Isolation of a soluble and membrane-associated Fe(III) reductase from the thermophile, Thermus scotoductus (SA-01)

The Fe(III) reductase activity was studied in the South African Fe(III)-reducing bacterium, Thermus scotoductus (SA-01). Fractionation studies revealed that the membrane as well as the soluble fraction contained NAD(P)H-dependent Fe(III) reductase activity. The membrane-associated enzyme was solubilized by KCl treatment and purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A combination of ion-exchange and gel filtration chromatography was used to purify the soluble enzyme to apparent homogeneity. The molecular mass of the membrane-associated Fe(III) reductase was estimated to be 49 kDa, whereas the soluble Fe(III) reductase had an apparent molecular mass of 37 kDa. Optimum activity for the membrane-associated enzyme was observed at around 75 degrees C, whereas the soluble enzyme exhibited a temperature optimum at 60 degrees C.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

Apoplastic pH and Fe(3+) reduction in intact sunflower leaves

It has been hypothesized that under NO(3)(-) nutrition a high apoplastic pH in leaves depresses Fe(3+) reductase activity and thus the subsequent Fe(2+) transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO(3)(-) nutrition significantly increased apoplastic pH at distinct interveinal sites (pH >/= 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO(3)(-)/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH(4)(+) or NH(4)NO(3) nutrition. Complementary to pH measurements, the formation of Fe(2+)-ferrozine from Fe(3+)-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe(3+) reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5. 0. In analogy to the monitoring of Fe(3+) reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe(3+) reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.     

The NADH-dependent Fe3+-chelate reductases of tomato roots

The NADH-dependent Fe³⁺-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe³⁺-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe³⁺-EDTA reductase assay, which was catalyzed by at least another reductase of 46 kDa. After high-speed centrifugation of tomato root membranes, high FeCitR activities were detected in pellets and lower activities in supernatants. After two-phase partitioning of microsomes, FeCitR activity (91 nmol · min⁻¹ · mg⁻¹) was less active in the upper phase (plasma membrane) than in the lower phase (277 nmol · min⁻¹ · mg⁻¹). However, only the activity of the plasma-membrane-associated NFCHR (FeCitR) was significantly enhanced (2.6-fold) in iron-deficient tomato plants, whereas that of NFCHR in non-plasma-membrane rich fractions was unaffected by this treatment. The NFCHR obtained from lysophosphatidylcholine-solubilized plasma membrane was present as a 200-kDa protein complex following fast protein liquid chromatography on Superdex 200, or as a 28-kDa form following Blue Sepharose CL-6B chromatography. Both preparations were more active following iron starvation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 28-kDa protein purified from solubilized tomato microsomes or supernatant fractions by a final Mono Q step consisted of a single band of 32 kDa. Tomato root NFCHR resembled the NFCHR of maize (a strategy II plant, P Bagnaresi and P Pupillo, 1995, J Exp Bot 46: 1497–1503) in several properties: relative molecular mass, hydrophilicity, chromatographic behaviour, sensitivity to mercurials, specificity for electron donors and acceptors (e.g. cytochrome c), and a ferricyanide reductase-to-FeCitR ratio of 2.5. Preincubation with NADH partially protected NFCHR from PHMB-induced inactivation. Our data show that strategy I and II plants seem to share similar NFCHR proteins, which appear to belong to the cytochrome b ₅ reductase flavoprotein group. Received: 6 November 1996 / Accepted: 21 January 1997     

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

Identification and characterization of a novel ferric reductase from the hyperthermophilic Archaeon Archaeoglobus fulgidus

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing Archaeon, contains high Fe(3+)-EDTA reductase activity in its soluble protein fraction. The corresponding enzyme, which constitutes about 0.75% of the soluble protein, was purified 175-fold to homogeneity. Based on SDS-polyacrylamide gel electrophoresis, the ferric reductase consists of a single subunit with a M(r) of 18,000. The M(r) of the native enzyme was determined by size exclusion chromatography to be 40,000 suggesting that the native ferric reductase is a homodimer. The enzyme uses both NADH and NADPH as electron donors to reduce Fe(3+)-EDTA. Other Fe(3+) complexes and dichlorophenolindophenol serve as alternative electron acceptors, but uncomplexed Fe(3+) is not utilized. The purified enzyme strictly requires FMN or FAD as a catalytic intermediate for Fe(3+) reduction. Ferric reductase also reduces FMN and FAD, but not riboflavin, with NAD(P)H which classifies the enzyme as a NAD(P)H:flavin oxidoreductase. The enzyme exhibits a temperature optimum of 88 degrees C. When incubated at 85 degrees C, the enzyme activity half-life was 2 h. N-terminal sequence analysis of the purified ferric reductase resulted in the identification of the hypothetical gene, AF0830, of the A. fulgidus genomic sequence. The A. fulgidus ferric reductase shares amino acid sequence similarity with a family of NAD(P)H:FMN oxidoreductases but not with any ferric reductases suggesting that the A. fulgidus ferric reductase is a novel enzyme.     

Anaerobic respiration using Fe(3+), S(0), and H(2) in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans

The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans has been known as an aerobe that respires on iron and sulfur. Here we show that the bacterium could chemolithoautotrophically grow not only on H(2)/O(2) under aerobic conditions but also on H(2)/Fe(3+), H(2)/S(0), or S(0)/Fe(3+) under anaerobic conditions. Anaerobic respiration using Fe(3+) or S(0) as an electron acceptor and H(2) or S(0) as an electron donor serves as a primary energy source of the bacterium. Anaerobic respiration based on reduction of Fe(3+) induced the bacterium to synthesize significant amounts of a c-type cytochrome that was purified as an acid-stable and soluble 28-kDa monomer. The purified cytochrome in the oxidized form was reduced in the presence of the crude extract, and the reduced cytochrome was reoxidized by Fe(3+). Respiration based on reduction of Fe(3+) coupled to oxidation of a c-type cytochrome may be involved in the primary mechanism of energy production in the bacterium on anaerobic iron respiration.     

A partially purified putative iron P type-ATPase mediates Fe3+-transport into proteoliposome

We report that two fractions containing proteins from rat hepatocyte nuclei, obtained by nondenaturing gel electrophoresis, were able to bind iron and ATP, and to hydrolyze ATP. Electroelution of these two active fractions followed by SDS-PAGE analysis showed an identical protein pattern, each one containing four proteins in a range of 62-80 kDa. Phosphorylated protein bands were also detected in acid gel and disappeared after treatment with hydroxylamine/acetate or KOH, and upon chasing with cold ATP. A proteoliposome system, made by the incorporation of these partially purified protein fractions into phosphatidylcholine vesicles, carried out Fe(3+)-citrate uptake in a Mg(2+)-ATP-dependent way; Fe(3+) accumulation increased with time reaching a plateau in 30 min. Iron uptake was not supported by AMP-PNP, was partially inhibited by orthovanadate and was not affected by a mix of specific inhibitors of known ATPases. These results support our previous hypothesis that a putative nuclear membrane Fe(3+)-ATPase is involved in nuclear iron homeostasis.     

Redox control of calcineurin by targeting the binuclear Fe(2+)-Zn(2+) center at the enzyme active site.

The interaction of protein serine/threonine phosphatase calcineurin (CaN) with superoxide and hydrogen peroxide was investigated. Superoxide specifically inhibited phosphatase activity of CaN toward RII (DLDVPIPGRFDRRVSVAAE) phosphopeptide in tissue and cell homogenates as well as the activity of the enzyme purified under reducing conditions. Hydrogen peroxide was an effective inhibitor of CaN at concentrations several orders of magnitude higher than superoxide. Inhibition by superoxide was calcium/calmodulin-dependent. Nitric oxide (NO) antagonized superoxide action on CaN. We provide kinetic and spectroscopic evidence that native, catalytically active CaN has a Fe(2+)-Zn(2+) binuclear center in its active site that is oxidized to Fe(3+)-Zn(2+) by superoxide and hydrogen peroxide. This oxidation is accompanied by a gain of manganese dependence of enzyme activity. CaN isolated by a conventional purification procedure was found in the oxidized, ferric enzyme form, and it became increasingly dependent on divalent cations. These results point to a complex redox regulation of CaN phosphatase activity by superoxide, which is modified by calcium, NO, and superoxide dismutase.     

Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E(h)) from positive values to negative ones (down to ～-200 mV). In this study, iron (III) ions (Fe(3+)) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe(2+)) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E(h) values during bacterial growth. It was revealed that ATPase activity with and without N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase, increased in the presence of even low Fe(3+) concentration (0.05 mM) but decreased in the presence of Fe(2+). It was established that Fe(3+) and Fe(2+) both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe(2+) with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F(0)F(1)) MS116 strains but they were different with Fe(3+) and Fe(2+). It is suggested that the effects of Fe(3+) might be due to interaction of these ions with F(0)F(1) or there might be a Fe(3+)-dependent ATPase different from F(0)F(1) in these bacteria that is active even in the presence of DCCD. Fe(2+) inhibits E. hirae cell growth probably by strong effect on E(h) leading to changes in F(0)F(1) and decreasing its activity.     

Purification and characterization of fermodulin, an Fe2+-dependent inhibitor protein of 3-hydroxy-3-methylglutaryl-CoA reductase

The activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase of rat liver microsomes was inhibited by the addition of FeSO4 and the cytosolic protein, fermodulin. Modulation of the activity was obtained only in the combined presence of Fe2+ and fermodulin. Using ammonium sulfate fractionation, heat treatment, and chromatography on CM-Sephadex and then on an Fe2+-Blue Sepharose affinity matrix, fermodulin was purified to homogeneity. The molecular weight of the purified protein, as determined by filtration through a Sephacryl S-200 column, was 58,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the protein resolved into two subunits of Mr 43,000 and 28,000. Fermodulin showed ultraviolet absorption and fluorescence spectra typical of tryptophan-containing proteins, and addition of FeSO4 quenched the fluorescence. Using the Millipore filter assay the binding of 1.6 mol 55FeCl2/mol fermodulin was observed in the presence of Tris-HCl buffer. The inhibitory effect of fermodulin at nonsaturating concentrations was potentiated by bicarbonate, ATP.Mg, and ADP.Mg.     

Hormone-stimulated Mg(2+) accumulation into rat hepatocytes: a pathway for rapid Mg(2+) and Ca(2+) redistribution

Many diseases such as cardiac arrhythmia, diabetes, and chronic alcoholism are associated with a marked decrease of plasma and parenchymal Mg(2+), and Mg(2+) administration is routinely used therapeutically. This study uses isolated rat hepatocytes to ascertain if and under which conditions increases in extracellular Mg(2+) result in an increase in intracellular Mg(2+). In the absence of stimulation, changing extracellular Mg(2+) had no effect on total cellular Mg(2+) content. By contrast, carbachol or vasopressin administration promoted an accumulation of Mg(2+) that increased cellular Mg(2+) content by 13.2 and 11.8%, respectively, and stimulated Mg(2+) uptake was unaffected by the absence of extracellular Ca(2+). Mg(2+) efflux resulting from stimulation of alpha- or beta-adrenergic receptors operated with a Mg(2+):Ca(2+) exchange ratio of 1. These data indicate that cellular Mg(2+) uptake can occur rapidly and in large amounts, through a process distinct from Mg(2+) release, but operating only upon specific hormonal stimulation.     

Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Fructose-induced modifications of myoglobin: Change of structure from met (Fe3+) to oxy (Fe2+) form

We studied structural modifications of metmyoglobin (Mb) after short-term (6 days) and long-term (30 days) glycation by fructose (fructation). Fructation caused gradual changes in the structure of the protein with respect to increased absorbance at 280 nm, enhanced fluorescence emission (with excitation at 285 nm), increased surface accessible tryptophan residues and reduced α-helix content and change in tertiary structure. However, long-term fructation changed Mb to oxymyoglobin (MbO2), as demonstrated by different spectroscopic (absorption, fluorescence, circular dichroic and electron paramagnetic resonance) studies and trifluoperazine-induced oxygen release experiment. Fructation appeared to modify Arg139 to arg-pyrimidine, which exhibited antioxidative activity and might be involved in the conversion of met (Fe3+) to oxy (Fe2+) form of myoglobin.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.