©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Anaerobic Escherichia coli Ribonucleotide Reductase
SUBUNIT STRUCTURE AND IRON SULFUR CENTER (*)

(Received for publication, November 15, 1995; and in revised form, February 5, 1996)

Sandrine Ollagnier (1) Etienne Mulliez (1) Jaques Gaillard (2) Rolf Eliasson (3) Marc Fontecave (1) Peter Reichard (3)(§)

From the  (1)Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, Université Joseph Fourier, CNRS URA 332, Chimie-Recherche, BP 53, 38041 Grenoble Cédex 09, France, the (2)CEA Department de Recherche Fondamentale sur la Matière Condensée, SESAM-SCPM, 17 Rue des Martyrs, 38054 Grenoble Cedex 09, France, and the (3)Department of Biochemistry 1, Medical Nobel Institute, MBB, Karolinska Institute, 17177 Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha(2)) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta(2)). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha(2)beta(2) structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.


INTRODUCTION

Anaerobically grown Escherichia coli synthesizes the deoxyribonucleoside triphosphates required for DNA replication via the action of a specific ribonucleoside triphosphate reductase, which is different from the ribonucleoside diphosphate reductase employed during aerobic growth(1) . In their active form, both enzymes are radical proteins. One of the amino acids of the polypeptide chain is an organic radical and initiates the radical chemistry required for the activation of the substrate ribonucleotide. To this purpose the aerobic enzyme contains a stable tyrosyl radical (Tyr-122) that is formed by an oxygen-dependent process(2) . The anaerobic enzyme contains a glycyl radical (Gly-681), stable during anaerobic conditions but rapidly destroyed by oxygen(3) .

The direct isolation of an active anaerobic reductase was for a long time prevented by the exquisite lability of the glycyl radical to oxygen. The enzyme was instead isolated in an inactive form that could be reactivated by preincubation with a complex activating system consisting of S-adenosylmethionine (AdoMet), (^1)a reducing system (NADPH, dithiothreitol, flavodoxin reductase and flavodoxin)(4, 5, 6, 7) , and an additional 17.5-kDa protein, provisionally called activase(7) . Activation occurred by ``radicalization'' of Gly-681. With time we could exclude oxygen more efficiently during enzyme purification and now can obtain a protein that has partially retained its glycyl radical and reduces ribonucleotides without prior activation(8) . However, the activity of the best preparations is increased by the activation system.

The anaerobic ribonucleotide reductase shows considerable similarities to pyruvate formate lyase (PFL), another E. coli anaerobic enzyme. Previous elegant work by Knappe and collaborators showed that active PFL contains a glycyl radical required for enzyme activity(9, 10) . This radical is generated in inactive PFL by an enzyme system consisting of AdoMet, the reducing flavodoxin system and an activating protein(11, 12, 13) . With the exception of the activating proteins, the components required for generation of the glycyl radical are identical for PFL and the reductase. The two activases also show some sequence homology(7) . In particular three cysteines occupy identical positions at the N terminus of both proteins(7) . In the case of the PFL activase, the cysteines were suggested to bind the iron required for the activation reaction. Iron had to be added during the course of the reaction, and the pure protein could bind close to 1 Fe/polypeptide (12) .

The protein required for the generation of the glycyl radical of the reductase was found to contain substoichiometric amounts of iron(7) . The pure protein was red-brown in solution, and its electronic spectrum showed three poorly resolved absorption bands between 380 and 620 nm, suggesting the possibility of an iron-sulfur cluster(7) . In further studies we now demonstrate that the protein after anaerobic treatment with sulfide and Fe(II) dimerizes and forms a labile 4Fe-4S cluster with considerable increase in enzyme activity. The dimer (beta(2)) forms a quite tight alpha(2)beta(2) complex with the protein (alpha(2)) containing the glycyl residue that undergoes radical formation. Originally this second protein was considered to be the reductase proper. It now appears more appropriate to look upon the complex as the complete anaerobic ribonucleotide reductase. In this paper we will use the term ``large protein'' for alpha(2) and the term ``small protein'' for beta(2).


EXPERIMENTAL PROCEDURES

Materials

Except as stated below, the sources and purification of enzymes and other materials have been described previously(4, 5, 6, 7) . The large protein used in the present work was obtained from a new mutant strain of E. coli with an insertion in the chromosomal gene for the large protein, carrying a plasmid with the gene for only the large protein. (^2)When prepared by the previously used procedure (8) the large protein obtained from this strain contained no iron and was not contaminated with the small protein. Pyruvate ferredoxin oxidoreductase from Clostridium pasteurianum was provided by J. M. Moulis (CENG, Grenoble, France) and ferredoxin IV from Rhodobacter capsulatus by Y. Jouanneau (CENG, Grenoble, France).

Analyses

Protein was determined by the method of Bradford (14) , standardized by amino acid analyses of the small and large subunits. Iron was determined as described by Fish(15) , and sulfide as described by Beinert(16) .

Purification of the Small Protein

Two different preparations of the small protein were used. In one case, the protein was purified in the presence of DTT as described earlier(7) . This method gave a nearly homogeneous protein preparation, contaminated with nucleotides that were removed by DEAE-cellulose chromatography. In a typical experiment, we first decreased the salt concentration of a solution of 34 mg of protein to 30 mM KCl, 30 mM Tris-HCl, pH 7.5, 2 mM DTT by exchange on Sephadex G25 and filtration through a 5-ml column of DE52 (Whatman), equilibrated with the same buffer. The active protein, free of nucleotides, passed directly through the column and was concentrated by centrifugation in Centriprep-10 to give a final yield of 25 mg. This material was used for the reactivation experiment described below.

In an alternative preparation that avoided DTT, extracts from a different bacterium, E. coli BL 21(DE3), carrying phage pN9 were used. This strain gave much higher protein expression. Cells were grown at 37 °C in LB medium with 200 µg/ml ampicillin and 0.2% glucose. When the A had reached 0.5, the culture was cooled rapidly to 25 °C and induced with 0.5 mM isopropyl thio-beta-D-galactoside. After 4 h of growth at this temperature, the cells were centrifuged, washed twice with 50 mM Tris-HCl, pH 7.5, and frozen in liquid nitrogen. Purification of the enzyme was made at approximately 4 °C. The cells (4 g) were suspended in 9 ml of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM DTT, 1% Nonidet P-40, and 1 mM phenylmethanesulfonyl fluoride and sonicated. After centrifugation at 14,000 rpm for 30 min, the pinkish supernatant solution (10 ml, 35 mg of protein/ml) was precipitated with streptomycin sulfate (final concentration 3%) during 1 h of stirring, and again centrifuged. MgCl(2) (final concentration 2 mM) was added to the supernatant solution, which was treated with 2-3 mg of pancreatic DNase for 60 min at 4 °C and then precipitated with solid ammonium sulfate (final saturation 40%). The pellet after centrifugation was dissolved in 50 mM Tris-HCl, pH 7.5 (2.8 ml, 100 mg/ml), and the solution was chromatographed at 0.8 ml/min on a column of Superdex 75, 16/60 (Pharmacia Biotech Inc.), equilibrated with 30 mM Tris-HCl, pH 7.5/1 M KCl. After 40 ml had passed through the column, fractions (2 ml) were collected and assayed for protein and enzyme activity. Enzyme activity (in a total of 150 mg of protein) was recovered in two well separated peaks, corresponding to the dimer and monomer of the protein, as shown under ``Results.'' Fractions corresponding to each peak were combined, and the protein was concentrated by centrifugation in Centricon-10 tubes.

Reconstitution with Iron and Sulfide

We describe here a typical experiment in which radioactive iron was introduced. A solution of 15 mg of enzyme (0.6 ml in three small tubes) was continuously purged with argon on a manifold and treated in an ice bath under strict anaerobiosis for 3 h with 4 µmol of DTT and 3 µmol each of Na(2)S and Fe(NH(4))(2)(SO(4))(2) (6 µCi), added in that order. After addition of iron the solution became immediately dark brown. After 3 h, EDTA (9 µmol) was added, the capped tubes were transferred to an anaerobic hood, and the solution was passed through a 15-ml column of Sephadex G25, equilibrated with 50 mM Tris-HCl, pH 8.0, with recovery of 9.1 mg of protein. The final material contained 2.1 atoms of Fe/protein monomer and initially had a specific radioactivity of 0.81 times 10^6 cpm/mg protein. In some of the samples used for EPR measurements, 10% glycerol was included during the last Sephadex G25 step to prevent the concentrated protein from precipitating.

Kinetic Experiments

Under standard conditions the small and large proteins (usually between 0.5 and 5 µg each) in a total volume of 20 µl were added to small tubes attached to a manifold and purged with moist argon during 30 min at room temperature to remove all air. An anaerobic preincubation mix (15 µl) was added with a syringe through the septum of the tubes and incubation was continued for 60 min at room temperature to generate the glycyl radical on the large protein. The reductase reaction was then started at room temperature by addition of 15 µl of substrate mix and allowed to proceed for between 5 and 20 min. The reaction was stopped by addition of 0.5 ml of 1 M HClO(4), and the solution was worked up as described earlier(4) . One unit of enzyme activity represents the formation of 1 nmol dCTP/min.

The preincubation mix was 2.5 mM NADPH, 1.0 mM AdoMet, 12.5 mM DTT, 12.5 mM sodium formate, 75 mM KCl, 75 mM Tris-HCl, pH 8.0, and contained flavodoxin (4 µg/ml) and flavodoxin reductase (2 µg/ml). The substrate mix contained 5 mM [^3H]CTP (9 cpm/pmol), 2.5 mM ATP, and 25 mM MgCl(2).

EPR Spectroscopy

EPR first-derivative spectra were recorded on a Varian E 109 (9.5 GHz) EPR spectrometer equipped with an Oxford Instruments ESR 900 helium flow cryostat. Double integrals of the EPR signals were evaluated by using a computer on-line with the spectrometer. Spin concentrations in the protein samples were determined by calibrating double integrations of the EPR spectra with a standard sample of a 2Fe-2S protein (62 µM of ferredoxin IV of R. capsulatus).

Power saturation experiments were carried out by measuring the amplitude of the central component of the EPR signal (g = 1.92) as a function of the incident microwave power in the 40 dB (20 microwatts) to 4 dB (82 milliwatts) range at fixed temperature. The amplitude at the lowest power value was taken as 100%. At each power increment, the amplifier gain was changed in order to keep the signal amplitude constant (non-saturating conditions). The 2Fe-2S center of ferredoxin IV and one of the 4Fe-4S centers of pyruvate ferredoxin oxidoreductase were used as references.

The iron signal of the small protein was recorded directly on solutions of the pure protein after reconstitution with iron and sulfide. Analyses of the reduced form of the small protein were carried out after anaerobic incubation of the reconstituted protein (200-300 µM) in the presence of 10 mM dithionite at 25 °C for 1 h in EPR tubes. To stabilize the small protein, all experiments were done in 10% glycerol.

Sucrose Gradient Centrifugation

Centrifugations (17) were made at 35,000 rpm for 15 h at 20 °C with a 5-20% sucrose gradient (4.5 ml) containing 30 mM Tris-HCl, pH 7.5, 30 mM KCl, 5 mM sodium formate, 10 mM MgCl(2), 2 mM DTT, 1 mM ATP, and 5.4 µMFe-labeled small protein. The large protein, dissolved in 0.1 ml of 30 mM Tris-HCl, pH 7.5, 30 mM KCl was layered on top of the gradient. After centrifugation, fractions of 2 drops (0.13 ml) were collected from the bottom of the tubes. The amount of protein and radioactivity present in each tube was determined.


RESULTS

Expression and Purification of the Small Protein

In a previous publication, the small protein was obtained from an extract of E. coli JM 109(DE3) carrying plasmid pN9, by direct chromatography on a column of Superdex-75 in the presence of 5 mM DTT(7) . Part of the experiments described here were done with an enzyme obtained by a slightly modified procedure. The protein chromatographed on Superdex-75 as a monomer of 17.5 kDa and contained 0.3-0.8 Fe/polypeptide chain.

A better yield of pure protein was obtained when the enzyme was prepared from E. coli BL21(DE3) carrying plasmid pN9, with the overproduced small protein constituting almost 40% of the total soluble protein. The recovery was highly dependent on growth conditions. When induction was made at 37 °C, most of the protein was present as inclusion bodies. However, when the growth temperature was decreased to 25 °C, the enzyme remained soluble.

After the preliminary purification with streptomycin and ammonium sulfate described under ``Experimental Procedures,'' the protein was chromatographed without DTT on Superdex-75 with the results shown in Fig. 1. Enzyme activity was present in the two last, well defined peaks with apparent molecular masses of 35 and 17.5 kDa, corresponding to the di- and monomer, respectively, of the small subunit. On denaturing gel electrophoresis both peaks gave only a single band at 17 kDa. Together, 150 mg of pure material was recovered from 4 g of wet weight bacteria.


Figure 1: Purification of the small protein on Superdex-75. The dialyzed ammonium sulfate fraction was chromatographed as described under ``Experimental Procedures.'' Enzyme activity was present in the last two peaks whose molecular masses correspond to the dimer and monomer of the small subunit. The lower panel shows analyses by denaturing gel electrophoresis of: A, molecular weight standards (94, 67, 43, 30, 20, and 14 kDa, from top to bottom); B, crude bacterial extract; C, ammonium sulfate fraction; D, dimer (fractions 13-16 combined); E, monomer (fractions 17-25 combined).



The crude bacterial extract and the ammonium sulfate fraction were brown. After gel filtration the dimer fraction was also colored, whereas the monomer fraction was almost colorless. The optical absorption spectrum of the colored solutions showed broad bands around 420, 460, and 550 nm, suggesting that the enzyme contained an iron-sulfur cluster.

In different preparations the dimer contained 0.10-0.25 Fe/polypeptide chain, whereas the monomer contained only 0.01-0.02 Fe. Both dimer and monomer thus contained less iron than the monomer preparation obtained in the presence of DTT(7) . Sulfide was present in 1:1 stoichiometric amounts throughout the preparation of the enzyme (Table 1).



These results indicate that the small protein may exist as a dimer or monomer in solution and that it contains a highly labile iron-sulfur cluster. Preparations obtained in the presence of DTT retained a larger fraction of iron, suggesting a stabilizing effect of DTT.

Reconstitution of the Iron-Sulfur Center of the Small Protein

Anaerobic incubation of either dimer or monomer with a slight excess of iron and sodium sulfide as described under ``Experimental Procedures'' resulted in the uptake of close to 2 Fe and 2 sulfide/polypeptide chain (Table 1). Reconstitution was a slow process and required more than 2 h of incubation (data not shown). The absorption in the 420 nm region increased proportionally to the iron uptake, but the bands were less distinct after (Fig. 2B) than before (Fig. 2A) reconstitution. The molar extinction coefficient for iron at 420 nm was 3700 before and 3400 after reconstitution. During anaerobic incubation with dithionite, the absorption decayed (data not shown).


Figure 2: Electronic spectra of the small protein before (A) and after (B) reconstitution with iron. A shows the spectrum of a solution of 2 mg/ml protein (0.4 Fe/polypeptide), prepared in the presence of DTT as described under ``Experimental Procedures.'' B shows the spectrum of the same protein after reconstitution with iron (0.95 mg of protein/ml, 2.1 Fe/polypeptide).



When the reconstituted monomer was rechromatographed on Superdex-75 in the absence of DTT, the protein eluted as a mixture of fractions of different sizes, including the monomer, dimer and more aggregated structures. Fig. 3shows such a chromatogram from an experiment in which a monomer preparation, reconstituted with Fe was chromatographed. The figure gives the tracing of the protein emerging mainly as dimer and monomer from the column as well as the amount of iron in the protein, calculated from the radioactivity in the fractions. Before chromatography, the protein contained 2.1 Fe/polypeptide. In the chromatogram, the dimer contains approximately 1.5 Fe/polypeptide, i.e. almost a full complement of the cluster, whereas the monomer has lost iron, with values starting at 0.8 Fe at the front of the peak and decreasing to 0.2 Fe at the end. The results indicate that the cluster was tightly bound in the dimer but was lost on dissociation to the monomer during chromatography. A plausible interpretation of these results is that the native small protein is a homodimer with the cluster holding together the two monomers. Further evidence on this point is presented below from EPR experiments.


Figure 3: Chromatography of Fe-labeled small protein on an analytical column of Superdex-75. The reconstituted labeled protein (0.2 ml, 1.0 mg/ml, 0.73 times 10^6 cpm/mg) was chromatographed at a flow rate of 0.4 ml/min on a column, equilibrated with 30 mM Tris-HCl, pH 8.0/0.2 M KCl. After the first 8 ml, fractions (0.2 ml) were collected and analyzed for protein (+) and radioactivity. Fe/polypeptide (circle) was calculated from these values.



EPR Studies of the Small Protein

The EPR spectrum recorded at 10K of the iron-sulfur cluster present in the fully reconstituted (2 Fe and 2 S/polypeptide chain) small protein is shown in Fig. 4A. The weak signal, centered at g = 2.03, is characteristic of an oxidized S = 1/2 (3Fe-4S) iron-sulfur cluster. The iron in this cluster represents only 1-5% of the total iron. Treatment with ferricyanide or formate did not change the shape or intensity of the signal.


Figure 4: X-band EPR spectra of the reconstituted small protein. A, oxidized form (150 µM); B, reduced form (85 µM) in the presence of 10% glycerol; C, reduced form (115 µM) without glycerol. g values are indicated for each spectrum. Samples B and C were obtained by reduction with 10 mM dithionite for 1 h at 25 °C in 0.2 M Tris-HCl, pH 8.0. Recording conditions: temperature, 10K; microwave power, 1 milliwatt; modulation amplitude, 1 millitesla; receiver gain, 2 times 10^4 (A) or 10 times 10^3 (B and C).



After anaerobic incubation with a 20-fold excess of dithionite, the 3Fe-4S signal disappeared and a new rhombic signal was detected with features at 2.02, 1.92, and 1.88 (Fig. 4B). This signal became more axial (Fig. 4C) when glycerol was omitted during the G25 step after reconstitution of the protein (g = 2.03, 1.92). The signal arises from a S = rapidly relaxing species, detectable only at low temperature. Increasing the temperature to 20K resulted in broadening, with disappearance of the signal above 30K. The power saturation properties of the signal are shown in Fig. 5and compared to those of well characterized 2Fe-2S and 4Fe-4S centers. Both temperature and power dependence values demonstrate that the EPR active species is a reduced 4Fe-4S cluster and not a 2Fe-2S cluster, as might be suspected from the Fe-polypeptide stoichiometry. Integration of the signal revealed that only 30% of the total iron was present in the EPR active reduced species. However, when a reduced EPR sample was chromatographed anaerobically on Sephadex G25 to separate protein-bound Fe from free Fe, we found that a large amount (from 20 to 50%) of the total iron had dissociated from the protein during reduction. Correcting for this loss, the EPR signal accounts for 50-80% of the remaining protein-bound iron.


Figure 5: Microwave saturation curves of the EPR signal of the reduced form of the small protein. Samples B (circle) and C (down triangle) were analyzed by EPR spectroscopy at 10K. The signal amplitudes were normalized to the maximum values. Standard samples were: ferredoxin IV (2Fe-2S) from R. capsulatus () and pyruvate:ferredoxin oxidoreductase (4Fe-4S) from C. pasteurianum (bullet).



Combined with the Fe/polypeptide stoichiometry, the EPR results strongly suggest that two polypeptide chains bind one 4Fe-4S cluster.

Interaction between the Small and the Large Proteins

The interaction between the two proteins was studied both by kinetic experiments and by sucrose gradient centrifugation.

The enzyme reaction consists of two steps. In the first, the enzyme is activated by generation of the glycyl radical. In the second, the active enzyme reduces CTP to dCTP. Our assay measures the second step. Both steps are carried out in the same anaerobic tube, but CTP is added only after preincubation of the enzyme with the activating system. We chose a preincubation period of 60 min, since this fully activated the enzyme, irrespective of the relative amounts of small and large proteins (data not shown). Addition of increasing amounts of the small protein to various amounts of the large protein generated the results shown in Fig. 6. The system became saturated with respect to the small protein and saturation was achieved later when the amount of the large protein was increased. From the molecular masses of the two proteins (160 kDa for the large and 35 kDa for the small protein) one can calculate that a 2-4-fold molecular excess of the small protein was required for saturation. The results can be explained by a tight but dissociable interaction between the two proteins.


Figure 6: Dependence of dCTP formation on the concentration of the two proteins of the reductase. Increasing amounts of the reconstituted small protein were incubated in the presence of various amounts of the large protein (+, 0.71 µg; times, 1.43 µg; circle, 2.85 µg). Conditions are described under ``Experimental Procedures.''



In a second experiment we compared the activity of the fully active small protein with its activity before reconstitution. The results of Fig. 7show that the protein containing the full complement of the FeS center saturates the large protein much more readily than the protein with an iron-deficit. This might indicate that the interaction with the large protein occurs with the dimer of the small protein, since dimer formation is favored by the presence of the iron cluster.


Figure 7: CTP reduction depends on the iron center of the small protein. Increasing amounts of the small protein before (+) or after (circle) reconstitution were incubated with 2.85 µg of the large protein. The small protein contained 0.5 Fe/polypeptide before and 2.1 Fe/polypeptide after reconstitution.



To measure the interaction more directly, we turned to sucrose gradient centrifugation, a method that earlier permitted the quantitation of proteins R1 and R2 in the complex of the aerobic reductase(18) . To overcome the problem posed by the dissociation of the complex during centrifugation, an excess of the Fe-labeled small protein was included in the gradient through which the large protein was sedimented. This protein sediments much faster than the small protein and forms the complex during centrifugation. Fig. 8illustrates such an experiment in which two different amounts of the large protein as well as a blank containing only the small protein were centrifuged. At the end of the centrifugation the complex was found in fractions 6-11, both by protein (Fig. 8A) and radioactivity (Fig. 8B) analyses. The concentration of small protein in each fraction can be calculated directly from radioactivity, since the protein did not lose iron during centrifugation. This was apparent from the finding that the specific radioactivity of the protein in the blank fractions 6-11 was virtually identical with the specific activity of the starting material. The concentration of the large protein was then obtained by subtracting the concentration of small protein from the total protein concentration. To obtain the fraction of bound small protein, the value from the corresponding blank centrifugation tube was subtracted.


Figure 8: Sucrose gradient centrifugation of the complex formed between the small and the large protein. Parallel 5-20% sucrose gradients containing 5.4 µMFe-labeled small protein throughout the whole gradient were centrifuged as described under ``Experimental Procedures.'' One tube (+) served as control. On the two others, 0.153 mg (circle) or 0.397 mg (times) of the large protein was layered before the start of the run. After the run, the tubes were punctured, fractions (0.13 ml) were collected from the bottom and analyzed for protein (A) and radioactivity (B). The arrow in A shows the position of aldolase (158 kDa) run in a separate gradient.



Table 2summarizes such calculations for fractions 6-11 for both experiments. The results show a clear 1:1 stoichiometry between the two proteins. Strikingly convincing results were obtained at the higher concentration of the large protein when calculations are less influenced by some of the uncertainties involved in the blank corrections. The complex sediments at a position slightly heavier than that of an aldolase marker (158 kDa), suggesting that it is formed by dimers from each protein.




DISCUSSION

The small protein of the ribonucleotide reductase has certain structural and functional similarities with the activase involved in the PFL reaction(9, 11, 12, 13) . Both proteins contain iron and generate a glycyl radical in a second protein with the aid of AdoMet, with similar requirements for NADPH and the flavodoxin system.

However, there are also important distinguishing features. The genes for the small and the large proteins are under coordinate control, whereas PFL and its activase are regulated separately. The two reductase proteins bind tightly in an alpha(2)beta(2) complex, whereas binding of the PFL-activase to PFL does not appear to be strong. In addition, the nature of iron seems to be different. The PFL-activase was reported to operate with loosely bound iron, one atom per polypeptide chain(12) , whereas we now show that the small protein of the reductase contains a 4Fe-4S center, proposed to link together two polypeptides.

As prepared, only a small fraction of the molecules contains the cluster. However, a full complement of 2 Fe and 2 S/polypeptide can be restored to the protein by treatment with ferrous ion and sulfide. EPR spectroscopy of the reduced, reconstructed protein then indicates the presence of a 4Fe-4S. Taken together with the stoichiometry of 2 Fe/polypeptide, this suggests that the iron cluster is bound by cysteines from both polypeptides. A precedence for the location of an FeS cluster at the interface of two polypeptide chains is provided by the Fe protein of nitrogenase(19) .

The original concept of an activase implied a catalytically acting protein that introduces a structural change in a second protein, leading to its activation. In the case of the ribonucleotide reductase, it seems more appropriate to view the small protein as an integral component of the enzyme. The function of this protein is probably related to radical generation from AdoMet. However, more work is required on this point.

A tight interaction between the two proteins is apparent already from previous work(7) . Here, we employed sucrose gradient centrifugation to demonstrate the formation of a 1:1 complex between dimers of the two proteins. It was necessary to centrifuge the large protein in a gradient containing an excess of of small protein to counteract dissociation. The kinetic experiments also speak for a tight interaction between the two proteins and suggest that the two proteins are bound together in a dissociating system that represents the active enzyme.

In the absence of the large protein, the small protein apparently occurs in a dimer-monomer equilibrium in solution. When the protein was chromatographed in the absence of DTT both species were recovered, but only the dimer contained some iron and sulfide, albeit in substoichiometric amounts. This is in accord with the idea that the cluster is bound by cysteines from both polypeptides. DTT was able to stabilize the binding of iron to the monomeric protein, apparent from the relatively high iron content in the monomer in the presence of DTT. It seems possible that under those circumstances the SH-groups of DTT help to anchor the cluster to the protein. Such preparations could also be fully reconstituted by treatment with iron and sulfide. The fact that, during chromatography of the reconstituted monomer on Superdex-75, both the dimer and the monomer were recovered and that the monomer had lost its iron again argues for the necessity of an interaction between the two polypeptides to maintain a stable cluster.

We reported earlier that the large protein prepared from bacteria overproducing both the large and the small proteins contains a 3Fe-4S cluster(20) . Later work showed that such preparations contained some tightly bound small protein(7) . With the demonstration of a 4Fe-4S cluster in the small protein, the question now arises whether the 3Fe-4S cluster might have been present in the contaminating protein as a degradation product of the 4Fe-4S cluster. When we now prepare the large protein from bacteria lacking a functioning gene for the small protein, iron is no longer present in the active large protein. The simplest explanation for these results is that only the small protein contains an iron-sulfur center, and that this center is a 4Fe-4S cluster. However, more complicated explanations cannot be excluded and these are being explored experimentally.

The following model summarizes our present concept concerning the structure and function of the anaerobic E. coli ribonucleotide reductase. The enzyme is a heterodimer with an alpha(2)beta(2) structure, containing two identical polypeptides of each of the large and the small proteins, with the peptides of the small protein held together by a 4Fe-4S center. The principle of its quaternary structure is thus identical to that of class I enzymes and differs from class II enzymes that have an alpha or alpha(2) structure(1) . Because of the structural similarity with the PFL activase, it seems likely, but remains to be proven, that the small protein interacts with AdoMet to generate a transient radical, which subsequently generates the stable radical on Gly-681 of the large protein. This radical then participates in the catalytic process, possibly via a transient thiyl radical(21, 22) .


FOOTNOTES

*
This work was supported by grants from CNRS (to E. M. and M. F.), the European Community (to M. F. and P. R.), and the Swedish Medical Research Council and the Wallenberg foundation (to P. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46-8-7287001; Fax: 46-8-333525; peter.reichard{at}mbb.ki.se.

(^1)
The abbreviations used are: AdoMet, S-adenosylmethionine; DTT, dithiothreitol; PFL, pyruvate formate lyase.

(^2)
A. Jordan, X. Garriga, and J. Barbé, manuscript in preparation.


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