Influence of Complex Structure on the Biodegradation of Iron-Citrate Complexes

The biodegradation of iron-citrate complexes depends on the structure of the complex formed between the metal and citric acid. Ferric iron formed a bidentate complex with citric acid, [Fe(III) (OH)₂ cit]^2- involving two carboxylic acid groups, and was degraded at the rate of 86 μM h^-1. In contrast, ferrous iron formed a tridentate complex with citric acid, [Fe(II) cit]^-, involving two carboxylic acid groups and the hydroxyl group, and was resistant to biodegradation. However, oxidation and hydrolysis of the ferrous iron resulted in the formation of a tridentate ferric-citrate complex, [Fe(III)OH cit]^-, which was further hydrolyzed to a bidentate complex, [Fe(III)(OH)₂ cit]^2-, that was readily degraded. The rate of degradation of the ferrous-citrate complex depended on the rate of its conversion to the more hydrolyzed form of the ferric-citrate complex. Bacteria accelerated the conversion much more than did chemical oxidation and hydrolysis.     

Influence of metal complexation on the metabolism of citrate byKlebsiella oxytoca

The uptake of ¹⁴C-labeled cadmium-, copper and zinc-citrate into cells of Klebsiella oxytoca was followed. The study was made in order to examine if the earlier reported disability of the bacterium to degrade these complexes was due to an inhibition in transport through the cell membrane. Citrate complexed to cadmium, copper or zinc was taken up at a similar rate to the free citric acid. However, the metal-citrate complexes were not metabolized as shown by the marked accumulation of ¹⁴C in the cells as compared with the ¹⁴C content in the cells incubated with free citric acid. This was confirmed by the results from trichloroacetic acid-precipitation showing that no ¹⁴C was incorporated into macromolecules when the citrate was complexed to the different metals. It is suggested that the inhibited degradation was due to effects on the interaction between enzyme (aconitase) and substrate in the conversion of citrate to iso-citrate. The role of complex configuration on the mineralization of metal-citrate is discussed and also tested in mineralization studies of other metal-citrate complexes (aluminum-, calcium-, cobalt-, manganese- and nickel-citrate).     

R161, K452 and R460 residues are vital for metal-citrate complex transport in Cit(Sc) from Streptomyces coelicolor

Recent discoveries have been made that demonstrate Gram-positive bacteria can transport metal-citrate complexes through the CitMHS family of proteins in symport with H(+) ions. The CitMHS family of transporters investigated to date have the ability to selectively transport only certain metal-citrate complexes. Despite sharing amino acid sequence similarity as high as 73%; predicting what complexes are transported remains difficult. The iron-citrate transporter from Streptomyces coelicolor has been mutated at three postulated critical sites (R161, K452 and R460) based on activity modeling against the LacY permease. All three mutants eliminate or greatly reduce uptake of metal-citrate complexes tested. The implications of this are discussed.     

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.     

Determination of element distribution between the symplasm and apoplasm of cucumber plant parts by total reflection X-ray fluorescence spectrometry

The distribution of Cd, Ni, Pb and Fe between the symplasm (cytoplasm) and apoplasm (cell wall) of cucumber roots and leaves was determined by total reflection X-ray fluorescence spectrometry following a special sample preparation procedure. The plants were grown in modified Hoagland nutrient solution containing Fe in chemical form of Fe(III)-citrate or Fe(III)-EDTA, as well as the heavy metal contaminants, each in concentration of 10 microM. In the roots the larger part of Pb was found in the apoplasm, while Ni and Cd were mostly in the symplasm. In the leaves however, about 50-60% of the Pb content and practically the total amount of Cd were detected in the symplasm. About 30-40% of the translocated Ni remained in the apoplasm of the leaves. The Cd-, Ni- and Pb-treatments resulted in higher total concentration of Fe in the roots, however, the relative amount of Fe in the symplasm decreased in all cases. In the leaves of the control plants the larger part (60-80%) of Fe occurs in the symplasm. Due to the heavy metal effects, the relative amount of Fe in the symplasm decreased except in the Pb-contaminated plants, where in the presence of Fe(III)-EDTA, the Pb treatment resulted in a moderate increment of Fe concentration in the symplasm.     

Functional characterization and metal ion specificity of the metal-citrate complex transporter from Streptomyces coelicolor

Secondary transporters of citrate in complex with metal ions belong to the bacterial CitMHS family, about which little is known. The transport of metal-citrate complexes in Streptomyces coelicolor has been investigated. The best cofactor for citrate uptake in Streptomyces coelicolor is Fe(3+), but uptake was also noted for Ca(2+), Pb(2+), Ba(2+), and Mn(2+). Uptake was not observed with the Mg(2+), Ni(2+), or Co(2+) cofactor. The transportation of iron- and calcium-citrate makes these systems unique among the CitMHS family members reported to date. No complementary uptake akin to that observed for the CitH (Ca(2+), Ba(2+), Sr(2+)) and CitM (Mg(2+), Ni(2+), Mn(2+), Co(2+), Zn(2+)) systems of Bacillus subtilis was noted. Competitive experiments using EGTA confirmed that metal-citrate complex formation promoted citrate uptake. Uptake of free citrate was not observed. The open reading frame postulated as being responsible for the metal-citrate transport observed in Streptomyces coelicolor was cloned and overexpressed in Escherichia coli strains with the primary Fe(3+)-citrate transport system (fecABCDE) removed. Functional expression was successful, with uptake of Ca(2+)-citrate, Fe(3+)-citrate, and Pb(2+)-citrate observed. No free-citrate transport was observed in IPTG (isopropyl-beta-d-thiogalactopyranoside)-induced or -uninduced E. coli. Metabolism of the Fe(3+)-citrate and Ca(2+)-citrate complexes, but not the Pb(2+)-citrate complex, was observed. Rationalization is based on the difference in metal-complex coordination upon binding of the metal by citrate.     

Accumulation and mobilization of arsenate by Fe(III) polyions trapped in a Ca-polygalacturonate network

The role of Fe(III) stored at the soil–root interface in the accumulation of arsenate and the influence of citric acid on the As(V) mobility were investigated by using Ca-polygalacturonate networks (PGA). The results indicate that in the 2.5–6.2 pH range Fe(III) interacts with As(V) leading to the sorption of As(V) on Fe(III) precipitates or Fe–As coprecipitates. The FT-IR analysis of these precipitates evidenced that the interaction produces Fe(III)–As(V) inner-sphere complexes with either monodentate or bidentate binuclear attachment of As(V) depending on pH.In the 3.0–6.0 pH range, As(V) diffuses freely through the polysaccharidic matrix that was found to exert a negligible reducing action towards As(V). At pH 6.0 citric acid is able to mobilize arsenate from the As–Fe–PGA network through the complexation of the Fe(III) polyions that leads to the release of As(V).     

Transport of L-glutamic acid in the fission yeast Schizosaccharomyces pombe.

Transport of L-glutamic acid into the fission yeast Schizosaccharomyces pombe grown to the early stationary phase and preincubated for 60 min with 1% D-glucose is practically unidirectional and is mediated by a single uphill transport system with a KT of 170 microM and Jmax of 4.8 nmol min-1 (mg dry wt.)-1. The system proved to be rather non-specific since all the amino acids transported into the cells acted as potent competitive inhibitors. It has a pH optimum at 3.0-4.0, the accumulation ratio of L-glutamic acid is highest at a suspension density of 0.6-1.0 mg dry wt. per ml and decreases with increasing L-glutamic acid concentrations in the external medium. The system present in the cells after preincubation with D-glucose is unstable and its activity decays after washing the cells with water or after stopping the cytosolic proteinsynthesis with cycloheximide, with a half-time of 24 min in a reaction significantly retarded by phenylmethylsulfonyl fluoride, a serine proteinase inhibitor. The synthesis of the transport protein appears to be repressible by ammonium ions.     

Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli.

Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).     

Permeability of Plant Young Root Endodermis to Cu Ions and Cu-Citrate Complexes in Corn and Soybean

The non-selective apoplastic passage of Cu and Cu-citrate complexes into the root stele of monocotyledonous corn and dicotyledonous soybean was investigated using an inorganic-salt-precipitation technique. Either Cu ions or Cu-citrate complexes were drawn into root through the apoplast from the root growth medium, and K₄[Fe(CN)₆] was subsequently perfused through xylem vessels or the entire root cross section. Based on microscopic identification of the reddish-brown precipitates of copper ferrocyanide in the cell walls of the xylem of corn and soybean roots, Cu²⁺ passed through the endodermal barrier into the xylem of both species. When the solution containing 200 μM CuSO₄ and 400 μM sodium citrate (containing 199.98 μM Cu-citrate, 0.02 μM Cu²⁺) was drawn via differential pressure gradients into the root xylem while being perfused with K₄[Fe(CN)₆] through the entire root cross-section, reddish-brown precipitates were observed in the walls of the stele of soybean, but not corn root. However, when a CuSO₄ solution containing 0.02 or 0.2 μM free Cu²⁺ was used, no reddish-brown precipitates were detected in the stele of either of the two plants. Results indicated that endodermis was permeable to Cu-citrate complexes in primary roots of soybean, but not corn. The permeability of the endodermal barrier to the Cu-citrate complex may vary between dicotyledonous and monocotyledonous plants, which has considerable implications for chelant-enhanced phytoextraction.     

Involvement of coenzyme A thioesters in anaerobic metabolism of 4-hydroxybenzoate by Rhodopseudomonas palustris.

The initial steps of anaerobic 4-hydroxybenzoate degradation were studied in whole cells and cell extracts of the photosynthetic bacterium Rhodopseudomonas palustris. Illuminated suspensions of cells that had been grown anaerobically on 4-hydroxybenzoate and were assayed under anaerobic conditions took up [U-14C]4-hydroxybenzoate at a rate of 0.6 nmol min-1 mg of protein-1. Uptake occurred with high affinity (apparent Km = 0.3 microM), was energy dependent, and was insensitive to external pH in the range of 6.5 to 8.2 Very little free 4-hydroxybenzoate was found associated with cells, but a range of intracellular products was formed after 20-s incubations of whole cells with labeled substrate. When anaerobic pulse-chase experiments were carried out with cells incubated on ice or in darkness, 4-hydroxybenzoyl coenzyme A (4-hydroxybenzoyl-CoA) was formed early and disappeared immediately after addition of excess unlabeled substrate, as would be expected of an early intermediate in 4-hydroxybenzoate metabolism. A 4-hydroxybenzoate-CoA ligase activity with an average specific activity of 0.7 nmol min-1 mg of protein-1 was measured in the soluble protein fraction of cells grown anaerobically on 4-hydroxybenzoate. 4-Hydroxybenzoyl-CoA was the sole product formed from labeled 4-hydroxybenzoate in the ligase reaction mixture. 4-Hydroxybenzoate uptake and ligase activities were present in cells grown anaerobically with benzoate, 4-hydroxybenzoate, and 4-aminobenzoate and were not detected in succinate-grown cells. These results indicate that the high-affinity uptake of 4-hydroxybenzoate by R. palustris is due to rapid conversion of the free acid to its CoA derivative by a CoA ligase and that this is also the initial step of anaerobic 4-hydroxybenzoate degradation.     

Catalysis of electron transfer across phospholipid bilayers by iron-porphyrin complexes

Phospholipid vesicles containing K₃Fe(CN)₆ were prepared from egg yolk phosphatidylcholine. Hemin dimethyl ester was incorporated into these vesicles during preparation in ratios of phospholipid to hemin dimethyl ester that varied from 200 : 1 to 45000 : 1. Electron transfer across the bilayer was measured anaerobically after injecting the vesicles into a solution containing reduced indigotetrasulfonic acid. Vesicles containing hemin dimethyl ester exhibited high rates of electron transfer (240 electrons/molecule hemin dimethyl ester per min). Conditions could be selected where the rate-limiting step for catalysis was either the bimolecular reaction between ferric hemin dimethyl ester and reduced indigotetrasulfonic acid or the movement of hemin dimethyl ester from interface to interface. The hemin dimethyl ester-catalyzed electron transfer went to completion within a few seconds, completely oxidizing the reduced indigotetrasulfonic acid. Valinomycin (in the presence of potassium) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were without effect on catalyzed electron transport. Thus, the electron transport is not electrogenic but is a coupled, neutral system. By specific assay, neither phosphate nor cyanide was significantly transported during electron transfer but evidence is provided to suggest that a coordinated hydroxide accompanies movement of Fe(III) hemin dimethyl ester from the inside surface to the outside surface of the bilayer. It was also demonstrated in a bulk phase transport system that hemin dimethyl ester readily catalyzes transfer of S¹⁴CN^? through a chloroform layer separating two aqueous phases. Another more hydrophobic iron-porphyrin complex, Fe(III) tetraphenylporphyrin, was found to be twice as effective as hemin dimethyl ester. Other porphyrin complexes were also tested as control systems. No significant catalysis was found for metal-free protoporphyrin IX dimethyl ester or Ni(II) tetraphenylporphyrin. The results are discussed in comparison with in vivo electron transport and the future usefulness of this model system.     

Fate of the Nitrilotriacetic Acid-Fe(III) Complex during Photodegradation and Biodegradation by Rhodococcus rhodochrous

Aminopolycarboxylic acids are ubiquitous in natural waters and wastewaters. They have the ability to form very stable water-soluble complexes with many metallic di- or trivalent ions. The iron complex nitrilotriacetic acid-Fe(III) (FeNTA) has been previously shown to increase drastically the rate of photo- and biodegradation of 2-aminobenzothiazole, an organic pollutant, by Rhodococcus rhodochrous. For this paper, the fate of FeNTA was investigated during these degradation processes. First, it was shown, using in situ ¹H nuclear magnetic resonance, that the complex FeNTA was biodegraded by Rhodococcus rhodochrous cells, but the ligand (NTA) alone was not. This result indicates that FeNTA was transported and biotransformed inside the cell. The same products, including iminodiacetic acid, glycine, and formate, were obtained during the photo- and biodegradation processes of FeNTA, likely because they both involve oxidoreduction mechanisms. When the results of the different experiments are compared, the soluble iron, measured by spectrophotometry, was decreasing when microbial cells were present. About 20% of the initial iron was found inside the cells. These results allowed us to propose detailed mechanistic schemes for FeNTA degradation by solar light and by R. rhodochrous.     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

Microbial Metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation

Benzene and toluene were biodegraded when chelated Fe(III) served as the terminal electron acceptor in aquifer sediments contaminated by a petroleum refinery. Benzene biodegradation ceased when Fe(III) was depleted but resumed upon reamendment. Microorganisms from the same sediments degraded toluene, but not benzene, under nitrate reducing conditions. However, the anaerobic oxidation of Fe(II) to Fe(III) was also observed in toluene-degrading incubations. Fe(II) oxidation was dependent on the presence of nitrate and enhanced when organic electron donors were provided. Microbial nitrate-linked Fe(II) oxidation was also documented in other petroleum-contaminated aquifer sediments, sludge from an oil–water separator, a landfill leachate-impacted aquifer and a garden soil. These observations suggest that some of the reported effects of nitrate on hydrocarbon biodegradation may be indirect through the reoxidation of Fe(II).     

Transepithelial transport of ferulic acid by monocarboxylic acid transporter in Caco-2 cell monolayers

Our previous study (Biosci. Biotechnol. Biochem., 66, 2449-2457 (2002)), suggested that ferulic acid was transported via a monocarboxylic acid transporter (MCT). Transepithelial transport of ferulic acid was examined in this study by directly measuring the rate of its transport across Caco-2 cell monolayers. Ferulic acid transport was dependent on pH, and in a vectorical way in the apical-basolateral direction. The permeation of ferulic acid was concentration-dependent and saturable; the Michaelis constant was 16.2 mM and the maximum velocity was 220.4 nmol min-1 (mg protein)-1. Various substrates for MCTs, such as benzoic acid and acetic acid, strongly inhibited the permeation of ferulic acid, demonstrating that ferulic acid is obviously transported by MCT. Antioxidative phenolic acid compounds from dietary sources like ferulic acid would be recognized and transported by MCT by intestinal absorption.     

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.     

MgATP-Dependent Transport of Phytochelatins Across the Tonoplast of Oat Roots

In Cd-exposed oat (Avena sativa) roots Cd was found to be associated primarily with the phytochelatin ([gamma]-glutamylcysteinyl)3-glutamic acid [([gamma]EC)3G], with a peptide to Cd ratio of 1:3 (cysteine to Cd ratio of 1:1), even though both ([gamma]EC)2G and ([gamma]EC)3G were present in the roots. Phytochelatins are known to accumulate in the vacuoles of plant cells on exposure to Cd, but the mechanism is not clear. Here we present evidence for the transport of the phytochelatins ([gamma]EC)3G and ([gamma]EC)2G as well as the Cd complex Cd-([gamma]EC)3G across the tonoplast of oat roots. Transport of ([gamma]EC)3G had a Km, for MgATP of 0.18 mM and a Vmax of 0.7 to 1 nmol mg-1 protein min-1. Transport of ([gamma]EC)3G was also energized by MgGTP and to a lesser extent MgUTP and was highly sensitive to orthovanadate, with a 50%-inhibitory concentration of 0.9 [mu]M. The Cd complex Cd-([gamma]EC)3G and ([gamma]EC)2G were also transported in a MgATP-dependent, vanadate-sensitive manner. Therefore, this process is a candidate for the transport of both phytochelatins, and Cd as its peptide complex, from the cytoplasm into the vacuole.     

The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putrefaciens. Part 2. Oxo-and hydroxo-bridged polynuclear Fe(III) complexes

The susceptibility to dissimilatory reduction of polynuclear oxo- and hydroxo-bridged Fe(III) complexes byShewanella putrefaciens intact cells and membranes has been investigated. These complexes were ligated by the potential tetradentates heidi (H₃heidi =N-(2-hydroxyethyl)iminodiacetic acid) or nta (H₃nta = nitrilotriacetic acid), or the potential tridentate ida (H₂ida = iminodiacetic acid). A number of defined small complexes with varied nuclearity and solubility properties were employed, as well as undefined species prepared by mixing different molar ratios of ida or heidi:Fe(III) in solution. The rates of Fe(III) reduction determined by an assay for Fe(II) formation with ferrozine were validated by monitoringc-type cytochrome oxidation and re-reduction associated with electron transport. For the undefined Fe(III) polymeric species, reduction rates in whole cells and membranes were considerably faster in the presence of heidi compared to ida. This is believed to result from generally smaller and more reactive clusters forming with heidi as a consequence of the alkoxo function of this ligand being able to bridge between Fe(III) nuclei, with access to an Fe(III) reductase located at the cytoplasmic membrane being of some importance. The increases in reduction rates of the undefined ida species with Fe(III) using membranes relative to whole cells reinforce such a view. Using soluble synthetic Fe(III) clusters, slow reduction was noted for an oxo-bridged dimer coordinatively saturated with ida and featuring unligated carboxylates. This suggests that sterically hindering the cation can influence enzyme action. A heidi dimer and a heidi multimer (17 or 19 Fe(III) nuclei), which are both of poor solubility, were found to be reduced by whole cells, but dissimilation rates increased markedly using membranes. These data suggest that Fe(III) reductase activity may be located at both the outer membrane and the cytoplasmic membrane ofS. putrefaciens. Slower reduction of the heidi multimer relative to the heidi dimer reflects the presence of a central hydroxo(oxo)-bridged core containing nine Fe(III) nuclei within the former cluster. This unit is a poor substrate for dissimilation, owing to the fact that the Fe(III) is not ligated by aminocarboxylate. The faster reduction noted for the heidi dimer in membranes than for a soluble ida monomer suggests that the presence of ligating water molecules may relieve steric hindrance to enzyme attack. Furthermore, reduction of an insoluble oxo-bridged nta dimer featuring ligating water molecules in intact cells was faster than that of a soluble monomer coordinatively saturated by nta and possessing an unligated carboxylate. This suggests that steric factors may override solubility considerations with respect to the susceptibility to reduction of certain Fe(III) complexes by the bacterium.Previous paper in this series: Dobbin PS, Powell AK, McEwan AG, Richardson DJ. 1995 The influence of chelating agents upon the dissimilatory reduction of Fe(III) byShewanella putefraciens.BioMetals 8, 163–173.     

The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cells was accompanied by an increase in whole cell nitrogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein:MoFe protein was 1.47 +/- 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen-input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed X min-1 X (mg Fe protein)-1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell-free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed X min-1 X (mg Fe protein)-1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis.