the journal of chemistry vol. 257, no. 11, june 10, pp ... · the recent discovery (1-3) of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Prlnted in U. S. A. Vol. 257, No. 11, Issue of June 10, pp. 6259-6267, 1982

Interconversions of [3Fe-3S] and [4Fe-4S] MOSSBAUER AND ELECTRON PARAMAGNETIC RESONANCE FERREDOXIN 11*

Clusters STUDIES OF DESULFOVIBRIO GIGAS

(Received for publication, January 12,1982)

Jose J. G. Moura+§, Isabel Moura+g, Thomas A. Kent+, John D. Lipscombl, Boi Hanh HuynhJI, Jean LeGall**, Antonio V. Xavier+§ and Eckard Munck+ From the $Gray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392, 8Centr-o de Quimica Estrutural das Universidades de Lisboa Znstituto Superior Technico, 10oO Lisbon, Portugal, ?Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455, JIDepartment of Physics, Emory University, Atlanta, Georgia 30322, and ‘*Department of Biochemistry, University of Georgia, Athens, Georgia 30602

We have shown previously that the tetrameric form of a Desulfovibrio gigas ferredoxin, Fd 11, contains a [3Fe-3S] cluster (Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Munck, E. (1980) J Biol. Chem. 255, 3242-3244). Here we report the results of further Mossbauer and EPR studies of samples derived from Fd II. We have reconstituted apoprotein obtained from Fd I1 by using either excess (5 Fe/apoferredoxin) or stoichiometric (3 Fe/apoferredoxin) amounts of iron and sulfide. When iron and sulfide were provided in excess the reconstituted hol- oprotein, termed FdR, was found to be spectroscopically pure and to contain a [4Fe-4S] cluster. The Mossbauer spectra of FdR reveal the presence of inequivalent cluster subsites, occurring in a 3:l and 2:2 ratio in the oxidized (diamagnetic) and reduced form of the cluster, respectively. When FdR, buffered at pH 7.6 in 50 mM Tris-HC1, was reduced with dithionite an intense g = 1.94 EPR signal typical of reduced ferredoxins was observed. At high ionic strength, however, a substantial amount (up to 40%) of [3Fe-3S] clusters were formed upon reduction. Treatment of FdR with ferricyanide led to the formation of [3Fe-3S] clusters similar to those observed for Fd 11.

We have also incubated Fd 11 with iron, using 95% enriched “Fe, in the presence of sulfide and dithiothreitol. This procedure converted the [3Fe-3S] cluster into a structure with a [4Fe-4S] core; the latter seems to be structurally identical with the cluster of FdR. The 57Fe Mossbauer spectra of the newly formed [4Fe-4S] cluster correspond to those observed for subsites of the FdR cluster. The data show that the externally provided iron occupies either one subsite or at most two structurally equivalent sites of the [4Fe-4S] cluster. The incubation technique described here can be used for isotopic labeling, providing, among other benefits, en- hanced spectral resolution in Mossbauer experiments.

The binding of both cluster types by the D. gigas ferredoxin is discussed in terms of amino acid sequence data.

* This work was supported by National Science Foundation Grant PCM-05610, by the National Institutes of Health through Grants GM 22701, GM 25879, and GM 24689, by the Instituto National de Investigacao Cientifica and the Junta Nacional de Investigacao Cien- tifica e Tecnologica, Portugal. and by the Calouste Gulbenkian Foun- dation, Portugal. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The recent discovery (1-3) of iron-sulfur clusters containing 3 iron atoms and acid-labile sulfur has stimulated research in biochemistry and bio-inorganic chemistry. Mossbauer spectroscopy used in conjunction with electron paramagnetic resonance has proven to be an indispensable tool for the char- acterization of these structures. The first center spectroscopically identified and characterized was that belonging to a ferredoxin from Azotobacter uzneZandiz (1). X-ray diffraction studies (4) of this protein, at 3 A resolution, reveal a ring-like [3Fe-3S] core.’ The ligands attaching to the core structure are thought to consist of 5 cysteinyl residues and one oxygen ligand, perhaps from water (4).

The [3Fe-3S] clusters can be stabilized in two oxidation states. In the oxidized form, a fairly isotropic EPR signal centered at g = 2.01 is observed. The Mossbauer spectra, recorded under standard conditions, of the oxidized cluster are not sufficiently distinct from those observed for the [4Fe- 4S] cluster of oxidized high potential iron protein to provide a firm basis for identification. In strong applied magnetic fields, however, the two cluster types have distinct signatures ( 5 ) . The Mossbauer spectra of the 1-electron reduced clusters exhibit two quadrupole doublets with an intensity ratio of 2:l (3, 6, 7). The more intense doublet reflects two identical iron sites at formal oxidation level Fe2.5+, whereas the site of lower occupancy is high spin ferric in character (3). At low temperatures, a weak applied magnetic field induces strong magnetic hyperfine interactions, proving that the reduced [3Fe-3S] centers are paramagnetic. These characteristic features have been observed for all reduced [3Fe-3S] clusters studied so far. Quite recently, Thomson and co-workers (8) have shown that the low temperature magnetic circular dichroism spectra of [3Fe-3S] clusters are highly structured and distinct from those observed for [4Fe-4S] structures, providing another tool for cluster identification. The shape of the low temperature magnetic circular dichroism magnetization curves suggests an electronic spin S = 2 for the reduced clusters (8). The magnetic properties of the oxidized [3Fe-3S] clusters have been explained with a model involving three exchange-coupled ferric

’Throughout this manuscript we will refer to these clusters as [3Fe-3S]. The stoichiometry of the cluster core is known from the X- ray diffraction studies of C.D. Stout and co-workers (4); these studies have demonstrated the presence of a ring-like [3Fe-3S] core structure in A . vinelandii ferredoxin single crystals. A recent extended X-ray absorption fine structure study of Fd I1 in frozen solution suggests Fe-Fe distances of 2.7 8, (W. H. Orme-Johnson, private communica- tion), in striking contrast to the 4.1 8, deduced from the X-ray data. It is possible that %iron clusters can exist in different conformations, possibly even with Merent iron/sulfur stoichiometries.

6259

6260 Interconversions between [3Fe-3SJ and [4Fe-4S] Clusters

sites (9); no such model has as yet been put forward for the reduced state.

Soon after [3Fe-3S] clusters were discovered, it was found by Mossbauer spectroscopy that beef heart aconitase, isolated under aerobic conditions, contains such a cluster (10). Studies of reductive activations of this enzyme yielded a surprising result: the [3Fe-3S] cluster can be converted, in the presence of iron, into a structure with a [4Fe-4S] core (5). The reverse process, namely the conversion of a cubane cluster into a 3-Fe center, by means of ferricyanide oxidation, has been demonstrated recently (11, 12) for the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

Some very interesting ferredoxins have been isolated from the sulfate-reducing bacterium Desulfovibrw gigas. These ferredoxins, termed Fd I and Fd 11, are different oligomeric forms built from one type of monomeric unit for which the sequence of 57 amino acids is known (13, 14). Fd I1 is a tetramer of molecular weight 24,000. Mossbauer and EPR studies (3) have shown that each monomer of Fd I1 contains one [3Fe-3S] cluster. These studies have recently been com- plemented by Resonance Raman (15) and magnetic circular dichroism (8) studies. It has been shown that Fd I1 is capable of mediating electron transfer between cytochrome c3 and the sulfite reductase system (16).

The other oligomeric form, the trimeric Fd I, seems to serve as an electron carrier in the phosphoroclastic reaction (16). A Mossbauer and EPR study (3) of Fd I has revealed that it contains, as a majority species, a [4Fe-4S] cluster. The Fd I trimer, however, can accommodate a [3Fe-3S] cluster as a minority species of variable proportions. Since the different oligomers of ferredoxin can accommodate both clusters it is natural to ask whether both forms are biologically active and whether they interconvert in vivo. Particularly intriguing in this regard has been the observation that Fd I1 stimulates the phosphoroclastic reaction after a long lag phase, when added to crude cell extracts depleted in Fd I (16). We have recently observed that this stimulation by Fd I1 is accompanied by a concomitant appearance of a g = 1.94 type EPR signal, suggesting that cluster conversions do take place in crude cell extracts2

Stimulated by the above observations, in particular the aconitase results, we initiated a program using the D. gigas ferredoxin to study the interconversions between the two cluster types. Mossbauer spectroscopy is well suited for this task (17) since a signal is obtained regardless of the spin and oxidation state of the iron. Since the recoilless fraction, the factor determining the signal strength, is virtually the same at 4.2 K for different protein iron sites, the technique allows one to quantify with good precision the various iron environments, i.e. clusters originally present, converted clusters, and adven- titiously bound iron. Of particular importance is the fact that signals are only obtained from the isotope 57Fe (2.2% natural abundance). Thus, by using highly enriched 57Fe to convert [3Fe-3S] to [4Fe-4S] clusters, one can achieve selective labeling of [4Fe-4S] cluster subsites. As demonstrated in this article, this technique allows us, on one hand, to selectively mark a certain cluster or, on the other hand, to study the hyperfine interactions of the labeled subsite with added precision. Using the advantages that Mossbauer spectroscopy can offer, we demonstrate here various ways by which facile interconversions between [3Fe-3S] and [4Fe-4S] clusters can be achieved.

MATERIALS AND METHODS

The culture of D. gigas and the isolation of Fd I1 were as previously

J. J. G. Moura, A. V. Xavier, and J. LeGall, manuscript in preparation.

described (18). All chemicals were reagent grade and water was glass- distilled. Except where noted, solutions were buffered in Tris-HC1 at pH 7.6 (henceforth referred to as buffer). Anaerobic atmospheres were created with argon gas which had been passed over BASF Inc. copper catalyst at 150 "C. Reduction of anaerobic samples was accom- plished by adding aliquots of 100 mM Na2S204 solution prepared in 0.5 M Tris-HC1 at pH 9 degassed with argon. The final pH of this solution was 7.5. Iron was determined by forming the ferrous complex with 2,4,6-tripyridyl-S-triazine as described by Fischer and Price (19). Anaerobic transfer of samples between EPR tubes and Mossbauer sample holders was performed using gas-tight Hamilton syringes and special glassware fitted with rubber serum stoppers.

Instrumentation-EPR spectra were recorded on a Varian E-I09 spectrometer fitted with an Oxford Instruments ESR-10 continuous flow helium cryostat. Temperatures near 8 K were measured with a Au(Fe) versus chrome1 thermocouple mounted directly below the sample. The g values in the g = 2 region were measured relative to the known values of cY,cY-diphenyl-/3-dipicryl-hydrazyl. Samples were frozen by slow immersion in liquid nitrogen. Data were recorded for integration procedures using a digital computer interfaced directly to the spectrometer. Quantitation of EPR active species was made relative to standard solutions of either Cu-EDTA or native Fd 11. The Fd I1 concentration was determined using an optical extinction coef- ficient of 15,700 M" cm" at 415 nm. Optical spectra were recorded at room temperature with a Beckman Model 35 spectrophotometer. The Mossbauer spectrometer was of the constant acceleration type. All isomer shifts, 6 ~ , are quoted relative to Fe metal at 298 K.

Reconstitution of Iron-Sulfur Centers in Fd IZ-The procedure for removing the [3Fe-3S] center from Fd I1 and rebuilding an iron-sulfur center in the resulting monomeric apoprotein was adapted from that proposed by Hong and Rabinowitz (20). The protein was precipitated at 4 "C with trichloroacetic acid (5% final concentration) in the presence of 0.5 M mercaptoethanol and under argon. After 2 h, the precipitate was collected by centrifugation and then dissolved in 0.5 M Tris base containing 60 mM mercaptoethanol to a concentration of 5 mg of protein/ml. A second precipitation was done under exactly the same conditions, and the apoprotein redissolved. The resulting apoprotein solution showed no optical absorption in the visible region and iron quantitation showed that essentially all of the iron had been removed. The apoprotein solution was kept under argon at 4 "C for 1 h; then 57Fe (enriched to 956) and sulfide were added simultane- ously. In this and all procedures described below, the iron and sulfide were added in equimolar concentrations. Reconstitutions were performed with an Fe2+/protein monomer ratio of either three or five to one. The mixture was then allowed to react under argon for 30 min. After exposure to air, the solution was passed through a Sephadex G- 25 column (1 X 15 cm) equilibrated with 10 m~ buffer. The resulting protein was loaded at 25 "C on a DEAE-cellulose column (0.5 X 10 cm) and eluted with 0.4 M buffer. The protein was then desalted on another Sephadex G-25 column and finally evaporated under argon to the desired concentration.

The monomeric nature of reconstituted ferredoxin and apoferredoxin was established by gel filtration on a Sephadex G-50 column, using the following standards: chymotrypsin, soybean trypsin inhibi- tor, horse heart cytochrome c, and Desulfouibrio vulgaris rubredoxin.

Treatment of Reconstituted Ferredoxin with Ferricyanide-A sample of reconstituted ferredoxin (350 p ~ ) was prepared using a 5- fold excess of 57Fe2+ and Sz- as described above and shown by EPR and Mossbauer spectroscopy to contain only [4Fe-4S] centers. 500 p1 of this solution in 100 mM buffer was incubated with a 5-fold excess of K3Fe(CN)6 at 4 "C for 3 h. No protein precipitation was observed. The solution was then passed through a Sephadex G-25 column (1 x 15 cm) equilibrated with 100 m~ buffer, and loaded on a Whatman DE-52 column (0.5 x 10 cm) equilibrated in 10 m~ buffer. The column was developed with a buffer step gradient (0.1, 0.2, 0.3, 0.4 M buffer). The ferredoxin band was eluted with 0.4 M buffer and readily separated from the band of Prussian blue which was retained by the column. The protein fraction was then desalted on a Sephadex G-25 column equilibrated in 10 mM buffer and concentrated by evaporation under argon to a final volume of 0.3 ml.

Incubation of D. gigas Fd I Z with Fe and Sulfide in the Presence of Dithiothreitol-A sample for Mossbauer spectroscopy was prepared by incubating 300 pl of dithionite-reduced, 1.4 m M , native Fd I1 with 5 m~ dithiothreitol, and 22.4 mM 57Fe2' and S2- for 6 h under anaerobic conditions. The sample was allowed to reoxidize in air and the reagents were separated on a Sephadex G-25 column equilibrated with 10 mM buffer. The ferredoxin was then loaded on a DEAE- cellulose column (0.5 X 10 cm) equilibrated with 10 m~ buffer and

- dX d!-

Interconversions between [3Fe-3S] and [4Fe-4S] CLusters

.lomT I

2.1 2.0 1.9 g-value

FIG. 1. X-band EPR spectra of D. gigas ferredoxin reconstituted by adding 3 eq of 6'Fe/apoferredoxin 11 monomer. The spectra were taken on dithionite-reduced (A) and oxidized (B) material. Since 57Fe was used for the reconstitution procedure, the spectra are broadened by magnetic hyperfine interactions. Temperature, 8 K; microwave power, 0.03 milliwatt; modulation amplitude, 4 G.

eluted with 0.4 M buffer. The sample was desalted using a Sephadex G-25 column equilibrated in 10 m~ buffer and concentrated by evaporation under argon. A 300-pl sample 1 nm in ferredoxin was obtained. Samples for EPR spectroscopy were prepared in a similar manner except that the Fe2+ and 5'- were supplied in either stoichiometric amounts with the protein monomer concentration or in a 5- fold molar excess. Also the incubation time was varied (see Fig. 5), and it was not necessary to separate the reagents or repurify the protein after treatment.

RESULTS

As described under "Materials and Methods," we have performed cluster reconstitution experiments using apoprotein obtained from D. gigas Fd I1 tetramers. These experiments employed either stoichiometric (3 Fe/monomer) or excess (5 Fe/monomer) amounts of iron and sulfide.

Reconstitution of Apoferredoxin 11 with Excess Iron- Samples reconstituted with an excess amount of 57Fe were EPR-silent in the oxidized state. Upon reduction with dithionite the samples developed an EPR-spectrum identical with that shown inFig. lA. The principal g values at g = 2.07, 1.94, and 1.91 are the same as those reported (21) for D. gigas Fd I.3 The observed g values are typical of a reduced [4Fe-4S] cluster rather than the [3Fe-3S] cluster of Fd 11. This conclusion is emphasized by the Mossbauer data.

Fig. 2B shows a 4.2 K Mossbauer spectrum of oxidized reconstituted ferredoxin (reconstituted with 5 Fe/monomer; we will refer to such materid as FdR). The spectrum consists of two sharp quadrupole doublets, labeled 1 and 2. The spectrum of Fig. 2C was taken in an applied magnetic field of 6 Tesla; the observed absorption pattern establishes that both doublets result from diamagnetic (S = 0) sites. The quadrupole splitting and the isomer shift of doublet 2, hEQ(2) = 1.32 mm/s and &,(2) = 0.45 mm/s, together with the observed diamagnetism leave no doubt that doublet 2 results from subsites of a [4Fe-4S] cluster in the +2 oxidation state, [4Fe- 4SI2+. Doublet 1 has the parameters m~(1) = 0.55 mm/s and &,(1) = 0.41 mm/s. Quantitative analysis of the spectra, employing least squares fitting and spectral decompositions, showed that doublets 1 and 2 occur with an intensity ratio of

The spectra of reconstituted ferredoxin contain a minor species with g values at g = 2.04 and g = 1.86. This species, which accounts for about 20% of the spin concentration is not observed in reduced Fd I. Such a component, however, can be elicited when Fd I is reduced in a dimethyl sulfoxide/water system. We suspect therefore that the g = 2.04 feature belongs to [4Fe-4S] clusters associated with partially unfolded protein.

z c cr

0 a

m % Q

LJ

2

6261

-4 -2 0 2 4 VELOCITY (rnrn/s)

FIG. 2. Mossbauer spectra of oxidized Fd I and oxidized reconstituted Fd (FdR) recorded at 4.2 K. A, zero-field spectrum of the oxidized [4Fe-4S] cluster of Fd I. The spectrum was prepared as described in the text. The solid line is a theoretical spectrum generated by adding two quadrupole doublets with an intensity ratio of 13, using the parameters h E & ) = 0.56 mm/s and S F A ~ ) = 0.40 mm/s, and 6 E ~ ( 2 ) = 1.37 mm/s and 6d2) = 0.45 mm/s for doublets 1 and 2, respectively. B, zero-field spectrum of oxidized FdR. The solid line was generated by superimposing two doublets with intensity ratio of 1:3, using AEe(1) = 0.55 mm/s and &.(I) = 0.41 mm/s, and hEQ(2) = 1.32 mm/s and h ( 2 ) = 0.45 mm/s. c, spectrum of oxidized FdR recorded in a 6 T magnetic field applied parallel to the observed Mossbauer radiation. Shown also are two theoretical spectra for site 1 and site 2. The spectra were computed by assuming a diamagnetic complex and added in a ratio of 1:3. In addition to the parameters quoted in B, we used for the asymmetry parameters of the quadrupole interaction, q(1) = 0 and q(2) = 0.7.

approximately 1:3. This suggests that the doublets reflect inequivalent subsites of the [4Fe-4SI2+ cluster. The solid curue drawn through the data points of Fig. 2C is the result of a spectral decomposition of the high field spectrum, assuming a site occupancy ratio of 1:3. The simulations show that the applied magnetic field accounts for the observed magnetic splitting, i.e. the material is diamagnetic. The shape of the high field spectrum suggests that the asymmetry parameters of the quadrupole interactions for the three doublet 2 irons are not axial; for the simulations we have used A E Q > 0 and TJ = 0.7. The shape of the site 1 component is too poorly resolved for a reliable determination of sign (AEQ) and TJ. For the calculation we have used A E Q > 0 and 9 = 0.

For comparison, we have displayed in Fig. 24 a spectrum of the oxidized [4Fe-4S] cluster of D. gigas Fd I. According to our Mossbauer studies, the Fd I sample, pure by chromato- graphic criteria, contained both a [3Fe-3S] and a [4Fe-4S] cluster, the latter contributing about 75% of the total intensity. The spectrum shown in Fig. 2A was prepared by subtracting the spectral contribution of the [3Fe-3S] cluster as observed for oxidized Fd 11. It is apparent that the [4Fe-4S] clusters of Fd I and FdR reside in essentially identical environments.

We have studied the zero field spectra of FdR in the temperature range from 4.2 K to 195 K. The value for A E Q of doublet 2 decreases with increasing temperature; A E Q (2) = 1.18 mm/s at 60 K, and A E Q (2) = 0.8 mm/s at 195 K. The quadrupole splitting of site 1 seems to be independent of

6262 Interconversions between [3Fe-3S] and [4Fe-4S] Clusters

I , 1 I , , , , , , , , j -4 -2 0 2 4

VELOCITY (rnrnis)

FIG. 3. Low-temperature Mossbauer spectrum of reduced FdR. The spectrum in A was recorded at 4.2 K in a parallel field of 0.06 T. The spectrum in B was prepared by subtracting from the raw data of A contributions from a reduced [3Fe-3S] cluster (10%) and a Fez+ impurity (5%). The solid line is a theoretical spectrum generated from the published parameters of the B. stearothermophilus ferredoxin (23).

temperature. At temperatures above 100 K, the doublets are not resolved, precluding a detailed analysis of the spectra:

Fig. 3A shows a Mossbauer spectrum of reduced FdR taken at 4.2 K in a 60 milliTesla parallel magnetic field. A study of the sample in the temperature range from 4.2 K to 200 K revealed that the spectrum contains two minority species. One species, accounting for about 5% of total Fe, is due to adven- titiously bound Fe2+; this species is easily discernible at higher temperatures and has parameters AI& 3.0 mm/s and = 1.2 mm/s. More importantly, the Mossbauer spectra show about 10% of the total Fe as reduced [3Fe-3S] clusters. This is not obvious from a cursory inspection of the spectrum shown in Fig. 3A since reduced [3Fe-3S] clusters yielded broad spectra under the employed experimental conditions. How- ever, by studying the sample in applied magnetic fields as well as zero field and by taking difference spectra as discussed previously (1, 7) even small amounts of [3Fe-3S] clusters can be detected. In Fig. 3B, we have displayed a spectrum of reduced FdR prepared by removing the two minority species from the raw data; to subtract the contribution of the [3Fe- 3S] cluster we have used the experimental spectrum of reduced Fd 11.

As anticipated from the EPR data, the spectrum of reduced FdR exhibits paramagnetic hyperfine structure. The magnetic features of the spectrum are similar, if not identical with those observed for reduced Fd I (22). This is not surprising since the electronic ground states of Fd I and FdR have the same g values. It is instructive to compare the spectrum of FdR with that reported for the [4Fe-4S] ferredoxin from Bacillus stearothermophilus (23). The solid line shown in Fig. 3B is a

*We would like to emphasize the importance of studying samples in strong applied fields. One can be misled by concluding from the zero field data alone that doublet 1 belongs to a subsite of the [4Fe- 4SI2+ cluster. We have encountered situations, in related projects (27), where species with parameters such as those observed for doublet 1 result from high spin (S = 5/2) ferric material with fast electronic spin relaxation rates (attributable to aggregation). High field studies can easily identify such species. Mossbauer spectroscopy can also be a valuable aid for purification. Our first reconstitution attempts yielded samples which after treatment on a Sephadex G-25 column contained as little as 30% of the iron as Fe-S clusters. The remainder was associated with ferric components which, however, did not yield EPR signals. The Mossbauer data suggested the presence of aggregated, magnetically ordered particles attached to the protein. A further purification step using a DEAE-column removed this material completely. We have found no evidence for any impurity in the spectra of Fig. 2, B and C (the presence of a 5% ferric impurity would be quite apparent in the high field spectrum).

theoretical curve computed with a set of parameters that fits the data of the B. stearothennophilus ferredoxin quite well (23). It is apparent that reduced FdR contains a [4Fe-4S] cluster.

We have noted above that the Mossbauer spectrum of reduced FdR contained a 10% contribution from a reduced [3Fe-3S] cluster. This observation is interesting since a [3Fe- 3s) cluster was not present in the oxidized sample. (We would have detected it in the Mossbauer spectra, and a g = 2.02 EPR signal would have been observed.) This was studied by varying the ionic strength of the buffer, retaining dithionite as a reductant. We observed that the concentration of [3Fe-3S] clusters increased with the ionic strength of the buffer. In 0.8 M buffer, as much as half of the clusters in reduced FdR were observed as [3Fe-3S]; we found no evidence for the presence of [3Fe-3S] clusters prior to reduction.

Reconstitution of Apoferredonin 11 with Stoichiometric Amounts of Iron-In the following, we present some results obtained from a sample produced by adding 3 Fe/monomer in the reconstitution procedure. Fig. 1B shows an EPR spectrum of the sample in the oxidized state. The observation of an intense EPR signal centered at g = 2.02 suggested that the reconstitution had yielded [3Fe-3S] clusters. After the sample was reduced with dithionite, we observed the spectrum displayed in Fig. L4. We have pointed out above that the [4Fe- 4S]'+ clusters of FdR yielded such an EPR signature. Thus, the EPR data indicate the presence of both cluster types.

Fig. 4 shows a low temperature Mossbauer spectrum of the oxidized sample (hash marks). It is apparent that the central region of the spectrum contains a spectral component such as that displayed in Fig. 2B, i.e. the sample contains a [4Fe-4S] cluster in the +2 state. The broader feature stretching over a velocity range from -3 mm/s to +3 mm/s is associated, as suggested by the EPR data, with an oxidized [3Fe-3S] center (see also Fig. 10 below). Quantitatively, the experimental spectrum can be represented by superimposing (full circles) the spectra of Fd I1 (with a weight corresponding to 40% of total Fe) and oxidized FdR (data of Fig. 2B). The perfect match of the spectra, considered together with the EPR results, allows us to conclude that 50% of the reconstituted clusters are of the [3Fe-3S] type. This is the f is t evidence that [3Fe-3S] clusters can be assembled in a chemical reconstitution procedure. It is obvious from the data that the observed spectra belong to a reconstituted [3Fe-3S] cluster since the Mossbauer spectrum results from iron highly enriched in 57Fe and the EPR signal is broadened by magnetic hyperfine interactions involving 57Fe nuclei (compare the spectrum shown in Fig. 1B with that of Fig. 5A).

Conversions of [3Fe-3S] to [4Fe-4S] Clusters-As men-

. . L ' :. ' ' 1

-4 -2 0 2 4 VELOCITY (mm/s)

tuted with a "Fe to monomer ratio of 3:l (hash marks). The FIG. 4. Mossbauer spectrum of D. gigas ferredoxin reconsti-

spectrum was recorded at 4.2 K in a parallel field of 0.06 T. Super- imposed is a spectrum (full circles) generated by adding the spectra of oxidized Fd I1 (according to 40% of total Fe) and oxidized FdR.

Interconversions between r3Fe-35'' and [4Fe-4S] Clusters

tioned above, activation experiments with beef heart aconitase have shown the facility of [3Fe-3S] to [4Fe-4S] cluster conversions (5). For aconitase, the presence of dithiothreitol accelerated the interconversion kinetics; we have therefore included dithiothreitol in the medium used for the D. gigas ferredoxin interconversions.

The EPR spectra shown in Fig. 5 are the results of a series of experiments with Fd 11. Fig. 5A shows the spectrum of oxidized Fd II; the observed g = 2.02 feature results from the [3Fe-3S] cluster. It was known from our Mossbauer studies (3) that this material contained no other iron than that associated with the [3Fe-3S] center. The spectra in Fig. 5, B to D, show the time course of an experiment in which Fd I1 was incubated with 1 eq each of iron and sulfide/[3Fe-3S] cluster and with a slight excess of dithionite. The appearance of a characteristic g = 1.94 EPR spectrum shows that [4Fe- 4S] clusters have been formed. Quantitation of the EPR spectra revealed that 36% of the [3Fe-3S] clusters had been converted into [4Fe-4SI1+ clusters after 10 h incubation. When a 5-fold excess of iron and sulfide was used, 72% of the clusters were found to be converted after 1 h incubation time (Fig. 5E); reoxidation of the sample in air revealed the presence of the unconverted [3Fe-3S] clusters (Fig. 5F). When dithiothreitol was replaced with mercaptoethanol only 5% rather than 72% of the clusters were found to be converted after 1 h incubation.

We have studied these cluster conversions with 57Fe Moss- bauer spectroscopy by incubating Fd I1 with iron isotopically enriched in either 56Fe or 57Fe. When 57Fe is added to the reconstitution medium the Mossbauer technique gives information about the sites into which the externally provided iron is incorporated, whereas the fate of the intrinsic iron is fol- lowed when @Fe (99.9% enriched) is used. Fig. 6A shows a Mossbauer spectrum of Fd I1 incubated anaerobically for 6 h with a 4-fold excess of 57Fe and sulfide, and then purified

21 2.0 1.9 g-value

FIG. 5. EPR spectra of native Fd I1 and of Fd I1 incubated with Fe2+ and Sa- in the presence of dithiothreitol and dithionite. A, spectrum of 0.47 m~ oxidized Fd 11. B to D, EPR spectra of 0.47 m~ Fd I1 incubated with 0.47 mM Fe2', 0.47 m~ S2-, 5 mM dithiothreitol, and about 0.7 mM dithionite for B, 1 h; C, 2.5 h; and D, 10 h. The spectrum shown in E was taken on a sample incubated for 1 h with both Fez+ and S2- at 2.3 mM; Fd 11, dithiothreitol, and dithionite concentrations were as in B. The spectrum in F was obtained after reoxidation of the sample used in E. Throughout this series, the conditions of EPR were as listed in the caption of Fig. 1.

I " " " " " '

I . .

6263

-4 -2 0 2 4 VELOCITY (rnm/s)

FIG. 6. Mossbauer spectra of Fd 11 activated with 95% enriched "Fe (sample A in the oxidized state). The spectrum shown in A was recorded at 4.2 K in zero field, while that shown in B was taken in a parallel applied field of 6 T. The solid line in B is the result of a spectral simulation, assuming diamagnetism, L W Q = 1.32 mm/s and q = 0.7.

under aerobic conditions. In the following, we refer to this material as sample A. The spectrum of sample A consists of a single quadrupole doublet with parameters identical with those obtained for doublet 2 of oxidized FdR. Thus, the externally provided iron is selectively incorporated into site 2.5 Since three indistinguishable subsites contribute to doublet 2, the spectrum shown in Fig. 6A gives no information about whether the 57Fe resides in one specific subsite or whether it is distributed among three sites.

The spectrum shown in Fig. 6B was recorded in an applied field of 6 Tesla. Analysis of the spectrum shows that it results from a diamagnetic species, as expected for the [4Fe-4SI2+ state. Moreover, the shape of the spectrum corresponds to that of component 2 of the spectrum shown inFig. 2C; in fact, the high field spectra can be described with identical parameters.

We have studied the sample in the temperature range from 4.2 K to 195 K. The quadrupole splitting was found to be strongly temperature-dependent; L W Q = 1.06 mm/s at 90 K and AEQ = 0.84 mm/s of 195 K. A least square fit to the spectrum displayed in Fig. 6A yielded a linewidth of r = 0.31 mm/s. Interestingly, the width increases to r = 0.40 mm/s at 90 K and becomes sharper again, I' = 0.35 mm/s at 195 K. This unusual behavior could be explained if the 57Fe would have been incorporated into at least two sites with different temperature dependences of A E Q . Alternatively, if only one site were occupied, the data suggest intermediate relaxation rates between the electronic ground state and one or more excited states; such a mechanism has been proposed to account for similar observations in an oxygenated heme compound (24).

After completing the experiments in the oxidized state, we reduced sample A with dithionite. Before presenting our data, we like to review briefly some pertinent features of the Moss- bauer spectra of [4Fe-4SI1+ clusters. In the +1 state, the tetranuclear clusters have electronic spin S = 1/2; the EPR signature is the well known "g = 1.94" signal. At 4.2 K, the electronic spin relaxation rate is slow compared to the 57Fe nuclear precession frequencies; consequently, the Mossbauer spectra exhibit magnetic features. Unfortunately, the component spectra resulting from the four iron nuclei are never well

We have assumed that the [4Fe-4S] clusters formed here are the same as those observed for FdR. This seems to be a valid assumption in view of the fact that both samples yield identical EPR spectra in the reduced state. The Mossbauer spectra presented in this section further justify this assumption. Moreover, preliminary experiments suggest that the proteins of FdR and sample A are in the monomeric state.

6264 Interconversions between [3Fe-3S] and L4Fe-45'1 Clusters

I

J -4 -2 0 2 4

VELOCITY (rnrn/s) FIG. 7. Mossbauer spectra of reduced FdR and of reduced

sample A recorded in zero field at 90 K. The spectrum of reduced FdR (hash marks) was prepared from the raw data (not shown) by subtracting the contributions of a reduced [3Fe-3S] cluster (10%) and of an Fez+ component (5%). Also shown in A is a spec'rum of reduced sample A (full circles). B shows a difference spectrum obtained by subtracting the spectrum of sample A (according to 50% of total Fe) from that of reduced FdR.

resolved. Nevertheless, by studying the clusters under a vari- ety of experimental conditions, in particular in strong applied fields, the spectra can be decomposed, not necessarily unambiguously, into subcomponents. Middleton and co-workers (23) have reported a data analysis of the low temperature spectra of the [4Fe-4S] ferredoxin from B. stearothermophilus. Two results of their study are especially noteworthy. First, the spectra could be fitted quite well by assuming that they consist only of two spectral components each represent- ing two equivalent iron sites. Second, high field data showed that the two components represented iron sites with internal magnetic fields, ant, of opposite sign, a manifestation of spin coupling. = -2- <S>/g,,& where A is the magnetic hyperfine tensor and <g> an appropriately taken expectation value of the electronic spin S; for details see Ref. 6.) Two pairs of equivalent sites with internal fields of opposite sign have also been observed for the [4Fe-4SI3+ state of oxidized high potential iron protein from Chromatium uinosum (25) and for the [4Fe-4SIL+ cluster of Escherichia coli sulfite reductase.6

At temperatures above 30 K, the electron spin relaxation rate of [4Fe-4SI1+ clusters is generally fasc consequently, the magnetic interactions are averaged out and quadrupole doublets are observed. The high temperature spectra of the [4Fe- 4S]'+ clusters of the B. stearothermophilus ferredoxin (23) and of E. coli sulfite reductase6 exhibit two well resolved doublets of equal intensity. This pattern is also indicated, albeit with poor resolution, for the [4Fe-4SI1+ cluster of FdR. A spectrum (hash marks) taken at 90 K is shown in Fig. 7A. (We have mentioned above that the spectra of the reduced sample contain a contribution from a reduced [3Fe-3S] cluster (10%) and a ferrous impurity (5%); we have removed their contributions from the spectrum shown in Fig. 7A.) We have labeled the two spectral components as doublet I and II; we will refer to the iron sites which give rise to these doublets as type-I and type-I1 sites.

A 90 K spectrum of the reduced sample A is shown also in Fig. 7A (full circles). Only one quadrupole doublet is observed. As pointed out in a preliminary account of these incubation experiments (26) , the 57Fe of sample A has been incorporated only into type-I1 sites; type-I sites are unoccu- pied. This observation allows us to correlate, for the fiist time, spectral components observed in different oxidation states of

J. A. Christner, P. A. Janick, L. M. Siegel, and E. Munck, manuscript in preparation.

a [4Fe-4S] cluster. It is clear that the site which gives rise to doublet 1 in the oxidized sample contributes to doublet I in the reduced state. We have fitted the doublet observed for sample A with two Lorentzian lines (0.36 mm/s full width) and obtained AEQ(II) = 1.67 mm/s and 6~,(11) = 0.60 mm/s. Next, we have subtracted the spectrum of sample A from the observed for FdR. The subtractions indicated that about 50% of the FdR spectrum is contributed by doublet 11. From the difference spectrum shown in Fig. 7B, we obtained AEQ(I) =

In its principal axes form, the electric field gradient tensor is characterized by the three components V,., V,, and Vzz. Since V., + V, + V,, = 0 the quadrupole interaction is generally expressed by quoting the two parameters

1.07 mm/~ and &+(I) = 0.51 -/s.

v = (V, - v , ) / v , *

In the absence of a strong applied field, only I A E Q I can be determined from the quadrupole spectra. In the limit of fast electronic relaxation, it is frequently possible to determine q and the sign of A E Q by studying the compound in strong applied fields. The presence of multiple sites has made such studies very difficult for [4Fe-4S]+' clusters. Encouraged by the observation of only one quadrupole doublet we have studied sample A at 90 K in an applied field of 4 Tesla. The spectrum shown in Fig. 8 displays a pattern typical of a compound with an axial symmetric field gradient, and A E Q > 0. The solid line shown in Fig. 8 is a spectral simulation for A E Q = +1.67 mm/s and q = 0, with the assumption that only one spectral component is present. The good match of the theoretical curve with the data supports this assumption. Thus, the spectra recorded at 90 K reveal the presence of one type of iron environment; they result either from a single site or from two equivalent subsites of the [4Fe-4S] cluster.

Fig. 9A shows a 4.2 K Mossbauer spectrum of sample A. Besides the expected magnetic component the spectrum contains a minority species (15% of total Fe) in the form of a quadrupole doublet, indicated in the figure. In Fig. 9B, we have displayed (full circles) a spectrum by removing the doublet from the raw data (we discuss this below). For comparison the appropriate spectrum of FdR (hash marks) is shown as well. It can be seen that the spectrum of sample A has a smaller magnetic splitting than that of FdR. This leads to the conclusion that the type-I1 sites have smaller magnetic hyperfine interactions than the type-I sites. Spectra obtained at 4.2 K in applied fields up to 6 Tesla (data not shown) revealed that the type-I1 sites have positive internal fields. This experimental result agrees with the conclusions reached from the data analysis of the B. stearothermophilus ferredoxin (23). Spectral simulations of the low temperature spectra of

I " " " " " ' l


FIG. 8. Mossbauer spectrum of Fd 11 activated with "Fe (reduced sample A) taken at 90 K in a parallel field of 4 T. The solid line is a theoretical spectrum computed with A E Q = 1.67 mm/s and q = 0.

Interconversions between [3Fe-3S] and [4Fe-4S] Clusters 6265

sample A suggest that they consist of only one magnetic component. This leaves open the question of whether the 57Fe has been incorporated into one site or two equivalent sites. The answer may be provided by EPR spin quantitation and measurements of the absolute Fe concentration and the 57Fe/

Fe ratio. We will perform such experiments after completion of further Mossbauer studies aimed at determining the hyperfine parameters more precisely.

Our data are compatible with the assumption that the “Fe has been incorporated into only one subsite. As mentioned above we cannot, however, exclude the possibility that two equivalent sites are occupied by 57Fe. Actually, fractional populations need to be considered as well since the mechanism of cluster conversion is not yet understood. Our data are quite clear on one point: 57Fe has not been incorporated into type- I sites (or site 1). The data also show that exchange between type-I and type-I1 sites does not occur at 25 “C within the incubation time of 6 h.

We have pointed out above that the spectrum of Fig. 9A contains a contribution from a quadrupole doublet. The presence of a doublet was quite obvious when the sample was studied at 4.2 K in zero field. Under these conditions, the spectra of [4Fe-4S] clusters are much less structured than those observed in weak applied fields and quadrupole doublets become more conspicuous. Furthermore, within the con- straints placed on the data analysis by the 90 K data, we could not fiid a set of hyperfine parameters explaining the shape of

56

I

- 4 -2 0 2 4 VELOCITY (mrn/s)

FIG. 9. 4.2 K Mossbauer spectra of reduced sample A and reduced FdR taken in parallel fields of 0.06 T. The spectrum of sample A shown in A contains a fast relaxing component in form of a quadrupole doublet, as indicated. The spectrum of the main, slow relaxing component shown in B (full circles) was prepared by subtracting the contribution (15%) of the fast relaxing species. For comparison the spectrum of reduced Fdn (hash marks) is also shown.

r“ ’ ’ ’ ’”


FIG. 10. Mossbauer spectrum of ferricyanide-treated FdR taken at 4.2 K in a parallel field of 0.06 T (hash marks). After the ferricyanide treatment the sample was purified as described under “Materials and Methods.” For comparison a spectrum of oxidized Fd I1 (full circles) is shown as well.

the spectrum shown in Fig. 9A. The zero field spectra show the presence of a doublet with A E Q =; 1.85 mm/s and =;

0.60 mm/s, which suggests that the doublet results from [4Fe- 4S]+’ clusters with fast electronic relaxation. (AEQ of doublet I1 is temperature-dependent; A&(II) = 1.80 mm/s at 40 K, AEQ(II) = 1.40 mm/s at 195 K). This would indicate that the clusters of a subpopulation of molecules experience strong spin-spin relaxation, probably caused by partial aggregation of molecules. We have demonstrated by Mossbauer and EPR spectroscopy (27) that such a phenomenon occurs in desulforedoxin samples. For that protein, the doublet component vanished, with a concomitant rise of the EPR signal, when the salt concentration was increased. The recognition that slow and fast relaxing components of the same species occur in desulforedoxin was based on the observation of only one Mossbauer spectral component when the sample was studied in an applied field of 4.5 Tesla. Likewise, the 6 Tesla kG spectrum of sample A seems to contain only one c~mponent .~

Conversion of [4-Fe-4S] to [3Fe-3S] Clusters by Ferricya- nide Treatment-As described under “Materials and Meth- ods,” we have incubated FdR with ferricyanide. The starting material, oxidized FdR, was EPR-silent, in accordance with the Mossbauer results discussed above. The EPR spectra of the ferricyanide treated samples were virtually identical with that shown in Fig. 1B. (Since a 57Fe-enriched sample was used, the EPR spectrum of the 57Fe-reconstituted protein is the proper reference.) After reduction with dithionite, the samples were found to be EPR-silent; these observations suggest the absence of [4Fe-4Slf’ clusters. In order to assess whether significant damage occurs when a sample is passed through a redox cycle, we have reoxidized a sample in air. A subsequent EPR study revealed that only 50% of the original g = 2.02 signal was recovered.

A Mossbauer spectrum of a ferricyanide-oxidized sample taken at 85 K exhibited two quadrupole doublets. The more intense doublet, accounting for 75 to 80% of total Fe, was virtually identical with that observed for the [3Fe-3S] cluster of oxidized Fd I1 (3). The remainder was a component with AEQ =; 0.8 mm/s and =: 0.45 mm/s. These parameters suggest that the minority doublet results most likely from Fe3+ sites not associated with a Fe-S cluster; it could possibly be a species similar to that removed from FdR preparations by a DEAE-column. This interpretation agrees with the fact that no EPR signal other than the g = 2.02 feature was observed at low temperature. Furthermore, the impurity (before DEAE treatment) in FdR yielded, at 4.2 K, broad spectra with magnetic features; because of its low concentration the putative Fe3+ impurity of the ferricyanide-treated sample would be hidden in the statistical noise of the low temperature spectrum shown in Fig. 10.

The Mossbauer spectrum (hash marks) of the ferricyanide- oxidized sample, purified on a Sephadex and a DEAE column, shown in Fig. 10 was taken at 4.2 K. For comparison, we have plotted also the corresponding spectrum of D. gigas Fd I1 (see also Fig. 2 of Ref. 3). It is apparent that the general features of the two spectra are the same. There are minor differences, barely above the noise, at Doppler velocities of -1.2 mm/s

For an isotropic S = 1/2 system, the argument is briefly stated. In the limit of slow relaxation the internal field is given by Hint(i) = - A<S,>,/g,P, where <S,>, and <S,>z are expectation values of the operator S, taken for the spin-down and spin-up states, respectively. In small applied fields, the Mossbauer spectrum depends on I HIntI; since = -<Sz>2, both states yield the same spectrum. For fast relaxation the <S,>, have to be replaced by the thermal average <Sz>th = (1 - exp x)/(l + exp x ) . For H = 6 Tesla, g = 2, and T = 1.5 K, the exponential terms can be neglected since x = -gpH/ KT = -5.4; thus, slow and fast relaxing species yield the same spectrum since <Sr>th G

6266 Interconversions between [3Fe-3S] and [4Fe-4S] Clusters

and +1.8 mm/s. However, the differences observed here are much less than the dissimilarities between the [3Fe-3S] cluster spectra of the A. vinetandii ferredoxin, beef heart aconitase, and D. gigas Fd 11. Taken together, the Mossbauer and the EPR spectra show clearly that the [4Fe-4S] cluster of FdR has been converted into a [3Fe-3S] center by ferricyanide oxidation.

DISCUSSION

We have demonstrated here that the protein derived from D. gigas ferredoxin can accommodate [3Fe-3S] clusters as well as [4Fe-4S] core structures. Moreover, we have shown that these clusters can be interconverted under appropriate conditions. In the following, we discuss the pertinent results of our studies. In a reconstitution experiment with excess iron and sulfide (5 eq each/protein monomer) we obtained a spectroscopically pure sample (FdR) containing [4Fe-4S] clusters. Since the apoprotein was derived from the [3Fe-3S]-protein Fd 11, our results persuasively indicate that the same polypeptide can accommodate both cluster types. Spectroscopi- cally, the [4Fe-4S] cluster of FdR is identical with that observed for the trimeric Fd I. As in the latter protein, the [4Fe- 4S] cluster is stabilized in the +1/+2 states. Preliminary experiments indicate that FdR is monomeric. The spectro- scopic similarity of the tetranuclear clusters of (chromato- graphically pure) Fd I and FdR suggests that the oligomeric state of the protein has little bearing on structural features as expressed by Mossbauer and EPR parameters. It will be interesting to learn whether the oligomeric state has influence on the redox potential.

This study has also demonstrated that [3Fe-3S] clusters can be reconstituted with substantial yields if the apoprotein is provided with less than 4 iron atoms/monomer in the reconstitution procedure. The percentage of [3Fe-3S] clusters can likely be increased by further lowering the concentration of iron and sulfide. Such conditions are suggested by the observation of [3Fe-3S] to [4Fe-4S] cluster conversions when Fd I1 is incubated with dithiothreitol and iron, i.e. excess amounts of iron seem to favor the formation of [4Fe-4S] clusters. Clearly, systematic reconstitution studies are desira- ble. Since both cluster types yield characteristic EPR spectra, although in different oxidation states, the technique of choice for the evaluation of such studies is EPR spectroscopy.

Mossbauer studies (5) of beef heart aconitase have demonstrated that a [3Fe-3S] cluster can be converted into a [4Fe- 4S] structure when the enzyme is activated with iron under reducing conditions. Furthermore, experiments with D. gigas crude cell extracts have shown that Fd I1 can develop intense g = 1.94 type EPR signals in the presence of pyruvate. We have demonstrated here that the 3-Fe cluster of Fd I1 can be converted into a [4Fe-4S] core structure by incubating the protein with iron and sulfide in the presence of dithiothreitol. Our studies show that the conversion kinetics depend critically on the relative concentrations of the reactants. When a stoichiometric amount of iron and sulfide was added, only 35% of the clusters were found to be converted after an incubation time of 10 h. With a 5-fold excess of iron and sulfide a 70% conversion yield was achieved with 1 h incubation time.

Thomson and co-workers (11) have reported that the low temperature magnetic circular dichroism spectra of ferricyanide-oxidized 2[4Fe-4S] ferredoxin from C. pasteurianum are very similar to those of Fd I1 (8). Johnson and co-workers (12) have extended these studies using the Resonance Raman technique; these authors have also reported that [4Fe-4S] clusters can be regenerated when the ferricyanide-treated material is incubated with excess sulfide. Using the [4Fe-4S] protein of reconstituted ferredoxin, we have extended the

ferricyanide oxidation studies to the D. gigas ferredoxin. The Mossbauer data show unambiguously that a [4Fe-4S] to [3Fe- 3S] cluster conversion can indeed be achieved by ferricyanide oxidation.

So far, we have discussed evidence that the [4Fe-4S] clusters of three different proteins can be converted by oxidative procedures, by ferricyanide treatment (11,12) or air oxidation (5), into [3Fe-3S] centers. Our studies have shown that such conversions can take place also under reducing conditions. We have found that dithionite reduction of FdR in 0.8 M Tris-HC1, pH 7.6, causes a substantial fraction of tetranuclear clusters to convert into 3-Fe centers. It thus appears that at high ionic strength the [4Fe-4S] cores of FdR are unstable under reducing conditions. We would like to add that we have obtained no evidence for the formation of [2Fe-2S] clusters in all manip- ulations we have performed with the D. gigas ferredoxin.

Although the influences of the different oligomeric states of the D. gigas ferredoxins need to be further explored, it is apparent that the basic polypeptide unit can accommodate [4Fe-4S] clusters as well as [3Fe-3S] cores. Comparison of the amino acid sequences of the D. gigas monomer (14) with those of some other ferredoxins provides some insights as to why both cluster types are accommodated with facility. As shown in Fig. 11, the B. stearothermophilus ferredoxin (28) has four cysteines. These residues are homologous with those linking one of the two [4Fe-4S] clusters of the Peptococcus aerogenes ferredoxin (29) to the polypeptide. It is instructive to compare these sequence patterns of cysteinyl residues with that reported for ferredoxin I from A. vinelandii. The latter protein contains a [3Fe-3S] and a [4Fe-4S] cluster; the points of cluster attachment deduced from X-ray crystallographic studies by Ghosh et at. (4) are shown in Fig. 11. According to the interpretation of the X-ray data 5 cysteinyl residues link the [3Fe-3S] core to the protein. A sixth binding site is thought to be occupied by an oxygen ligand, presumably water. Re- markably, the D. gigas sequence is constructed such that both cluster types can be fitted into the protein matrix: cysteinyl residues 8, 11, 14, 18, and 51 (or 41) could ligate to a [3Fe-3S] core whereas residues 8, 11, 14, and 51 (or 41) could form the linkages to the [4Fe-4S] cluster. Experiments designed to identify the cysteines involved in cluster binding are in process.

Besides information about the cluster type, the Mossbauer studies have provided some interesting information about structural features of [4Fe-4S] clusters. The spectrum shown in Fig. 2B reveals the presence of two types of iron sites with an occupancy ratio of 3:l. We have recently observed virtually identical spectra for ferredoxin I of A. vznelandii (1) and for

c

I 1

FIG. 11. Comparison of the cysteine distribution for the ferredoxins from D. gigas (D.g.), A. vinelandii (A.u.), P. aemgenes (Pa), =dB. steumthennophilus (B. st.). The cluster attachments for the A. uinezandii and P. aerogenes ferredoxins have been determined by X-ray crystallography (4, 29); those indicated for the D. gigas and B. stearothermophilus ferredoxin are speculative.

Interconversions between [3Fe-3S] and [4Fe-4S] Clusters 6267

aconitase (5). The [4Fe-4S] clusters of these proteins exhibit, like FdR, a pronounced temperature dependence of the quadrupole splitting. In contrast, the [4Fe-4SI2+ clusters of phos- phoribosylpyrophosphate amidotransferase from Bacillus subtilis (30) and E. coli sulfite reductase (31) exhibit one sharp quadrupole doublet with a splitting nearly independent of temperature. Clearly, the influence of the protein matrix on the [4Fe-4SI2+ core is expressed in the Mossbauer spectra. We would like to point out that the Mossbauer data obtained for some [4Fe-4SI1' clusters show two sites with a 2:2 site ratio; well resolved spectra have been observed for the ferredoxin from B. stearothermophilus (23) and for E. coli sulfite reductase? A 2:2 site ratio is also suggested, but not resolved, for reduced FdR. It is noteworthy that the observed site ratios do not correspond to the ratios which one might, somewhat naively, expect from the notion that the clusters are formally 2 Fe(I1) + 2 Fe(II1) and 3 Fe(I1) + 1 Fe(II1) in the +2 and +1 states, respectively.

The cluster conversion performed with Fd I1 demonstrates that subsites of [4Fe-4S] clusters can be labeled with 57Fe. The data obtained here on sample A show that the externally supplied iron occupies either one subsite or two equivalent subsites. The [4Fe-4SI2+ clusters of sample A and of beef heart aconitase yield virtually the same spectra, both suggesting a 3:l site occupancy. We have shown here with sample A that the externally provided iron is incorporated into a site which yields doublet 2. Curiously, when beef heart aconitase is activated, the iron atom is incorporated into the (single) site which yields doublet 1 (5). Presently, it is not clear what structural features distinguish these sites. The observed differences in A E Q need not imply that the site 1 and site 2 iron atoms are attached to different amino acid residues; small asymmetries in the protein environment may be accentuated by the Mossbauer parameters. Kinetic accessibility may be at the root of the site selection.

The 4.2 K spectra of reduced sample A reflect the paramagnetic hyperfiie structure of the type-I1 sites. Studies in strong applied fields revealed that these sites have positive internal magnetic fields. Preliminary data analyses suggest that the magnetic hyperfine tensor of a type-I1 site is quite anisotropic. Since we have been able to determine sign (AEQ) and 7, it should be possible to determine the whole set of hyperfine parameters with added precision. The achievement of isotopic labeling of subsites should allow us to characterize the electronic structure of [4Fe-4S] clusters in more detail than has been possible so far. Such labeling will benefit the analysis of electron nuclear double resonance spectra as well.

Finally, two questions need to be asked: Are the cluster interconversions, observed here in uitro, of physiological rel- evance and, do [3Fe-3S] clusters have a physiological role or does their occurrence result from oxidative damage of [4Fe- 4S] clusters? Present evidence is not sufficient to provide the answers. Fortunately, the metabolic pathways of D. gigas seem to be suitable for experimental tests of these questions.

REFERENCES 1. Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-

Johnson, W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796

2. Stout, C. D., Gosh, D., Pattabhi, V., and Robbins, A. H. (1980) J. Biol. Chem. 255, 1797-1800

3. Huynh, B. H., Moura, J. J . G., Moura, I., Kent, T. A,, LeGall, J., Xavier, A. V., and Munck, E. (1980) J. Biol. Chem. 255,3242- 3244

4. Ghosh, D., Furey, W., Jr., O'Donnell, S., and Stout, C. D. (1981) J. Biol. Chem. 256,4185-4192

5. Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H., Emp- tage, M. H., Beinert, H., and Miinck, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1096-1100

6. Munck, E. (1982) in Iron-Sulfur Biochemistry (Spiro, T. G., ed) Chap. 4, pp. 147-175, John Wiley & Sons, New York

7. Miinck, E. (1981) in Mossbauer Spectroscopy and Its Chemical Applications (Stevens, J . G., and Shenoy, G. K., eds) pp. 305- 328, American Chemical Society, Washington, D.C.

8. Thomson, A. J., Robinson, A. E., Johnson, M. K., Moura, J. J. G., Moura, I., Xavier, A. V., and LeGall, J . (1981) Biochim. Bio- phys. Acta 670,93-100

9. Kent, T. A., Huynh, B. H., and Munck, E. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,6574-6576

10. Kent, T. A., Dreyer, J.-L., Emptage, M. H., Moura, I., Moura, J. J. G., Huynh, B. H., Xavier, A. V., LeGall, J., Beinert, H., Orme- Johnson, W. H., and Munck, E. (1982) in Symposium on Znter- action between Iron and Proteins in Oxygen and Electron Transport (Ho, C., ed) Elsevier/North Holland, Amsterdam, in press

11. Thomson, A. J., Robinson, A. E., Johnson, M. K., Cammack, R. Rao, K. K., and Hall, D. 0. (1981) Biochim. Biophys. Acta 637, 423-432

12. Johnson, M. K., Spiro, T. G., and Mortenson, L. E. (1982) J. Biol. Chem. 257,2447-2452

13. Travis, J., Newman, D. J., LeGall, J., and Peck, H. D., Jr. (1971) Biochem. Biophys. Res. Commun. 45,452-458

14. Bruschi, M. (1979) Biochem. Biophys. Res. Commun. 91,623-628 15. Johnson, M. K., Hare, J. W., Spiro, T. G., Moura, J . J. G., Xavier,

A. V., and LeGall, J . (1981) J. Biol. Chem. 256.9806-9808 16. Moura, J. J. G., Xavier, A. V., Hatchikian, E. C., and LeGall, J.

(1978) FEBS Lett. 89, 177-179 17. Munck, E. (1978) Methods Enzymol. 54,346-379 18. Bruschi, M., Hatchikian, E. C., LeGall, J., Moura, J. J. G., and

Xavier, A. V. (1976) Biochim. Biophys. Acta 449,275-284 19. Fischer, D. S., and Price, D. C. (1964) Clin. Chem. 10,21-31 20. Hong, J.-S., and Rabinowitz, J . C. (1970) J. Biol. Chem. 245,

21. Cammack, R., Rao, K. K., Hall, D. O., Moura, J. J. G., Xavier, A. V., Bruschi, M., LeGall, J., Deville, A., and Gayda, J . P. (1977) Biochim. Biophys. Acta 490,311-321

22. Xavier, A. V., Moura, J. J . G., and Moura, I. (1981) Struct. Bond.

23. Middleton, P., Dickson, D. P. E., Johnson, C. E., and Rush, J. D. (1978) Eur. J. Biochem. 88, 135-141

24. Spartalian, K., Lang, G., Collman, J. P., Gagne, R. R., and Reed, C. A. (1975) J. Chem. Phys. 63,5375-5382

25. Middleton, P., Dickson, D. P. E., Johnson, C. E., and Rush, J . D. (1980) Eur. J. Biochem. 104,289-296

26. Kent, T. A., Moura, I., Moura, J. J. G., Lipscomb, J. D., Huynh, B. H., LeGall, J., Xavier, A. V., and Munck, E. (1982) FEBS Lett. 138, 55-58

27. Moura, I., Huynh, B. H., Hausinger, R. P., LeGall, J., Xavier, A. V., and Munck, E. (1980) J. Biol. Chem. 255,2493-2498

28. Hase, T., Ohmiya, N., Matsubara, H., Mdinger, R., Rao, K. K., and Hall, D. 0. (1976) Biochem. J. 159.55-63

29. Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973) J. Biol. Chem. 248,3987-3996

30. Averill, B. A., Dwivedi, A., Debrunner, P., Vollmer, S. J., Wong, J. Y., and Switzer, R. L. (1980) J. Biol. Chem. 255, 6007-6010

31. Christner, J . A., Munck, E., Janick, P. A., and Siegel, L. M. (1981) J. Biol. Chem. 256, 2098-2101

6574-6581

43, 187-213

the journal of chemistry vol. 257, no. 11, june 10, pp ... · the recent discovery (1-3) of...

Documents