, iron assimilation, iron uptake, unicellular green algae, Chlamydomonas.      

Fe isotope fractionation caused by translocation of iron during growth of bean and oat as models of strategy I and II plants

Background

The determination of the plant-induced Fe-isotopic fractionation is a promising tool to better quantify their role in the geochemical Fe cycle and possibly to identify the physiological mechanisms of Fe uptake and translocation in plants. Here we explore the isotope fractionation caused by translocation of Fe during growth of bean and oat as representatives of strategy I and II plants.

Methods

Plants were grown on a nutrient solution supplemented with Fe(III)-EDTA and harvested at three different ages. We used the technique of multi-collector ICP-MS to resolve the small differences in the stable iron isotope compositions of plants.

Results

Total bean plants, regardless of their age, were found to be enriched in the light iron isotopes by ?1.2‰ relative to the growth solution throughout. During growth plants internally redistributed isotopes where young leaves increasingly accumulated the lighter isotopes whereas older leaves and the total roots were simultaneously depleted in light iron isotopes. Oat plants were also enriched in the light iron isotopes but during growth the initial isotope ratio maintained in all organs at all growth stages.

Conclusions

We conclude that isotope fractionation in bean as a representative of strategy I plants is a result of translocation or re-translocation processes. Furthermore we assume that both uptake and translocation of Fe in oat maintains the irons’ ferric state, or that Fe is always bound to high-mass ligands, so that isotope fractionation is virtually absent in these plants. However, in contrast to our previous study in which strategy II plants were grown on soil substrate, oat plants grown on Fe(III)-EDTA contain iron that enriches ⁵⁴Fe by 0.5 permil over ⁵⁶Fe. A possible explanation for the enrichment is the prevalence of a constitutive reductive uptake mechanism of iron in the nutrient solution used which is non-deficient in iron.     

Leaching behavior of iron from simulated landfills with different operational modes

The aim of the present study was to investigate the leaching behavior of iron from simulated landfills with different operation modes, with an emphasis on the variation of iron in different oxidation state, ferrous Fe(II) and ferric Fe(III) percentage and the distribution of iron content in different landfill leachate fractions. The leaching behavior and accumulated amounts of iron leached out by leachate from conventional landfill (CL) and leachate recirculated landfill (RL) exhibited decidedly different trends except for the initial 28 days. In addition, the percentage of iron leached from CL and RL accounted 1.00% and 0.14% for the total amount in landfills, respectively. No correlations between iron and selected characteristics in leachate were found were observed in the two simulated landfills. Significant positive correlations between particulate bound iron and Fe(III) were found in the leachates from RL (R² = 0.748) and CL (R² = 0.833).     

Mono- and dinuclear Fe(III) complexes with the N2O2 donor 5-chlorosalicylideneimine ligands; synthesis, X-ray structural characterization and magnetic properties

Two novel monomeric [C₁₈H₁₇Cl₃N₂O₂Fe] (1) and dimeric [C₃₈H₃₆N₄O₄Cl₆Fe₂] (2) Fe(III) tetradentate Schiff base complexes have been synthesized and their crystal structures have been determined by single crystal X-ray diffraction analysis. In complex (1) the Schiff base ligand coordinates toward one iron atom in a tetradentate mode and each iron atom is five coordinated with the coordination geometry around iron atom which can be described as a distorted square pyramid. The presence of a short (2.89 Å) non-bonding interatomic Fe···O distances between adjacent monomeric Fe(III) complexes results in the formation of a dimer. Structural analysis of compound (2) shows that the structure is a centrosymmetric dimer in which the six coordinated Fe(III) atoms are linked by μ-phenoxo bridges from one of the phenolic oxygen atoms of each Schiff base ligand to the opposite metal center. The variable-temperature (2-300 K) magnetic susceptibility (χ) data of these two compounds have been investigated. The results show that for both complexes Fe(III) centers are in the high spin configuration (S = 5/2) and indicate antiferromagnetic spin-exchange interaction between Fe(III) ions. The obtained results are briefly discussed using magnetostructural correlations developed for other class of iron(III) complexes.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.