THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 11, Issue of June 10, pp. 6871-6881,lW Printed in U.S.A.

Mossbauer Studies of Aconitase SUBSTRATE AND INHIBITOR BINDING, REACTION INTERMEDIATES, AND HYPERFINE INTERACTIONS OF REDUCED 3Fe AND 4Fe CLUSTERS*

(Received for publication, January 10, 1985)

Thomas A. Kent," Mark H. Emptage,"." Hellmut Merkle,"' Mary Claire Kennedy,b Helmut Beinert,"" and Eckard Munck",' From the "Gray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392 and the bZnstitute for Enzyme Research and Department of Biochemistv, College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

Active beef heart aconitase contains a [4Fe-4S] clus- ter. One iron of the cluster, Fen, is labile and can be removed easily by oxidation in air to yield the [3Fe- 4S]'+ cluster of inactive aconitase. We have previously shown that substrate binds to Fen. We have continued our Mossbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4SlZ+, is mixed with substrate, two species (substrates or intermediates bound to Fe.) la- beled Sl and Sz are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species Sz, but not S1, is observed, suggesting that SZ represents a carbanion transition state complex. We have prepared Mossbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 "C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 O C . Details of these experiments are given, al- though in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4SI1+; EPR at g = 1.94) in the presence of substrate with material selectively en- riched with "Fe in either Fen or the other three cluster sites. The spectra were analyzed with a spin Hamilton- ian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the re- duced [3Fe-4SIo cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase con- taining a 3Fe cluster is not active in either oxidation state.

* This work was supported by Research Grants GM 12394 (to H. B.) and GM 22701 (to E. M.) from the National Institutes of Health and Grant PCM 830964 (to E. M.) from the National Science Foun- dation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

e Present address: E. I. du Pont de Nemours and Company, Central Research and Development Department, Wilmington, DE 19898.

Present address: National Biomedical ESR Center, Medical Col- lege of Wisconsin, Milwaukee, WI 53226.

e Recipient of Research Career Award 5-K06-GM-18442 from the National Institutes of Health.

'To whom correspondence should be addressed Gray Freshwater Biological Institute, University of Minnesota, P. 0. Box 100, Navarre, MN55392.

In the work to be reported below, we have consolidated our Mossbauer spectroscopic studies on aconitase, extended them toward the resolution of thus far unresolved problems, and have explored the usefulness of Mossbauer spectroscopy in attempts to resolve early events in enzyme catalysis.

Aconitase catalyzes the reversible hydration of cis-aconitate to either citrate or isocitrate. We have shown previously (1- 4) that the enzyme isolated under aerobic conditions (inactive aconitase) contains a [3Fe-4S] cluster. Upon activation in the presence of ferrous ion, the [3Fe-4S] cluster is converted (1, 5), by incorporation of Fez+, into a diamagnetic [4Fe-4S} cluster. This finding, together with the observation that highly active aconitase does not contain any single Fe site (6), has cast serious doubts on the earlier proposed mechanism of aconitase action (7, 8). Addition of substrate to active aconi- tase causes a dramatic change in the Mossbauer spectra of the [4Fe-4SJ2+ cluster. This change is mainly confined to Feat the site generated by incorporation of Fez+ into the inactive [3Fe-4S] cluster core. The Mossbauer data show that sub- strates bind at Fe. which develops a localized Fez+ state by expansion of the ligand sphere from a tetrahedral to a five- or six-coordinated environment (6). In the presence of 170H- labeled citrate, the EPR spectrum of the reduced [4Fe-4SI1+- citrate complex exhibits transferred hyperfine interactions, suggesting that Fe. accepts the hydroxyl group of the sub- strate, thus facilitating its removal (6).

Aconitase is most active when the [4Fe-4S] cluster is in the diamagnetic 2+ state. Although the reduced state, [4Fe-4SI1+, appears to exhibit some activity (3), this state is probably less important because the redox potential, 5-500 mV, is too low to render this state physiologically relevant. Johnson and co-workers (9) have recently suggested that there may be significant catalytic activity associated with enzyme containing the reduced [3Fe-4S] cluster. These authors, how- ever, caution that the spectroscopic and activity assays are performed at widely differing enzyme concentrations (-10,000-fold) and that cluster transformation to [4Fe-4S] may occur under the conditions of the activity assay. We will address this question below.

Mossbauer spectroscopy has proven to be well-suited for the study of cluster transformations and substrate binding of aconitase. In particular, its utility has been demonstrated by the discovery of two novel cluster interconversions (1, 10). Since the spectra of the Fe. site are substantially changed in the presence of substrates, this technique can also be used to probe with good sensitivity the effects of substrate and inhib- itor binding. Schloss and co-workers (11, 12) have studied the binding of the nitro analogs of citrate and isocitrate and have suggested that these extremely tight-binding inhibitors are appropriate transition state analogs, serving as models of

6871

6872 Mossbauer Studies of Aconitase

carbanion intermediates of the aconitase reactions. Thus, a Mossbauer study with these inhibitors promised to yield val- uable information.

When either of the three substrates is added to the enzyme at 25 "C, with enzyme and substrate concentrations ( ~ 0 . 5 mM) as required for Mossbauer studies, equilibrium is at- tained before the samples can be frozen. Therefore, it was desirable to use techniques which allow one to freeze the samples before the enzyme has turned over. In the present study, we have explored two approaches to this problem. By adapting the well-developed rapid mix/rapid freeze technique to the preparation of Mossbauer samples, we have been able to achieve good time resolution of the reactions with the three substrates. In an alternative approach, we have attempted to expand the range of mechanistic studies by dissolving the enzyme at subzero temperatures in suitable cryosolvents. We will show below that new spectral components are observed when substrates are added at subzero temperatures.

Finally, activation and exchange reactions have allowed us to label iron subsites of the [4Fe-4S] cluster with 57Fe in either the Fe, site or the three complementary sites, Feb. This selective labeling has made it possible to study the paramag- netic hyperfine structure of the reduced [4Fe-4SI1+-substrate complex with a spectral resolution substantially increased over that previously achieved (6).

MATERIALS AND METHODS

Nitrocitrate (2-hydroxy-3-nitro-l,2-propanedicarboxylate), nitro- isocitrate (3-hydroxy-2-nitro-l,3-propanedicarboxylate), a-methyl- cis-aconitate (cis-2-butene-1,2,3-tricarboxylate), and threO-D.-a- methylisocitrate (3-hydroxybutane-1,2,3-tricarboxylate) were the kind gifts of Dr. J. V. Schloss (E. 1. du Pont de Nemours & Co.). (-)-erythro-2-Fluorocitrate was the kind gift of Dr. E. Kun (Univer- sity of California, San Diego). Other materials used and their sources are those listed in a previous publication (5).

67Fe-enriched Aconitase-Aconitase was isolated in its inactive form from beef heart mitochondria, activated anaerobically with iron and dithiothreitol, and assayed as previously described (5). Activation with 90% enriched 67Fe generated [4Fe-4S] clusters with a single iron site, Feat enriched. To enrich the other three iron sites, Feb, with 90% '?Fe, the procedure of Ref. 10 was modified in the following way. After ferricyanide treatment of the 67Fe-exchanged aconitase, the sample was desalted and concentrated to about 100 pl. In an alter- native method, an enzyme solution, 4-5 ml (-40 mg protein/ml), purified up to the final step of the usual purification procedure (5), i.e. before passage through the CM-Sephadex C-50 column, was concentrated to 1.5-2.0 ml, converted to purple aconitase, and acti- vated with "Fe as described previously (10). (In one experiment, "Fe was added to the activation medium to determine the extent of iron incorporation.) The ferricyanide-inactivated material was then de- salted into 8 mM phosphate, pH 7.4, and put on a CM-Sephadex C- 50 column and eluted as in the final step of the purification procedure. The enzyme was then concentrated for Mossbauer studies or it was activated as mentioned above with 100% 6BFe for Mossbauer spec- troscopy of the Fet, sites without interference from the Fe. site. Dithionite-reduced samples were prepared anaerobically as previously described (1).

tures of -22 "C were performed with a Beckman DU whose cell Low Temperature Assays-Continuous assays down to tempera-

compartment was cooled with a Neslab refrigerated circulating bath. Better thermal isolation of the cell compartment was achieved by addition of polyethylene plates between it and the optical and detector housings and by attachment of polyurethane foam sheets to the outer surfaces. Dry thermostated nitrogen was admitted through the bottom of the compartment to prevent moisture condensation. Mixing of the assay materials was done with a pre-cooled polyethylene paddle.

Rapid Freeze Samples-Rapid freeze Mossbauer samples were pre- pared by rapid mixing of 67Fe activated aconitase (150-200 mg/ml) with 5 mM substrate, using an Update Instruments ram and Model 725 controller. The system was essentially that described by Arm- strong et al. (13), except that a specially designed combination mixing chamber with an integrated spray nozzle was used for the fastest quenching time of about 4 ms (14). For longer mixing times, delay hoses of appropriate length were used in conjunction with an 0.008-

inch diameter nozzle. The reaction was quenched by shooting into a wide-stem Pyrex funnel which had an attached nylon Mossbauer cup at the bottom and which contained isopentane at -140 "C. The temperature of the syringes and mixing chamber was at 0 or 20 "C.

After shooting the sample, ice crystals were allowed to settle for about 1 min. Then vacuum from a water aspirator was applied to pull the isopentane out of the funnel through the bottom of the Mossbauer cup. The cup had a perforated bottom and a Whatman No. 1 filter paper covering it to allow the passage of isopentane but retain the crystals of frozen material. After the liquid had disappeared, a pre- cooled polyethylene packer was used to compress the ice crystals into the bottom of the cup. Subsequently, precooled filter paper and cotton (as filling material) were added into the cup before it was sealed with a nylon lid and placed into a brass fitting for shipment in a cryogenic container.

A second procedure for rapidly freezing equilibrium samples in less than a second was used. This entailed spraying the sample by hand from a glass syringe through a fine polyethylene nozzle onto a rotating aluminum pan, which was cooled by liquid nitrogen. The syringe was either kept at room temperature or cooled to -30 'C in a metal tube immersed in a dry ice/ethanol bath. The small ice beads were then scraped off and packed into a precooled Mossbauer cup. Filter paper and cotton were placed on top as mentioned above to maintain a compact pellet.

Cryosamples-All sample manipulations were done either in glass vessels under argon or in a nitrogen-filled glove bag, or under a stream of argon. Liquids were degassed and flushed with argon before use. Two different solvent systems were used. The first allowed the incubation of samples at -30 "C by the addition of an equal volume of ethylene glycol to an aqueous Mossbauer sample in 100 mM HEPES,' pH 7.5. We have measured the activity in the water/ ethylene glycol system over the temperature range from 45 to -30 "C. The activity follows an Arrhenius equation with an activation energy of 48 kJ/mol.

A second solvent system was used for cryoenzymology at -60 "C. It contained approximately 50% H20, 30% methanol, and 20% eth- ylene glycol; the mixture has a freezing point near -60 "C. (At -30 and at 0 "C, the activity measured in this solvent was essentially the same as observed in water/ethylene glycol.) Because methanol tends to precipitate the protein near 0 "C and above, ethylene glycol was used to help stabilize the enzyme, and methanol was added in steps at lower temperatures: The procedure used was as follows. 75 pl of ethylene glycol is added to 150 p1 of activated aconitase (150 mg/ml) in 100 mM HEPES, pH 7.5. Methanol is added in two 50-pl aliquots, the first at 0 "C and the second at -30 'C. The sample is then incubated in a stirred isopentane bath maintained at -55 to -57 'C. After 15 min of incubation, 20 p1 of precooled 40 mM substrate in the same solvent system is added, and the mixture is stirred for 1 min with a precooled motor-driven polyethylene paddle and quickly frozen in liquid nitrogen. The shortest time between addition of substrate and complete freezing of the sample was about 2 min.

RESULTS

Studies of the Reduced 13Fe-4SI Cluster-Recently, we have described a procedure for enriching the various cluster types that have been identified in aconitase with the Mossbauer isotope 67Fe. For the linear[3Fe-4SI1+ cluster of purple acon- itase, we have shown (10) that the three cluster subsites become homogeneously labeled (see also "Materials and Methods"). Using the same procedure, but omitting the sub- sequent conversion to either the linear 3Fe or the 4Fe cluster, we have prepared 67Fe-enriched aconitase containing the [3Fe- 4S]'+ cluster in the g = 2.01 form. We have studied the sample with Mossbauer spectroscopy and have obtained the same results as reported (1) for the unenriched enzyme. Here we report some new results on the dithionite-reduced inactive enzyme.

By providing a suitable Fez+ concentration, the [3Fe-4Slo cluster is readily converted into the active [4Fe-4SI2+ cluster (1, 5). In fact, under reducing conditions, the [4Fe-4S12+

I The abbreviation used is: HEPES, N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid.

Mossbauer Studies of Aconitase 6873

cluster is even formed in the absence of externally added Fez+, presumably by transfer of Fez+ between Fe-S clusters. In order to ensure that no Fez+ is available for the cluster interconver- sion, we have added an excess of EDTA to the sample. EDTA is neither bound to the enzyme nor does it inhibit aconitase (5). A 4.2 K Mossbauer spectrum of a dithionite-reduced sample containing excess EDTA is shown in Fig. lA (hatch- marks). The solid line drawn above the spectrum shows a spectrum which we have observed for Fe(II).EDTA; about 20% of the 57Fe of our sample belongs to this species. Sub- traction of the Fe(II).EDTA component from the raw data yields the spectrum shown in Fig. 1B. The latter spectrum, a typical signature of a [3Fe-4SIo cluster, can be decomposed into three quadrupole doublets of equal intensity. The left absorption band contains the low-energy lines of all three doublets. The sharp line at +0.6 mm/s Doppler velocity is the high-energy line of one doublet (belonging to the Fe3+ com- ponent of the cluster, see Ref. 1). The rightmost absorption line has twice the area as the line at +0.6 mm/s and is distinctly broader, suggesting that the two contributing lines are absorbing at different velocities. Thus, the three iron sites of the reduced [3Fe-4S] cluster are inequivalent, in contrast to the [3Fe-4S] clusters studied thus far which exhibit one equivalent pair of iron atoms at the oxidation level and one Fe3+ site. The solid line of Fig. 1B is the result of decomposing the spectrum into three doublets of equal inten- sity, fixing the widths of all six lines at 0.28 mm/s. By pairing the absorption lines in such a way as to yield the best match in isomer shifts with Desulfouibrio gigas ferredoxin 11, we obtained hEQ(l) = 0.57 mm/s and 6(1) = 0.30 mm/s for the Fe3+-type site and h E ~ ( 2 ) = 1.16 mm/s, 6(2) = 0.44 mm/s and h E ~ ( 3 ) = 1.47 mm/s and 6(3) = 0.47 mm/s for the two type sites. This is in good agreement with h E Q ( 1) = 0.47 mm/

-4 -2 0 2 4 VELOCITY ["/SI

FIG. 1. Mossbauer spectra of the reduced [3Fe-4S] cluster of aconitase (0.5 IUM). The spectra were recorded at 4.2 K in zero magnetic field. A, spectrum (hutchmarks) of the dithionite-reduced [3Fe-4S] cluster in the presence of 2 mM EDTA. The solid l ine indicates the spectrum of Fe(I1). EDTA. After subtracting the con- tribution (20%) of the Fe(II).EDTA complex from the data, the spectrum shown in B was obtained. This spectrum represents the [3Fe-4SIo cluster; the solid line is the result of least-squares fitting the spectrum to three quadrupole doublets, with parameters given in the text. Addition of 5 mM citrate to the sample results in a spectrum (dots in A ) which is practically identical to that of the substrate-free material.

s, 6(1) = 0.30 mm/s and U Q ( 2 ) = hEQ(3) = 1.47 mm/s, 6(2) = 6(3) = 0.46 mm/s reported for D. gigas ferredoxin I1 (15).

In an applied field of 60 millitesla, the spectrum of Fig. 1B is smeared out because of the onset of magnetic hyperfine interactions (see Fig. 1 of Ref. 1). Under these conditions, one can assess very well whether the sample contains [4Fe-4SI2+ clusters (a diamagnetic species). Our data show that not more than 8% of the molecules, if any, of our sample contain the [4Fe-4Slz+ cluster. After completing our studies, the sample was thawed under anaerobic conditions, and an excess of citrate was added. After removal of an aliquot for activity measurements, the sample was frozen. A Mossbauer spectrum of the sample is shown in Fig. L4 (dots). It can be seen that the spectrum of the [3Fe-4S] cluster is identical to that of the citrate-free material. Thus, there is no evidence that citrate binds to the [3Fe-4SIo+ cluster (as reported earlier (3, 6) and as shown below, the addition of citrate to active aconitase causes drastic changes in the spectra of the [4Fe-4S] cluster). The activity of the sample was less than 5% of that obtainable for fully active material. Thus, we conclude that citrate is not bound to the [3Fe-4S] cluster and, furthermore, that aconitase containing a [3Fe-4S] cluster is catalytically not active.

Studies with Substrates-Aconitase as isolated under aero- bic conditions contains a [3Fe-4S] cluster. Upon activation of the enzyme with Fez+, the added iron is incorporated into the [3Fe-4S] cluster core to yield a [4Fe-4S] cluster. The specific site to which the external iron is added is referred to as the a site or Feat the remaining three sites are Feb. For the studies described in this section, we have used 57Fe, enriched to >go%, for the activation. Fig. 2.4 shows a Mossbauer spectrum of activated aconitase (cluster in the diamagnetic 2+ state) with 57Fe in Fe,. Upon addition of substrate to active aconitase, two new species are observed. We have labeled these species SI and Sz in our earlier studies (6) and have emphasized that SI and Sz have distinct ferrous character (by this we mean that A E Q and 6, in particular 6, are typical of high-spin Fez+. Note that SI and Sz result from Fe. which is a subsite of the diamagnetic [4Fe-4SI2+ cluster.).

When we freeze-quenched aconitase 5 ms after mixing at 20 "C with any of the three substrates, we obtained a Moss- bauer spectrum essentially identical to that seen previously after long-term incubation with substrate. Thus, the three substrates, citrate, cis-aconitate, and isocitrate, have attained their equilibrium concentrations on the enzyme in 5 ms or less. Since the turnover time at 20 "C for the substrates is 25 to 75 ms (€4); these results strongly suggest that product release is the rate-limiting step in the catalytic cycle of acon- itase. In order to study in more detail the binding of the three substrates, we have used a rapid freeze technique (after mixing at 0 " C ) and a cryobiochemical approach.

Before discussing our results, we would like to make two comments. First, our studies with substrates have shown that the three b sites of the [4Fe-4S] cluster are very little affected when substrate is added to the enzyme. A E Q changes from 1.34 mm/s to 1.13 mm/s upon addition of substrate (the three b sites have the same ~ E Q ) ; the isomer shift, 6 = 0.45 mm/s at 4.2 K, is the same with and without substrate. In Figs. 2 and 3 we have therefore subtracted from the raw data the contributions (7% of total 57Fe) of the b sites (which contain 57Fe in natural abundance, 2.2%). Second, we have consist- ently observed that some of the original spectrum remains (see Table I) and conclude that some of the enzyme does not bind substrates or inhibitors at Fe,. Thus, all spectra exhibit a component apparently identical to the doublet of Fig. 2A

M. H. Emptage, H. Merkle, M. C. Kennedy, and H. Beinert, unpublished results.

6874 Mossbauer Studies of Aconitase

I I . . .

I . * . .

I - . . . . . . . - ms .I . . * . f

i I *.

E 0.5 - CITRRTE

CT m

" : : y

li v VELOCITY [ MM/S I

FIG. 2. Mossbauer spectra of the a site of the [4Fe-4SI2+ cluster of active aconitase. Spectra were recorded at 4.2 K in zero field. In all spectra the contributions of the b sites have been removed. A, spectra of the a site of the [4Fe-4SJ2+ cluster. B and C, spectra observed after 2.5 mM cis-aconitate was mixed with the enzyme, and the sample was quenched in isopentane at -130 "C after 5 ms B or 35 ms C. The spectra in U and E were obtained when 2.5 mM citrate D and 2.5 mM isocitrate E were used and the samples were quenched after 35 ms. The brackets in D designate components SI and Sz. The spectrum in F was obtained after 5 mM nitrocitrate was added to the enzyme; the sample was frozen in liquid nitrogen. The solid lines in D-F are least-squares fits. The relevant spectral parameters are listed in Table 1.

(in the following we refer to this doublet as Feeo). Presently, we do not understand the meaning of this observation. It is possible that some of the enzyme is "inactive." Alternatively, some enzyme molecules may be in a conformation which is not receptive or the substrates or inhibitors may be bound in conformations or orientations which may not involve coordi- nation to the cluster and therefore do not change the signal of Fe.. For instance, the competitive inhibitor tricarballylate has no effect on the Mossbauer spectra. Furthermore, we have observed two spectral forms in reduced aconitase (no sub- strates present) both with EPR and Mossbauer spectroscopy, when Tris . HC1 is used as buffer. Thus, buffer molecules could block access to the active site. Finally, the amount of Fe." material does not depend on the freezing rate; freezing in liquid NP (=5 s) and rapid freezing in isopentane (=5 ms) after addition of cis-aconitate produced the same distribution of species as when the material was equilibrated at 25 "C after addition of cis-aconitate.

Rapid Freeze Studies-As described under "Materials and

0. c

1.0

,".

bp u

0. e z 0 l-4

0.5 [r 0 ul CT m

0.0

1.0

I

-4 -2 0 2 4 VELOC I TY [ MM/S I

FIG. 3. 4.2 K Mossbauer spectra of active aconitase ( 1 mM) in water/methanol/ethylene glycol after addition of 2.5 mM citrate at subzero temperatures. A, citrate was added at -60 "C and allowed to react with the enzyme for 5 min before freezing in liquid NP. C, the sample of A was kept for 5 h at -30 "C and was then refrozen. The spectrum shown in B was obtained by subtracting 40% of the spectrum of C from that of A . The solid l ine in B represents a least-squares fit. Contributions of the b sites have been removed from the spectra prior to fitting.

Methods," we have added citrate, cis-aconitate, and isocitrate to active aconitase at 0 "C and quenched the reactions in isopentane at -140 "C at 5, 15, or 35 ms. After 5 ms, approx- imately 35% of the material has reacted with citrate, with 18% of the a sites yielding doublet SI and 17% yielding S,. 35 ms after mixing, 69% of the absorption belongs to S1 and Sz; a spectrum is shown in Fig. 2 0 . The absorption lines of all three doublets are exceedingly sharp; the full width at half- maximum is 0.26 mm/s for each of the doublets, close to our instrumental width of 0.23 mm/s. Our studies thus show that citrate is bound rather slowly; both SI and SP, however, are present in roughly equal proportions after 5 ms. After 35 ms, the equilibrium of ~ 4 0 % for Sl and ~ 4 0 % for SP has not been attained? In contrast, cis-aconitate and isocitrate react very

The uncertainties are difficult to estimate in some cases because of the presence of minor, unresolved components. To analyze the data of Figs. 2-5, we have used a computer program which allows one to merge (for comparison, addition and subtraction) various data files. The program can also generate a single absorption line of

over 300 aconitase spectra since 1981) and generated line shapes, a Lorentzian shape. Using the various data files (we have accumulated

components successively. Only after we had understood the principal given experimental spectrum can be analyzed by stripping spectral

features of a spectrum did we analyze it further by a least-squares fitting procedure. In some cases, such as for the data of Fig. 2F, the uncertainties in the determinations of relative concentrations of species are quite small (about fl%). In other cases, the uncertainties are significant despite the fact that the spectra appear to be fitted well. Thus, the concentrations of SI and SP in the spectrum of Fig. 2 0 are each +4%; these uncertainties were estimated by fitting and stripping spectra of the data with different assumptions about the line shapes.

Mossbauer Studies of Aconitase

TABLE I

6875

Results of rapid quench and cryobwchemical studies Line 1 gives a typical result for activated aconitase. Approximately 8% of the total absorption belongs to

unidentified species (see shoulder in Fig. 2.4). Line 2 was obtained on a sample equilibrated at 0 "C and then frozen in liquid nitrogen. Lines 3-10 are rapid quench experiments. Substrates were mixed with active enzyme at 0 "C, and the reaction was quenched in isopentane at -130 to -140 'C at times indicated (e.g. 35 ms after mixing). Lines 11-16 refer to samples prepared in water/methanol/ethylene glycol (503020, % v/v). The percentages listed do not in general add up to 100% because of uncertainties of analyses and because minor unidentified species may have been present. Substrate and inhibitor concentrations were 2-10 mM while enzyme concentrations were 0.5-1 mM.

Substrate Conditions mQ 6 % AEQ 6 % Fe. Comments

1 2

3 4 5

6 7 8 9

10 11 12

13 14 15 16 17

18

19 20

21

None Citrate

Citrate Citrate cis-Aconitate

cis-Aconitate Isocitrate Isocitrate Isocitrate Isocitrate Citrate Citrate

cis-Aconitate cis-Aconitate Isocitrate Isocitrate a-Methyliso-

citrate a-Methyliso-

citrate Nitrocitrate Nitroiso-

citrate

Fe.": Equilibrated s1: Rapid quench, 5 ms S1: Rapid quench, 35 ms Rapid quench, 5 ms SI:

Rapid quench, 35 ms Rapid quench, 5 ms S1': Rapid quench, 5 ms Rapid quench, 15 ms Rapid quench, 35 ms

at 0 "C

5 min at -60 "C S3: 5 h at -30 "C

18 min at -60 "C Sa: 1 h at -30 "C 5 min at -60 "C

15 min at -30 "C HEPES, pH 7.5

HEPES, pH 5.5

Fluorocitrate Desalted

mm/s 0.81 1.23

1.21

1.35

1.43

1.67

1.68

1.53

1.51

1.51

1.35

mm/s 0.45 0.85

0.86

0.85

0.85

0.69

0.71

0.87

0.87

0.87

0.83

>92 40 S2:

18 Sp: 32 42 Sp:

48 58 Sp: 59 59 57 48 Sp:

25

91

56

66

Sa: S2:

10 s2:

mm/s mmls

1.80 0.90 40

1.83 0.91 17 37

1.83 0.91 28

36 1.86 0.91 23

19 24 27

1.76 0.89 32 66

-1.68 -0.88 74

1.64 0.88 95 -0.95 0.70 26

-0.95 0.70 20

1.79 0.89 70 1.91 0.90 67

1.79 0.89 80

%

>92 17

62 31 15 Additional species

at 13%, see text 15 18 23 17 16 14 8 20% in A E Q = 1.20

mm/s form 73 26 9 5

12

10

30 33

10

fast. For instance, 5 ms after mixing the enzyme with cis- aconitate, 85% of the [4Fe-4SI2' clusters have changed their spectroscopic state (see Fig. 2B); 42% of the 57Fe is in SI, 28% in S p , and about 13% of the material contributes to a quad- rupole doublet with a high-energy line at +1.2 mm/s; the associated low-energy line of the doublet is at 0.2 f 0.1 mm/ s. This yields a species with A E Q = 1 mm/s and 0.65 < 6 < 0.75 mm/s. During the subsequent 30 ms, SI and S p increase essentially by depletion of the A E Q = 1 mm/s species. The spectrum of Fig. 2C (cis-aconitate, quenched after 35 ms) corresponds closely to those observed when the samples are allowed to equilibrate at 0 "C before freezing.

Fig. 2E shows a spectrum obtained by quenching a sample 35 ms after addition of isocitrate. The 5-ms spectrum was very similar except that S p was somewhat less intense (see Table I). Thus, isocitrate binds rapidly, with SI being the dominant species. Equilibrium has not been reached within 35 ms.

For the rapid-quench samples, the Mossbauer parameters of S2 are the same within the uncertainties, independent of the substrate used. On the other hand, there is some variabil- ity in AEQ of species S1 which is outside the experimental uncertainties.

Studies in Water/Methanol/Ethykne Glycol-In order to attempt a further resolution of the aconitase reactions, we have performed cryobiochemical studies by preparing the enzyme, with 57Fe in Fe,, in a hydro-organic mixture of 50% water, 30% methanol (to reduce viscosity), and 20% ethylene

glycol. Since nothing was known about the subzero kinetics, we have allowed the enzyme thus prepared to react at -60 "C (see "Materials and Methods") with the three substrates for a few minutes before freezing the samples in liquid Nz.

A 4.2 K Mossbauer spectrum of a sample prepared at -60 "C with citrate (Table I, line 11) is shown in Fig. 3A. After completion of the Mossbauer studies, the sample was allowed to warm up to -30 "C, incubated for 5 h, and then refrozen. The resulting Mossbauer spectrum is shown in Fig. 3C. (For clarity, we have subtracted 8% of the Fe.O doublet.) The spectrum of Fig. 3C contains two doublets. The dominant one (66% of all a sites in the sample) has A E Q = 1.76 mm/s and 6 = 0.89 mm/s; this is essentially Sa. Note that SI is absent. The second doublet (indicated by the solid l ine above the data and accounting for 20% of all a sites) has A E Q = 1.20 mm/s and 6 = 0.48 mm/s. Inspection of the rightmost absorption feature of Fig. 3A shows that two species contribute. By subtracting 40% of the spectrum of Fig. 3C from that of Fig. 3A, we have obtained the spectrum of Fig. 3B. The solid line is a least-squares fit to this difference spectrum, yielding A E Q

= 1.67 mm/s and 6 = 0.69 mm/s. This doublet, accounting for 48% of all Fe. of the spectrum of Fig. 3A, represents a new species which we label S3. Thus far, SI has only been observed when samples are prepared or frozen at subzero temperatures.

Fig. 4A shows a spectrum obtained after cis-aconitate was added to the enzyme, and the solution was allowed to incubate for 18 min at -60 "C (virtually the same spectrum was ob- served after a 5-min incubation time). The spectrum is a

Mossbauer Studies of Aconitase 6876

0.1

1.1 - be u

z 0

a. t-

0 m

U

0.1

m a 1.1

I , , , , ,

I , , , , ,

-4 -2 0 2 4 VELOCITY (MM/S I

FIG. 4. 4.2 K Mossbauer spectra of active aconitase (1.1 mM) in water/methanol/ethylene glycol after addition of sub- strates. 2 mM cis-aconitate A and 2 mM isocitrate B were added at -60 "C and allowed to react with the enzyme for 18 and 5 min, respectively. The solid lines in A and B are least-squares fits, assuming two ( A ) or one ( B ) symmetric quadrupole doublets.

superposition of unreacted material (73%) and species S3. Further incubation for 15 min at -30 "C yielded a spectrum containing species Sz at approximately 75% of total Fee, with the remainder of the absorption belonging to the Fe," doublet.

Fig. 4B shows a spectrum obtained with a sample incubated with isocitrate (Table I, line 15) at -60 "C for 5 min. (For clarity, we have subtracted 9% of the Fe," doublet). The doublet of Fig. 4B has A E Q = 1.53 mm/s and 6 = 0.87 mm/s. A 15-min incubation of the sample at -30 "C produced a spectrum containing essentially 95% of a doublet with A E Q = 1.64 mm/s and 6 = 0.88, i.e. a component with Sz character.

Since the spectra shown in Figs. 3 and 4 were obtained with enzyme prepared in hydro-organic mixtures, the question arises whether some of the new spectral components reflect the properties of the solvent rather than a new species trapped at subzero temperature. As is apparent from the spectrum of Fig. 4A, the parameters of the Fe." doublet are identical in aqueous and hydro-organic solvents. This is true for all cry- obiochemical preparations studied. We have also prepared a sample in water/ethylene glycol (50:50 v/v, freezing point -44 "C) in the presence of excess amounts of substrate. The material was equilibrated at 20 "C and then rapidly frozen by spraying the solution onto a metal surface kept at 77 K. The resulting Mossbauer spectrum exhibited the Fe," species (70%) and SI and Sz, the latter two in roughly equal concen- trations. Thus, the spectral parameters of the observed species are not affected by the presence of ethylene glycol. The equilibrium concentrations of Fee-bound species are, however, different. On the other hand, when an identical sample was (slowly) frozen by plunging the Mossbauer sample cuvette into liquid nitrogen, we obtained a spectrum exhibiting the Fea0 doublet and doublet S3 in approximately equal amounts. Thus, slow freezing produces a species, S3, which is also

observed in significant concentrations when citrate is added as -60 "C (see above). (In an aqueous medium the equilibrium concentration at 25 "C is 88% citrate, 4% cis-aconitate, and 8% isocitrate (8)).

Finally, we have rapidly frozen two of the cryo-samples by equilibrating at about -25 "C and then spraying the material onto a cold metal surface. In one case, using cis-aconitate as substrate, we observed a spectrum identical to that obtained after slowly freezing in liquid nitrogen. The citrate sample of line 12 of Table I, however, showed pronounced changes. First, the intensity of doublet Sz decreased from 71% of total Fe. to about 45% and, second, the A E Q = 1.20 mm/s doublet had almost disappeared in favor of the Fe." species which now accounted for about 40% of Fe,. (Rapid quenching by spraying onto a cold metal surface results in a substantial loss of material, yielding spectra with poor signal-to-noise which are difficult to quantitate.) It is obvious that the results obtained to date, although acquired with considerable expense in effort and material, can only be considered as exploratory. Thus, our limited experience does not allow us to assess the effects of the freezing rate in detail. It appears, however, that the freezing rate is an important parameter, the effect of which needs to be explored and eventually controlled.

Studies with Methyl-substituted Substrate-Aconitase con- verts a-methyl-cis-aconitate (cis-2-butene-1,2,3-tricarboxy- late) into a-methylisocitrate. Interestingly, the enzyme does not catalyze the conversion of the substrate into a-methyl- citrate. Schloss et al. (16) have studied the pH profiles and isotope effects for the methyl substrates and have proposed a catalytic mechanism (see also Ref. 11) which involves the formation of a carbanion intermediate and product release by substrate displacement.

The Mossbauer spectrum of active aconitase, buffered in HEPES at pH 7.5 and incubated with a-methylisocitrate, exhibits, besides component Fe.", two doublets with A E Q = 1.51 mm/s and 6 = 0.87 mm/s and A E Q = 0.95 mm/s and 6 = 0.70 mm/s (see Table I). Neither of these doublets matches those observed when the natural substrates were added to the enzyme in aqueous solution. The A E Q = 1.51 mm/s component matches, however, the species observed when isocitrate is added at -60 "C to the enzyme (see line 15 of Table I).

The equilibrium constant, [a-methyl-cis-aconitate]/[a- methylisocitrate], changes with a pK = 6.2 from a value of 1.5 at low pH to a value of 0.75 at high pH (16). At pH 5.5, the Mossbauer spectra show the same species as observed at pH 7.5, except that the concentration of the A E Q = 1.51 mm/ s species has increased relative to the A E Q = 0.95 mm/s species (Table I).

Studies with Inhibitors-Schloss and co-workers (11) have studied the binding of the nitro analogs of citrate and isocit- rate (see "Materials and Methods") to aconitase. (We will refer to these compounds as "nitrocitrate" and "nitroiso- citrate."). The Ki values of the citrate and isocitrate analogs are 59 and 72 nM, respectively, when these competitive inhib- itors are fully ionized; the Ki values increase to 1.7 mM and 58 p ~ , upon protonation of the carbon acid (11). The fully ionized nitro analogs have a trigonal carbon adjacent to the carbon with the hydroxyl group. This structural feature to- gether with extremely low Ki values suggest that these com- ponents can be considered analogs of carbanions formed from citrate or isocitrate during the catalytic reaction (11). Fig. 2F shows a Mossbauer spectrum of active aconitase, 67Fe in Fe, of the [4Fe-4SI2+ cluster, to which 5 mM nitrocitrate was added. 70% of the total absorption belongs to a doublet with UQ = 1.79 mm/s and 6 = 0.89, parameters identical to those of species Sz (line 2 in Table I). The remainder of the spectrum

Mossbauer Studies of Aconitase 6877

is the doublet of Fig. 2 A , i.e. Fe,". We do not know the shape of the spectra of the b sites (7% of total 57Fe) in the present case. We suspect that they are unresolved from Fe,". Remark- ably, despite the low Ki, a substantial fraction ( ~ 2 3 % ) of the Fe, sites remains uncoordinated. A spectrum very similar to that shown in Fig. 2F was observed with nitroisocitrate; again, a large portion of Fe. seemed uncoordinated.

Among the four stereoisomers of 2-fluorocitrate, only (-)er- ythro-2-fluorocitrate shows strong inhibition (17). The inter- action of this compound with aconitase is rather complex. The inhibitory isomer is initially a competitive inhibitor, but then a time-dependent inactivation of the enzyme sets in (18). Furthermore, the enzyme removes fluoride ion with a stoichi- ometry of 1 F-/enzyme molecule (19)'; thus, fluorocitrate is a substrate as well which is transformed into a tight-binding inhibitor. Addition of a 10-fold excess of fluorocitrate to active aconitase and incubating for 5 min before freezing produced a Mossbauer spectrum exhibiting components SZ, an SI type species (aE, - 1.35 mm/s and 6 = 0.83 mm/s), and Fe,", accounting for approximately 60, 25, and 15% of total 67Fe, respectively. Desalting of the sample on a Sephadex column removed most of the S1 type doublet and also decreased the amount of Fe."; the resultant species and their concentrations are listed in Table I. After the Mossbauer spectrum was recorded the sample was thawed. Addition of 10 mM citrate and 75 min of incubation at 25 "C produced no further change in the Mossbauer spectrum, showing that the dissociation of the defluorinated inhibitor is exceedingly slow.

In summary, the Mossbauer spectra of the transition state analogs nitrocitrate and nitroisocitrate exhibit doublet Sz. The same species is also observed with fluorocitrate. Neither of the nitro analogs produced component SI.

Studies of Reduced Active Aconitase Complexed with Sub- strute-Upon reduction, the [4Fe-4SI2+ cluster of active acon- itase attains the EPR-active S = 1h state which yields the characteristic g = 1.94 type EPR signal (1, 3). Since the reduced cluster has half-integral electronic spin and is there- fore in a Kramers state, the low-temperature Mossbauer spectra will exhibit paramagnetic hyperfine structure. Such spectra are rich in information, and we have therefore studied some of the reduced states of aconitase, with and without substrates present. Thus far we have not succeeded in pro- ducing a reduced aconitase with the desired spectral purity. The reduction process, using a 10-fold excess of dithionite, yielded either only partially reduced material ( ~ 4 0 % ) or, upon longer exposure (30 min) of the sample to dithionite, a sub- stantial amount of clusters were destroyed. Increasing the pH to 8.5 by desalting into Tris-HC1 and using the redox mediator methyl viologen yielded virtually fully reduced material. How- ever, both Mossbauer and EPR studies showed that the pres- ence of Tris produced an additional g = 1.94 type species. On the other hand, when aconitase is reduced in the presence of substrate, only one species, with g values at 2.04, 1.85, and 1.78 (3), is produced in fairly good spectral purity. In the following, we report Mossbauer spectra of the reduced en- zyme-substrate complex.

We will present two data sets for the reduced enzyme- substrate complex. One set was obtained for samples which had the a site enriched with 57Fe to more tnan 90%. A complementary set was obtained on material which was highly enriched with 57Fe in the b sites, the a site containing =Fe (>99%). For all Fe, spectra, we have removed the contribu- tions (-7%) of the b sites.

At temperatures above 40 K, the electronic spin of the reduced cluster, [4Fe-4SI1+, relaxes fast, and the Mossbauer spectra consist of quadrupole doublets. Two representative

spectra are shown in Fig. 5. The spectrum in Fig. 5A was obtained at 100 K on material enriched with S7Fe in Fe., whereas that of Fig. 5B was recorded at 60 K with a sample enriched in F6. The majority component (solid line) in Fig. 5A, accounting for 80% of the absorption, is a doublet with a E Q = 2.24 mm/s and 6 = 0.99 mm/s. Previously (6), we have labeled this component SR (we relabel it now Sa), and we have emphasized that it is high-spin ferrous in character (but a structural part of the [4Fe-4S] cluster). The remainder of the spectrum consists of 10% Fe," and some contribution of reduced uncomplexed cluster. The solid line in Fig. 5B is the result of simulating two quadrupole doublets and adding them in the ratio (Sbz + sb3)/&(1) = 2:l. (The reader may keep in mind that 57Fe enrichment was achieved by iron exchange rather than reconstitution. Our result thus suggests homoge- neous labeling of the three b sites. We have independent proof of homogeneous labeling from the spectrum of Fig. 1. Also, in an experiment where 55Fe was present to measure the extent of incorporation of added iron, the results indicate essentially total exchange. Added iron constituted 95.8% of the total iron in the experiment while 92.8% of the iron in the reisolated enzyme, as determined by chemical analysis and measure- ments of radioactivity, originated from the added iron.)

We have studied both samples in the temperature range from 1.5 to 180 K. In Fig. 6, we have plotted uersus 6 for each of the four sites (we have corrected the isomer shifts to account for the small temperature dependence of the second order Doppler shift, -0.05 mm/s/100 K). It can be seen that

I I , , , , , , , ,

-4 -2 0 2 4 VELOC I TY [ MM/S I

FIG. 5. Mossbauer spectra of the [4Fe-4SI1+ cluster with bound substrate. A, spectrum recorded at 100 K with a sample (1.2 mM) enriched with 5'Fe in Fe.. Doublet Sa is indicated by the solid l ine. B, sample (0.3 mM) enriched with 57Fe in the three b sites. The spectrum was recorded at 60 K, and a 10% contribution of oxidized material was subtracted from the data. The resulting spectrum is shown in B. The solid l ine is a least-squares fit using two doublets constrained to have an intensity ratio of 2:1, i.e. assuming one pair (SbZ) and a single (Sbl) b site. See also Fig. 6. Before reduction 10 mM citrate (A) or 2 mM citrate ( B ) was added to the sample.

6878 Mossbauer Studies of Aconitase

r"---- 'bl

150

' 0 X 50

11 7 7

ill00

:l150

x195

u 0.5 1 .o

6 (mm/s) FIG. 6. Graph of AEQ versus 6 for the reduced [4Fe-4S]

cluster with bound substrate. All isomer shifts, 6, are referred to 4.2 K relative to Fez+ metal at 298 K. The temperatures in K are given by the numbers next to the data points. Within the uncertain- ties, AEQ is independent of temperatures for the two equivalent sites Sb2 and Sb3.

0.0

0.5

- as u

z 0

I" W

& 0.0 0 m r n a

0.5

I I I I I I I I

-4 -2 0 2 4 V E L O C I T Y (MM/S I

FIG. 7. 4.2 K Miissbauer spectra of the a site of the dithio- nite-reduced [4Fe-4S] cluster with bound substrate. The spec- tra were taken in a magnetic field of 0.06 tesla applied parallel (A) and perpendicular ( B ) to the observed y-radiation. Contributions of the b sites (7%) and of oxidized material (8%) were subtracted from the data. The solid lines are spectral simulations based on Equation 1 using the parameters quoted in Table 11. In searching for an optimal parameter set, we have ignored the range from 0 to +2 mm/s Doppler velocity; this range contains absorption from species other than the desired ones.

the sites are quite different; we will discuss this point under "Discussion and Conclusions".

Low temperature Mossbauer spectra of Fe. and Feb are shown in Figs. 7-9. We have analyzed the spectra of each site

0. I

0.:

- as v

0 z

I- [L r x 0 m

H

m a 0.4

0.;

I , , , , , , , , , ,

- 4 -2 0 2 4 V E L O C I T Y (MM/S I

FIG. 8. 4.2 K Miissbauer spectra of the b sites of the dithi- onite-reduced [4Fe-4S] cluster with bound substrate. The spec- tra were taken in a 0.06 tesla parallel (A) and perpendicular ( B ) field. We have subtracted from the raw data a 10% contribution of oxidized, i.e. [4Fe-4SI2+, material. The solid lines are theoretical spectra com- puted from Equation 1 with the parameter set of Table 11. Indicated above the data is the contribution of the b l site spectrum.

with the spin Hamiltonian (S = %)

H = p s.g.fi + 9.A.i - g,$, fi.i+ - (3 I,2 - 1(1 + 1)) (1) eQ V,, 12

where g is the electronic g tensor (known from EPR), A is the magnetic hyperfine tensor, and A E B = eQVJ2 is the quad- rupole splitting. (We have assumed that the electric field gradient tensor is axially symmetric around the z axis, i.e. V,, = Vw) The solid lines in Figs. 7-9 are theoretical spectra computed from the parameter set listed in Table 11. We have previously described in some detail (20) the methodology of analyzing spectra as shown in Figs. 7-9. Therefore, we confine our discussion here to only some general remarks. As pointed out above, reduction and binding of substrate is not complete and, in addition, the spectra of Fe, contain a contribution of the b sites. We have subtracted all these contributions ac- cording to our understanding of the total data set. Although we believe that these subtractions are reasonably good, and mostly confined to the center portion of the spectra in Figs. 7-9, they introduce some uncertainties which influence the analyses. From a series of spectral simulations we realized that, within the experimental uncertainties, the data could be fitted reasonably well by restricting the analyses to axial symmetry. Since a E Q is quite large for Fe., we have explored the possibility that the electric field gradient tensor is tilted relative to the A tensor, without any improvement of the fits.

Mossbauer Studies of Aconitase 6879

FIG. 9. 6.0 tesla spectra of the a site (A) and the b sites (B) of the reduced [4Fe-4S] cluster with bound substrates. Spectra were recorded at 4.2 K in parallel field. The solid lines are theoretical curves computed from Equation 1 with the parameter set of Table 11. The spectrum of the b l site is separately drawn above the data.

TABLE I1 Hyperfine parameters of the substrate complex of the [4Fe-4SI1+

cluster of reduced aconitase In the last two columns the average A values, A, = (A, + A, +

A,)/3 of the [4Fe-4SI1+ clusters of E. coli sulfite reductase (20), and the B. stearothermophilus ferredoxin (27) are listed for comparison.

ilus

MHz MHz mm/s mm/s MHz MHz MHz Fe. +34 +26 +2.6 1.00 +31.4 Febl +12 +22 -2.6 0.64 +15.3 +17 +16 Febl = Febo -39 -32 +1.15 0.49 -36.7 -33 -31

Furthermore, since the g values are fairly isotropic, the Moss- bauer spectra are-practically insensitive to the relative orien- tations of g and A; we therefore have evaluated the data with g = 2. Since we assumed the field gradients to be axial, the quantity eQVz. is determined, at 4.2 K, from an extrapolation sf the quadrupole splittings to low temperature. The sign of A was determined, as discussed previously (ZO), by comparing the 0.06-tesla spectra with the 6.0-tesla data. Overall, the theoretical spectra agree quite well with data. Some improve- ment can certainly be made. For instance, the rightmost feature of Febl, in Fig. 9B, is too sharp; this line can easily be broadened by allowing some deviation from axial symmetry. Similarly, by allowing the spectra of Febl to be different from those of Febz, a better match with the data can be achieved.

DISCUSSION AND CONCLUSIONS

The results described above further illustrate the potential of Mossbauer spectroscopy in the elucidation of the structure and function of the active site of aconitase. Our results address

quite different aspects of these, and we will therefore partition this section accordingly.

Enzymatic Activity of Reduced [3Fe-4S] Cluster-On the basis of magnetic circular dichroism experiments and subse- quent activity measurements on diluted samples, Johnson and co-workers (9) and again Ramsay and Singer (21) have argued that, contrary to our experience, the reduced [3Fe-4S] cluster may have significant catalytic activity. The work described here suggests that this is not the case. First, the spectra (Fig. 1) of the reduced 3Fe cluster in the presence and absence of substrates are identical, indicating that the sub- strates do not coordinate to any of the iron sites of the reduced [3Fe-4S] cluster. This is to be contrasted with the pronounced changes which substrates and inhibitors induce at Fe, of the [4Fe-4S] cluster. Binding of substrates and inhibitors to the [4Fe-4S] cluster, in both the 2+ and 1+ oxidation states, is clearly evident from the data shown in Figs. 2-5. Second, by using EDTA, which does not bind to either active or inactive aconitase (5), we remove contaminating iron which presum- ably is generated by destruction of clusters in the reduction process. Thus, the presence of EDTA should prevent the formation of [4Fe-4S] clusters for both the purposes of spec- troscopy and the activity assays. The activity of our sample (as used for Fig. lA) was less than 5% of that obtainable for active aconitase, and spectroscopically, the sample did not contain any discernible amount (<8%) of [4Fe-4SI2+ clusters. We thus consider it unlikely that aconitase containing re- duced [3Fe-4S] clusters has significant activity and conclude that whatever activity is observed is due to spontaneous activation of the enzyme in the sample by cluster intercon- versions during manipulations or delay preceding the assay. Analogous experiments by magnetic circular dichroism spec- troscopy support these conclusion^.^

Binding of Substrates and Inhibitors-Our studies show that a variety of substrates and inhibitors bind to Fe. of the [4Fe-4SI2+ cluster of active aconitase. Before we discuss our results, a few comments might be in order. The isomer shifts of the four iron sites of [4Fe-4SI2+ cubanes are centered around 6 = 0.45 mm/s, within k0.03 mm/s. Replacement of the thiolates in Fe4S4(RS), synthetic complexes by phenols, i.e. replacement of four sulfur ligands by four phenolic oxy- gens, increases 6 only marginally from 0.45 mm/s to 0.49 mm/ s. Thus, the large isomer shifts of SI (6 = 0.83 mm/s) and S2 (6 = 0.90 mm/s) indicate that major structural rearrangements have taken place at the labile iron, Fe,. We have previously (6) suggested that these large shifts result from an expansion of the coordination sphere of Fe. from tetrahedral to a five- or, perhaps, six-coordinate ligand structure. This suggestion is supported by recent studies of synthetic complexes with [4Fe-4SI2+ cores (22, 23). A second comment regards the question of what structural features yield different values for A E Q for SI and SZ. Presently, this question cannot be answered since suitable model complexes do not yet exist. Unfortu- nately, theoretical calculations of A E Q values are unreliable even in cases where the coordination geometry is known.

When the natural substrates of aconitase are added to active enzyme and the samples are frozen, species SI and S, are observed. Inspection of Table I shows that S, type species are observed under a variety of conditions. Although the A E Q

values vary somewhat beyond the experimental uncertainties, we will take the view that all species with 6 2 0.90 mm/s and 1.75 mm/s 5 A E Q 5 1.91 mm/s are Sz. Interestingly, the nitro analogs of citrate and isocitrate exhibit doublet S2, but not

'Stephens, P. J., Morgan, T. V., Burgess, B. K., Stout, C. D., Kennedy, M. C., Emptage, M., and Beinert, H., submitted for publi- catiah.

,

6880 Mossbauer Studies of Aconitase

SI; according to Schloss et al. (11) these tight-binding inhibi- tors are analogs of carbanion transition state complexes of aconitase. This, then, suggests that it is reasonable to asso- ciate SZ with a carbanion transition state complex. We further suggest that the substrates and inhibitors are coordinated to Fe. with the hydroxyl group. This is supported by the obser- vation of EPR line broadening when 170H-labeled substrates are used (6). Furthermore, we have recently observed' that 170H-labeled nitroisocitrate broadens the EPR signals of the [4Fe-4SI1+ cluster as well. Further support for OH coordina- tion comes from the observation that the competitive inhibi- tor tricarballylate (which lacks a hydroxyl group) does not coordinate to Fe. at all.

The results obtained from the studies with 2-fluorocitrate are interesting because they show that the product of 2- fluorocitrate (after defluorination) binds to the active site in the same fashion as the nitro analogs and is not easily displaced. A good candidate for the product would be 4- hydroxy-trans-aconitate. This molecule is similar in structure to the carbanion of nitroisocitrate (see Ref. 11). The identi- fication of the product and its inhibitory properties are pres- ently being pursued.

Early Euents in Catalysis-Our rapid mix/rapid quench experiments are not yet complete. We have, however, made some interesting observations. Our studies show that one can resolve certain phases of the reactions. Some species are already present within 5 ms after mixing whereas others appear at a slower rate, and 35 ms after mixing, equilibrium is not attained with any of the substrates. Species Sz is observed with essentially the same spectral parameters inde- pendent of the substrate used. SI, on the other hand, appears in different spectral forms, indicating different coordination to Fe. reflecting either different conformations of substrate- derived reaction intermediates or different conformations of the protein.

Since virtually nothing was known about the reactions of aconitase at subzero temperatures, we have selected some temperatures and reaction times and have studied the samples with Mossbauer spectroscopy. Our results suggest that a cry- obiochemical approach is potentially rich in new information. For instance, at -60 "C a new species, SI, is observed when either citrate or cis-aconitate is used as substrate. This species is characterized by an isomer shift, 6 z 0.70 mm/s, which is just between those of Fe.' and S1 and Sz, suggesting that Ss represents a coordination environment of Fe. quite different in geometry, and perhaps in coordination number, from those of S1 and Sz. Interestingly, S, was not observed in any of the subzero temperature experiments. Furthermore, the quadru- pole splitting of Sz seems to depend on the substrate used. Since, however, the citrate sample, after incubating for 5 h at -30 "C, exhibits an Sz species quite similar to those observed in aqueous solution samples, we suggest that the cis-aconitate and isocitrate samples, lines 14 and 16 of Table I, have not attained equilibrium. It is also noteworthy that isocitrate, after 5 min of incubation at -60 "C, has almost completely reacted with enzyme to yield a species very similar to the majority of species observed in the aqueous solution sample to which a-methylisocitrate was added. We are confident that further Mossbauer spectroscopy of aconitase prepared and exposed to substrates at subzero temperatures may expand the range of mechanistic studies of this interesting enzyme. We have recently observed that substrate and inhibitor bind- ing can be monitored by the circular dichroism spectra of the iron-sulfur chromophore. Although this technique has inferior

M. C. Kennedy, M. Emptage, H. Merkle, and H. Beinert, unpub- lished data.

resolution as compared to Mossbauer spectroscopy, we should be able to use it as a monitoring device to explore subzero temperature kinetics. This should allow us to select the tem- peratures and reaction times more judiciously for the prepa- ration of the Mossbauer samples. By combining cryospectros- copy with cryo-kinetic studies, it should be possible to asso- ciate Mossbauer spectral components with intermediates of the enzymatic reactions.

Mossbauer Spectra of [4Fe-4S11+ Cluster with Bound Sub- strates-Fig. 6 shows that the quadrupole splittings and iso- mer shifts of three of the four iron sites are distinct. For the [4Fe-4SI1+ clusters reported thus far, we can distinguish two situations. The quadrupole splittings and isomer shifts of all four sites are either the same (although the lines are often heterogeneously broadened) or one observes two quadrupole doublets of equal intensity, i.e. the sites occur in equiva- lent pairs. The former case occurs, for instance, for the 2[4Fe-4SI1+ ferredoxin from Clostridium pasteurianum (24, 25) and the Fe protein of the nitrogenase system of Azotobac- ter uinelandii6 and synthetic complexes (26), whereas the latter situation is observed in the ferredoxin from Bacillus stearotherrnophilus (27) and in Escherichia coli sulfite reduc- tase (20). Furthermore, Mossbauer studies in strong applied fields (20, 27) have shown that the A tensors are divided into two sets. One pair of iron sites has a fairly isotropic A tensor with negative components whereas the other pair has an anisotropic A tensor with positive components. Although [4Fe-4SI1+ clusters show a high degree of electron delocaliza- tion, we may classify the two types of sites as ferric type ( A < 0. smaller AEa and 6) and ferrous type (A > 0, larger A E Q and 6). In the complex studied here, the two ferrous type sites, s. and Sbl, are clearly distinct. The observed inequivalence is, of course, attributable to the fact that the substrate binds to Fe. and changes the coordination geometry of this site. We have, however, some evidence that Fe. and F%, are intrinsi- cally different for the aconitase cluster. In the absence of substrate, the a site of the reduced [4Fe-4S] cluster has h E Q

and 6 values as indicated in Fig. 6, and preliminary evidence suggests that the b sites have the same quadrupole pattern with and without ~ubstrate .~ One further point is noteworthy. The Febl site has A E Q and 6 values quite similar to those observed for the Fez+ component of the P clusters of the MoFe protein of nitrogenase (see Fig. 4, doublet 11, of Zimmermann et al. (28)). In Table 11, we listed for comparison the average A values, A,, = (A, + A, + A,)/3, for the clusters of aconitase, B. stearothermophilus ferredoxin (27), and E. coli sulfite re- ductase (20). It can be seen that the b site A values are in the range observed for other [4Fe-4SI1+ clusters. In contrast, the A values of the a site are larger, by a factor 2, than the corresponding A values of the normal clusters. This increase in A reflects on one hand the increased ferrous character of Fen, towards a localized, more ionic site. On the other hand, some of the increase in A may be attributable to changes in

unpublished data. 6P. Lindahl, E. P. Day, W. H. Orme-Johnson, and E. Munck,

We have studied a sample of reduced aconitase, [4Fe-4SI1+, en-

pattern almost identical to that of the spectrum of Fig. 5B, i.e. reduced riched with "Fe in the b sites. We have observed at 60 K a quadrupole

aconitase has spectral component Sbl. We learned later, however, that two species are observed by EPR when Tris.HC1 is used as buffer. The Mossbauer spectrum also contained a line at about +1.5 mm/s, belonging to an unresolved doublet (<20% of the total 57Fe). This doublet belongs presumably to the second spectral species seen in EPR. However, until we have studied a sample yielding only one EPR active species, our conclusions are tentative. These comments do not apply to the spectrum of the a site of reduced aconitase. Only one species with parameters as shown in Fig. 6 was observed in two different preparations.

Mossbauer Studies of Aconitase 6881

the details of spin-coupling, i.e. the projections of the local spin of Fen onto the system spin S may have changed. Some changes in the spin-coupling scheme are evident in the g values, g = (2.04, 1.85, 1.78), which are more anisotropic than those observed for other [4Fe-4SI1+ clusters. By using the spin-coupling model developed for [2Fe-2S] clusters (29) as a guide, we suspect that the increased anisotropy of g reflects essentially the electronic structure of Fen. This is supported by the observation of a strong temperature-dependent h E Q ,

indicative of low-lying excited orbital states for Fe.. Such low- lying states can be mixed effectively with the ground state by spin-orbit coupling, thus partly unquenching orbital angular momentum and therefore contributing to the increased ani- sotropy of the system g tensor.

Acknowledgments-We thank Drs. J. V. Schloss and W. W. Cleland for helpful discussions concerning the inhibitor studies, Dr. W. H. Orme-Johnson for suggestions to improve the rapid freeze techniques, and E. Ruzicka for assistance in preparing aconitase.

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