J Biol Chem, Vol. 274, Issue 51, 36715-36721, December 17, 1999

Identification and Characterization of a Novel Ferric Reductase from the Hyperthermophilic Archaeon Archaeoglobus fulgidus^*

Alexander Vadas, Harold G. Monbouquette, Eric Johnson^§, and Imke Schröder^§¶

From the  Department of Chemical Engineering and the ^§ Department of Microbiology and Molecular Genetics, UCLA, Los Angeles, California 90095

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing Archaeon, contains high Fe³⁺-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 Fe³⁺-EDTA. Other Fe³⁺ complexes and dichlorophenolindophenol serve as alternative electron acceptors, but uncomplexed Fe³⁺ is not utilized. The purified enzyme strictly requires FMN or FAD as a catalytic intermediate for Fe³⁺ 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 °C. When incubated at 85 °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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Acquisition of iron for assimilation into cellular protein is a universal trait of life. Iron, in its many inorganic and organic forms, is the fourth most abundant element on earth (1). However, at physiological pH and under aerobic conditions, iron forms hydroxides and oxyhydroxides at exceedingly low solubilities of 10¹⁷ to 10¹⁸ M (1 µM being the threshold concentration to support life) (2, 3). For this reason, organisms acquire insoluble Fe³⁺ by complexing it with ferric-specific chelators, i.e. siderophores (2). Depending on the organism, the reduction of complexed Fe³⁺ is accomplished either before or after transport into the cell. Ferric reductase catalyzes the reduction of complexed Fe³⁺ to complexed Fe²⁺ using NAD(P)H as the electron donor (4). The resulting Fe²⁺ is subsequently released and incorporated into iron-containing proteins (5).

Assimilatory ferric reductase activities have been identified and characterized in animals, plants, yeast, and bacteria. In general, these enzymes are monomeric to homotetrameric with molecular weights ranging from 26,000 to 69,000 per monomer. The majority of the eukaryotic ferric reductases is membrane-bound and contains cytochrome b as well as covalently or non-covalently bound flavin (6-9). In contrast, prokaryotic ferric reductases lack cytochromes and bound flavin but require exogenous FMN for optimal activity (10).

Dissimilatory ferric reductases catalyze the reduction of Fe³⁺ as the terminal electron acceptor in energy metabolism (11). Iron-reducing organisms are found among the Gram-negative and Gram-positive genera of the Proteobacteria. They include the following: Shewanella putrifacience, strain GS-15, Geobacter metallireducens, Geobacter sulfurreducens, Desulfuromonas acetoxidans, Bacillus species, Thermotoga maritima, and other isolated bacteria (12-14). Two cytochrome c-type proteins with metal reductase activity were purified from G. sulfurreducens and D. acetoxidans. They also reduced complexed Fe³⁺ (15, 16). Both c-type cytochromes are located in the periplasm of the respective bacterium. Recently, Vargas et al. (17) reported iron-reducing activities in cell suspensions of certain hyperthermophilic Archaea including Archaeoglobus fulgidus. The authors (17) demonstrated that the Archaeon, Pyrobaculum islandicum, is capable of utilizing Fe³⁺-citrate as electron acceptor for growth.

Our knowledge about dissimilatory and assimilatory iron reduction in Archaea is scarce. Since little is known about the metabolism of iron by the Archaea, the purpose of this study was to investigate ferric iron reduction in the Archaeon A. fulgidus. A. fulgidus is an anaerobic, sulfate-reducing Archaeon first isolated from marine thermal vents in Southern Italy (18). In this paper, we describe the purification and characterization of a ferric reductase from this extremely thermophilic Archaeon. The A. fulgidus enzyme is the first reported ferric reductase isolated from an Archaeon.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Growth Conditions-- A. fulgidus VC-16 (DSM 4304) was grown anaerobically with 10 mM sodium lactate and 30 mM sodium sulfate at 83 °C. The medium was prepared according to published methods (18) with the following modifications: the carbonate buffer system was replaced by 20 mM PIPES,¹ pH 7.0 (pH adjusted at room temperature), and sodium acetate was omitted. Large scale cultures were grown in 70-liter batches using a custom-built 100-liter glass-lined steel fermentor (Pfaudler, Rochester, NY). The cells were harvested in late log phase (24 h after inoculation) at an optical density (OD₆₆₀) of 0.5-0.6 by concentrating with an A/G Technology hollow fiber unit (Needham, MA, nominal molecular weight cut-off 500,000). The concentrated cells were then pelleted by centrifugation for 20 min at 16,000 × g. Cell yields were approximately 80 g wet weight per 70 liters of culture.

Purification of Ferric Reductase-- All purification procedures were performed aerobically at room temperature unless indicated otherwise. Frozen cells were resuspended in 150 ml of 50 mM Tricine/KOH, pH 7.5, 2 mM MgCl₂, 2 mg DNase II. The cells were lysed by passing the cell suspension twice through a French pressure cell (Amicon) at 10,000 pounds/square inch. The insoluble fraction was removed by centrifugation at 130,000 × g for 90 min. A part of the soluble fraction (10 ml, 180 mg of protein) was loaded onto a 5-ml Q-Sepharose column (Amersham Pharmacia Biotech) equilibrated with 20 mM PIPES, pH 7.0. The column was washed with 25 ml of PIPES buffer containing 10 mM NaCl, and ferric reductase was eluted with PIPES buffer containing 60 mM NaCl. The eluted protein was applied to a 5-ml Red-agarose affinity column (Sigma) equilibrated with 20 mM PIPES, pH 7.0. After washing the column with 25 ml of the PIPES buffer containing 0.7 M NaCl, the ferric reductase was eluted with 0.9 M NaCl in PIPES buffer. Ammonium sulfate was added to the eluent to a concentration of 1.7 M, and the proteins were applied onto a 1-ml butyl-Sepharose hydrophobic interaction column (Amersham Pharmacia Biotech) equilibrated with PIPES buffer containing 1.7 M (NH₄)₂SO₄. The column was washed with buffer containing 1.25 M (NH₄)₂SO₄, and protein containing ferric reductase activity was eluted with buffer containing 1.15 M (NH₄)₂SO₄. The purified protein was stored either at 4 °C or at 20 °C for long term.

Ferric Reductase Assay-- The ferric reductase assay was adapted from the procedure described by Berczi et al. (6). The assay was performed anaerobically in stoppered quartz cuvettes at 85 °C. The assay mixture contained 50 mM sodium phosphate buffer, pH 7.0, 0.25 mM Fe³⁺-EDTA, 5 µM flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), and 0.18-36 µg of protein. The reaction was initiated by the addition of NADH or NADPH to a final concentration of 0.1 mM, and the oxidation of NADH or NADPH was monitored at 340 nm using a DU 640 spectrophotometer equipped with a high performance temperature controller (Beckman). Electron acceptors other than Fe³⁺-EDTA were used at a concentration of 0.25 mM. NAD(P)H:flavin oxidoreductase activity was measured with 50 µM FMN or FAD, and Fe³⁺-EDTA was omitted. One unit of activity is defined as 1 µmol of NAD(P)H oxidized per min.

Characterization of the pH Optimum and Stability-- The pH optimum of the ferric reductase enzyme was determined using the following buffers: 50 mM piperazine at pH 6.0, 6.5, and 6.7, 50 mM sodium phosphate at pH 6.8, 7.0, 7.2, 7.4, and 50 mM Tricine at pH 7.9. The pH stability of ferric reductase was determined by incubating the enzyme at room temperature in the buffers listed above. Samples were taken after 1 and 24 h, and ferric reductase activity was determined.

Determination of the Temperature Optimum and Stability-- The temperature optimum was determined by performing the ferric reductase assay at the indicated temperatures. The temperature stability of the purified enzyme was determined by incubating the enzyme in stoppered serum vials at the indicated temperatures. Samples were removed at the indicated times, and the activity was measured at 85 °C.

Protein Determination-- The protein concentration was measured with the Bradford assay (Bio-Rad) using bovine serum albumin as the standard.

Gel Electrophoresis-- Proteins were separated on 20% homogenous polyacrylamide gels under denaturing conditions with the Amersham Pharmacia Biotech Phastsystem. Prior to loading, the protein samples were incubated in SDS gel-loading buffer for 30 min at 100 °C (19). Precast SDS-polyacrylamide gels were purchased from Amersham Pharmacia Biotech.

Molecular Weight Determination-- The apparent molecular weight of the purified, denatured ferric reductase was determined from the mobility of the protein in SDS-PAGE. The molecular weight standards were bovine albumin (M_r 66,000), chicken egg albumin (M_r 45,000), glyceraldehyde-3-phosphate dehydrogenase (M_r 36,000), bovine carbonic anhydrase (M_r 29,000), bovine pancreas trypsinogen (M_r 24,000), soybean trypsin inhibitor (M_r 20,000), and bovine milk -lactalbumin (M_r 14, 200) (Sigma). The apparent molecular weight of the native ferric reductase was determined by size exclusion chromatography with a Superose 6 column from Amersham Pharmacia Biotech. The column was run in 50 mM PIPES, pH 7.0, 200 mM NaCl at a flow rate of 0.4 ml/min. The following molecular weight markers from Amersham Pharmacia Biotech were used: ribonuclease A (M_r 13, 700), chymotrypsinogen A (M_r 25,000), ovalbumin (M_r 43,000), and bovine serum albumin (M_r 67,000).

N-terminal Amino Acid Sequence Analysis-- The purified protein was run on a 20% polyacrylamide SDS gel and transferred to a Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad). The N-terminal sequence analysis was performed by the Protein Microsequencing Facility at UCLA.

Redox Titration of Complexed Fe³⁺-- The redox potentials of Fe³⁺-EDTA, Fe³⁺-NTA, and Fe³⁺-Citrate were determined with an Omni 90 potentiostat set to cycle the voltage at 50 mV/s (Cypress Systems Inc.). The potentiostat was equipped with an Ag/AgCl₂ reference electrode, a platinum auxiliary electrode, and a gold working electrode. Voltammetry was were performed under anaerobic conditions in 20 mM PIPES, pH 7.0. The Fe³⁺ complexes were at a concentration of 0.4 mM.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ferric Reductase Activity-- By using a modified ferric reductase assay adapted to 85 °C, high ferric reductase activity was detected in the soluble protein fraction of A. fulgidus VC-16 grown on lactate as carbon and energy source and sulfate as the electron acceptor. With NADPH as the electron donor and Fe³⁺-EDTA as the electron acceptor, the enzyme activity in the soluble fraction was determined to be 1 unit/mg protein. Addition of synthetic FMN to the assay stimulated the activity in the soluble fraction by 20-fold (i.e. 20 units/mg protein) (Table I). In contrast, the purified protein strictly required FMN as a catalytic intermediate for activity (see below). All assay components were stable at 85 °C for the duration of the assay (at least 2 min). The membrane fraction did not contain any measurable ferric reductase activity (<0.05 units/mg). Cell extract prepared from the hyperthermophilic Archaeon, Pyrobaculum aerophilum, did not exhibit ferric reductase activity (data not shown).

Purification of the Ferric Reductase-- Ferric reductase was purified to homogeneity using Q-Sepharose ion exchange, Red-agarose affinity, and butyl-Sepharose hydrophobic interaction chromatography (Fig. 1 and Table I). After the final purification step, the enzyme had been enriched 175-fold with a yield of 16%. Based on this yield, ferric reductase was calculated to comprise about 0.75% of the total soluble A. fulgidus protein (w/w). By using SDS-PAGE, it was determined that the purified ferric reductase consists of a single polypeptide with an apparent molecular weight of 18,000 (Fig. 1). The molecular weight of the native enzyme was determined to be 40,000 by size exclusion chromatography, suggesting that ferric reductase exists as a homodimer (data not shown). After purification, ferric reductase did not display any significant absorption in the visible range indicating that chromophores such as flavin, heme, and/or Fe-S centers were absent (data not shown).

View larger version (53K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE analysis of the purified A. fulgidus ferric reductase. Lane 1, 1 µg of ferric reductase; lane 2, molecular weight markers (see under "Experimental Procedures").

Flavin Dependence of Ferric Reductase-- During protein purification, it was noted that ferric reductase activity was gradually lost. When the soluble A. fulgidus protein was subjected to a desalting column, the activity was almost abolished suggesting that a low molecular weight compound was required to retain activity. A yellow-colored fraction that had been eluted from the desalting column in the column volume restored ferric reductase activity. This yellow compound exhibited a visible absorption spectrum characteristic of flavins (data not shown). The preparation was not further characterized. Synthetic FMN, FAD, but not riboflavin, also restored ferric reductase activity (Table II, data for FAD and riboflavin not shown). Without added flavin, no activity was observed demonstrating that purified ferric reduction was strictly dependent on either FMN or FAD.

To examine whether flavin could serve as the electron acceptor in place of Fe³⁺-EDTA, activity of the purified enzyme was measured in the absence of Fe³⁺-EDTA using a 10-fold higher concentration of FMN, FAD, or riboflavin than in the ferric reductase assay (Table II and Table III). Both FMN and FAD, but not riboflavin, served as electron acceptors with NADH or NADPH as electron donors. Therefore, the ferric reductase also functions as an NAD(P)H:flavin oxidoreductase similar to the ferric reductases of Saccharomyces cerevisiae and Escherichia coli (7, 20-22). Fig. 2A shows the reduction of FAD by ferric reductase with NADH as the electron donor. In contrast, FAD remains oxidized in the presence of Fe³⁺-EDTA consistent with the notion that FAD serves here as an electron mediator (Fig. 2B). Unlike many bacterial ferric reductases, the A. fulgidus enzyme does not require Mg²⁺ for optimal activity (data not shown) (2, 3, 23).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Absorption spectrum of FAD reduction with NADH by purified ferric reductase (FeR) in the absence (A) and presence of 200 µM Fe3+-EDTA (B). The reaction buffer contained 50 mM NaH2PO4-Na2HPO4, pH 7.0, 100 µM NADH, and 40 µM FAD. Spectra were recorded after anaerobic incubation for 5 min at 80 °C without (FeR) and with ferric reductase (0.25 µg) (+FeR).

Since the A. fulgidus ferric reductase lacked any associated flavin, a reconstitution of the enzyme with flavin was attempted. Ferric reductase was incubated with 1 mM FMN for 10 min at 60 °C. After incubation, the enzyme was subjected to a desalting and subsequently to an ion exchange chromatography column (i.e. Q-Sepharose). Whereas free FMN eluted in the total volume of the desalting column and did not bind to the Q-Sepharose column, the ferric reductase enzyme emerged brightly yellow-colored from the void volume of the desalting and the NaCl eluent of the Q-Sepharose column. This result demonstrates that the ferric reductase could be reconstituted with FMN. The FMN is, therefore, non-covalently bound to the enzyme. Based on the extinction coefficient for free FMN (12.2 mM¹ cm¹ at 450 nm) the amount of FMN per monomer was determined to be 1.4 mol/mol.

Kinetic Properties of the Ferric Reductase-- The kinetic properties of the ferric reductase were determined at 85 °C and are summarized in Table III. With Fe³⁺-EDTA as the electron acceptor, the purified ferric reductase exhibited a slightly higher affinity and V_max for NADH than for NADPH as the electron donor. Based on the molecular weight of the dimeric ferric reductase deduced from the gene sequence (i.e. 37, 318, see below) turnover numbers of 3069 and 2180 s¹ for NADH and NADPH oxidation, respectively, were calculated. The K_m for FMN as catalytic intermediate in ferric iron reduction and as electron acceptor in place of Fe³⁺-EDTA were identical (data not shown) and more than 200-fold lower than that for Fe³⁺-EDTA (Table III). The V_max values with FMN and FAD were about 12-fold slower than that determined with Fe³⁺-EDTA. The corresponding turnover numbers were 174 s¹ for FMN reduction and 216 s¹ for FAD reduction.

Electron Acceptors of Ferric Reductase-- Ferric reductase also exhibited activity with other complexes that contained Fe³⁺ as the electron acceptor (Table IV). The highest activity was obtained with sodium ferricyanide; it was 2.5 times higher than the activity measured with Fe³⁺-EDTA. Activities of 12% or less were obtained with other ferric complexes such as Fe³⁺-NTA and Fe³⁺-citrate. With dichlorophenolindophenol, an artificial electron acceptor of many flavoproteins, ferric reductase activity was half that with Fe³⁺-EDTA. All electron acceptors were reduced only when catalytic amounts of FMN were present. No activity was obtained with uncomplexed Fe³⁺, i.e. FeCl₃ and Fe(OH)₃ (data not shown), with the menaquinone analog dimethylnaphthoquinone or with Fe²⁺-EDTA (Table IV). The latter compound served as a control to demonstrate that the Fe³⁺ and not the EDTA part of the complex was reduced by the enzyme. Finally, ferric reductase did not reduce EDTA-chelated Ag⁺ and Cu²⁺ (data not shown).

pH and Temperature Optima and Stability of Ferric Reductase-- The A. fulgidus ferric reductase exhibited a pH optimum of 7.0 (Fig. 3). To ensure that the decrease of enzyme activity seen below and above pH 7.0 was not due to enzyme instability, ferric reductase was incubated in buffers at the different pH values for 24 h. The enzyme activity was unchanged under all conditions suggesting that the loss of activity below and above the pH optimum is due to protonation and deprotonation of active site residues.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   pH dependence of the A. fulgidus ferric reductase activity. The activity was measured as described under "Experimental Procedures."

Ferric reductase activity was highest at 88 °C (Fig. 4). This temperature is close to the optimal growth temperature of A. fulgidus (i.e. 83 °C). To evaluate the enzyme's temperature stability, ferric reductase was incubated at various temperatures, and its activity was measured at 85 °C (Fig. 5). The enzyme was highly stable at room temperature retaining more than 90% of its activity over 4 weeks, but it denatured with a half-life of 4 min when incubated at 100 °C. At incubation temperatures of 65 and 85 °C the half-life was 6.5 and 2 h, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Temperature dependence of the A. fulgidus ferric reductase activity. The activity was measured at 85 °C as described under "Experimental Procedures."

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Thermal stability of the A. fulgidus ferric reductase. The enzyme was incubated at the temperatures indicated in the figure. At various times samples were withdrawn, and ferric reductase activity was measured at 85 °C as described under "Experimental Procedures."

Amino Acid Sequence Analysis of the Ferric Reductase-- The 19 N-terminal amino acids of the A. fulgidus ferric reductase were determined to be Met-Asp-Val-Glu-Ala-Phe-Tyr-Lys-Ile-Ser-Tyr-Gly-Leu-Tyr-Ile-Val-Thr-Ser-Glu. The amino acid sequence was used to search the A. fulgidus genomic sequence data base of The Institute for Genomic Research. A 100% match was found to a hypothetical protein encoded by the predicted coding region AF0830 (24). The hypothetical protein contains 169 amino acids, which corresponds to a molecular weight of 18,659. This molecular weight is consistent with that of the purified enzyme as determined by SDS-PAGE (Fig. 1). The gene product of AF0830 is thus identified as a ferric reductase or, alternatively, as a NAD(P)H:flavin oxidoreductase. The corresponding gene is designated fer for ferric reductase. The enzyme has a theoretical isoelectric point of 5.84.

Various data bases were searched for similar proteins. However, no homology existed to any known ferric reductase. Homology of the A. fulgidus ferric reductase was found to members of a family of NAD(P)H:flavin oxidoreductases and to other hypothetical proteins from a variety of prokaryotic organisms (Fig. 6). The NAD(P)H:flavin oxidoreductase family includes the HpaC proteins from E. coli, Photorhabidus luminescence, Klebsiella pneumoniae (here called HpaH), and the E. coli NmoB protein all of which are involved in oxidative cleavage of 4-hydroxyphenylacetate (25-27). The C-terminal domain of the Chelatobacter heintzii NtaB protein is involved in the hydroxylation of nitrilotriacetate (28-30). Other family members are the Pseudomonas sp. StyB protein involved in styrene hydroxylation, as well as the Streptomyces pristinaespiralis SnaC protein and the ActVB proteins from Streptomyces coelicolor and Streptomyces roseofulvus involved in the synthesis of antibiotics (31-35). The sequence identity between these proteins and the A. fulgidus ferric reductase extends to about 26% with a similarity of 47%.

View larger version (101K): [in this window] [in a new window]   Fig. 6.   Multiple sequence alignment of A. fulgidus ferric reductase-related proteins generated by pileup and edited by GeneDoc. Consensus residues are indicated by white letters on black background for 100% homology and white letters on gray background for 80% homology. Bars above the sequence indicated the location of the conserved regions I, II, and III. Abbreviations and accession numbers for proteins are as follows: Af.FeR, A. fulgidus ferric reductase (gi 2649784); Ph.Ph0856, Pyrococcus horikoshii hypothetical protein (gi 3257267); Af.AF1786, A. fulgidus hypothetical protein (gi 2648759); Sc.Orf1, Synechococcus sp. (strain PCC 7942) hypothetical protein (gi 310858); Ss.Slr0001, Synechocystis sp. (strain PCC 6803) hypothetical protein (gi 1001553); Ss.Sll0550, Synechocystis sp. (strain PCC 6803) putative flavodoxin (gi 1001242); Kp.HpaH, K. pneumoniae coupling protein (gi 974147); Ec.HpaC, E. coli component C of the 4- hydroxyphenylacetate-hydroxylase (gi 757835); Pl.HpaC, Photorhabdus luminescens 4-hydroxyphenylacetic acid hydroxylase putative coupling protein (gi 3002549); Ec.NmoB, E. coli 4-hydroxyphenylacetate 3-monooxygenase small chain (gi 4062570); Mt.Rv3567c, M. tuberculosis hypothetical nitrilotriacetate monooxygenase component B (gi 1877298); Re.DszD, Rhodococcus erythropolis NADH:FMN oxidoreductase (gi 2944380); Ch.NtaB, C. heintzii nitrilotriacetate monooxygenase component B (gi 2507085); Rp.RP149, Rickettsia prowazekii hypothetical protein (gi 3860716); Stc.ActVB, S. coelicolor actinorhodin polyketide dimerize (gi 2498304); Str.ActVB, S. roseofulvus ActVB homolog (gi 3170573); Stp.SnaC, S. pristinaespiralis NADH:FMN oxidoreductase (gi 1711412); Stv.GraOrf Streptomyces violaceoruber putative FMN:NADH oxidoreductase (gi 4218572); Ps.StyB, P. fluorescens StyB protein (gi 2154928).

Visual inspection of the amino acid sequences of the A. fulgidus ferric reductase and related proteins shown in Fig. 6 revealed 3 short regions that appear to be significantly conserved. These regions are now defined as regions I, II, and III. Region I includes the amino acid residues 36-39, (T/S)XXP, of the A. fulgidus ferric reductase, where X indicates a variable amino acid residue. Regions II and III include amino acids 62-67, FX(L/I/V)X(L/I/V)L, and amino acids 124-128, G(T/D)HX(L/I/V) of the A. fulgidus ferric reductase sequence, respectively. None of the regions is homologous to any known flavin- or NAD-binding sites. However, these regions might be involved in flavin and/or NAD coordination.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A. fulgidus, when grown on lactate and sulfate, contained the enzyme, ferric reductase, in its soluble protein fraction. The ferric reductase was purified to homogeneity and found to be very abundant in A. fulgidus (0.75% of the soluble fraction) suggesting that it may have an important function in the A. fulgidus metabolism. The enzyme catalyzes the reduction of Fe³⁺-EDTA and other Fe³⁺ complexes with NADH or NADPH as the electron donors and requires the addition of free flavin for maximal activity.

The ferric reductase from A. fulgidus exhibits its highest activity at 88 °C, which is close to the temperature optimum for growth. Other enzymes isolated from A. fulgidus, i.e. the isocitrate dehydrogenase, the NADP-specific glutamate dehydrogenase, and the L-malate dehydrogenase exhibit an even higher temperature optimum for activity of 90 °C or greater (36-38). The latter enzymes have been reported to be very thermostable with a half-life of 80 min at 101 °C for the L-malate dehydrogenase, and 140 min at 100 °C for the NADP-dependent glutamate dehydrogenase (37, 38). In contrast, the ferric reductase exhibits a half-life of 4 min at 100 °C and is thus more similar to the A. fulgidus isocitrate dehydrogenase (36).

Ferric reductases have been described previously in eukarya and bacteria. Almost all of these enzymes are involved in iron assimilation metabolism. This work presents the first characterization of an archaeal ferric reductase. The A. fulgidus ferric reductase lacks any prosthetic groups after purification but strictly requires free FMN or FAD for the reduction of complexed Fe³⁺. In this respect, the A. fulgidus ferric reductase resembles the assimilatory bacterial ferric reductase enzymes isolated from Rhodobacter sphaeroides, Neisseria gonorrhoeae, and E. coli that also require free flavin for ferric reduction. (23, 39, 40). In addition, the A. fulgidus enzyme utilizes flavin as a substrate and thus functions also as NAD(P)H:flavin oxidoreductase. Since it was possible to reconstitute the A. fulgidus ferric reductase with FMN, this enzyme clearly classifies as a flavoprotein, and we suggest that the enzyme-bound flavin is involved in catalysis. This is substantiated by the very high affinity of the purified enzyme for FMN (i.e. 0.3 µM). Therefore, for the reduction of free flavin by NAD(P)H, Reactions 1-6 are postulated as follows:





Since the affinity for FMN is significantly higher (i.e. 220-fold) than for Fe³⁺-EDTA, it is likely that enzyme-bound flavin is also involved in the catalysis of Fe³⁺-EDTA reduction, which is described by Reactions 4-6.



or

The different k_cat values for flavin versus ferric reduction can be explained by Reaction 3 being rate-limiting for flavin reduction, whereas Reactions 4 and 5 (or 6) are much faster than Reaction 3 and rate-determining for the ferric reductase activity.

Whereas the affinities for NAD(P)H, Fe³⁺ complexes, and FMN among the bacterial ferric reductases and the archaeal enzyme are similar, the A. fulgidus enzyme differs strikingly by its very high specific activity. The activity of the A. fulgidus enzyme is at least 1000-fold higher than all other bacterial ferric reductase activities that have been measured thus far (Table I), and it is 40 times more active than the fastest eukaryotic ferric reductase isolated from spinach (6). Enzymes from extremeophiles usually exhibit enzyme activities that are in the same range as their mesophilic counter parts. Examples for thermophilic enzymes from A. fulgidus that have specific activities like their mesophilic counter parts include the isocitrate, L-malate, and NADP-dependent glutamate dehydrogenases (36, 37, 38).

E. coli contains three distinct enzymes that serve as assimilatory ferric reductases (42). Like the major ferric reductase in E. coli, the A. fulgidus enzyme also functions as an NAD (P)H: flavin oxidoreductase (see above) (42). However, the A. fulgidus enzyme has a specific activity more than 750-fold higher than the major E. coli flavin reductase, Fre. Whereas the A. fulgidus enzyme also utilizes FAD and FMN as electron acceptors, riboflavin does not serve as a substrate. This is unlike the E. coli major NAD(P)H:flavin oxidoreductase, which reduces FMN, FAD, and riboflavin (20). This NAD(P)H:flavin oxidoreductase has been demonstrated to activate ribonucleotide reductase in E. coli by reducing the enzyme's [Fe-S] center, which subsequently generates a tyrosyl radical (39). Coves and Fontecave (20) suggested that the reduced flavin produced by the E. coli NAD(P)H:flavin oxidoreductase serves to reduce chemically several suitable electron acceptors. It is, therefore, feasible that the A. fulgidus ferric reductase may also have a dual function in the metabolism of A. fulgidus.

N-terminal amino acid sequence analysis of the purified ferric reductase resulted in the identification of a hypothetical protein in A. fulgidus with no previously known function (i.e. encoded by gene AF0830). This protein is now assigned the function of a ferric reductase and an NAD(P)H:flavin oxido- reductase. Interestingly, no homology was found between the A. fulgidus ferric reductase and the ferric reductases from S. cerevisiae or from E. coli (21, 22). However, significant homology of the A. fulgidus ferric reductase exists to a family of NAD(P)H:flavin oxidoreductases that are members of a two-enzyme system, in which the NAD(P)H:flavin oxidoreductase generates reduced FMN for a monooxygenase (Fig. 6). In general, these monooxygenases are involved in the oxidative degradation of aromatic compounds, hydrocarbons, or in polyketide biosynthesis. Although the deduced amino acid sequence for the A. fulgidus ferric reductase did not reveal any known motif conclusive for the coordination of a prosthetic group, three conserved regions were noted (Fig. 6). These regions may be involved in the coordination of flavin and/or NAD. The three regions noted in this study are also present in the C-terminal domains of 4 Synechocystis sp. A-type flavoproteins of unknown function (43). The latter proteins contain a partially conserved flavodoxin signature within their N-terminal domain, which is speculated to be responsible for the binding of FMN. One of the Synechocystis flavoproteins was demonstrated to bind both FAD and FMN (43); however, it is not known whether this flavoprotein interacts with NADH or Fe³⁺.

The A. fulgidus ferric reductase is distinct from the dissimilatory cytochrome c-type metal reductases isolated from the iron-reducing bacteria D. acetoxidans and G. sulfurreducens (15, 16). These proteins have broad substrate specificity for complexed and uncomplexed Fe³⁺ compounds, manganese dioxide, elemental S⁰, and other compounds. In contrast, the A. fulgidus ferric reductase does not use uncomplexed iron and has its highest specificity for Fe³⁺-EDTA, although the redox potentials for Fe³⁺/Fe²⁺-NTA and Fe³⁺/Fe²⁺-citrate are almost identical to that of Fe³⁺/Fe²⁺-EDTA (Table IV).

The physiological role of ferric reductase in the sulfate-reducing A. fulgidus is uncertain. Functionally, the enzyme is more similar to the assimilatory ferric reductases. However, because of its high cellular abundance (see above), it seems possible that this enzyme serves a dissimilatory rather than an assimilatory function. Vargas et al. (17) previously reported on ferric reductase activity in A. fulgidus. In their study, the activity was measured with cell suspensions using hydrogen as the electron donor and Fe³⁺-citrate as the electron acceptor. The authors concluded that this reaction could potentially be used to generate energy for growth, although growth of A. fulgidus with Fe³⁺-citrate was not demonstrated. The ferric reductase purified in this study could be the key enzyme of this possible electron transfer pathway since the purification yielded only one type of this enzyme. A speculative electron transfer chain could contain a hydrogenase that oxidizes hydrogen to protons on the outside of the cell. The electrons could be transferred across the cytoplasmic membrane to an NADH dehydrogenase that would consume protons on the cytoplasmic side to generate NADH. NADH would then be regenerated by the cytoplasmically located ferric reductase. The scalar proton translocation would be sufficient to generate the proton motive force. This electron transfer pathway resembles somewhat the sulfate reduction pathway, which also involves soluble terminal reductases, i.e. the APS reductase and the sulfite reductase (44-46). Whether an electron transport chain involving the ferric reductase characterized in this study exists in A. fulgidus still has to be established.

	ACKNOWLEDGEMENT

We thank the anonymous reviewer of this manuscript for the helpful suggestions regarding the flavin reconstitution and the flavin and ferric reduction mechanism, which greatly strengthened our manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL-16251 and National Sciennce Foundation Grant MCB-9631006 (to I. S.) and a grant from the United States Department of Commerce/NIST Cooperative Research Agreement No. 70NANB7H0009 (to H. G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, 1602 Molecular Sciences Bldg., UCLA, Los Angeles, CA 90095. Tel.: 310-825-8085; Fax: 310-206-5231; E-mail: imkes@microbio.ucla.edu.

	ABBREVIATIONS

The abbreviations used are: PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES