Proc. Natl. Acad. Sci. USAVol. 80, pp. 4674-4678, August 1983Biochemistry

Mossbauer and EPR studies of activated aconitase: Development ofa localized valence state at a subsite of the [4Fe-4S] cluster onbinding of citrate

(isomer shift/H2170 effect/transferred hyperfine interactions/aconitase mechanism)

MARK H. EMPTAGE*, THOMAS A. KENTt, MARY CLAIRE KENNEDY*t, HELMUT BEINERT*, ANDECKARD MUNCKt*Institute for Enzyme Research and the Department of Biochemistry College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706;tGray Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392; and tDepartment of Chemistry, Villa Maria College,Erie, Pennsylvania 16505

Contributed by Helmut Beinert, April 29, 1983

ABSTRACT During activation of aconitase a ferrous ion is in-corporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or '"Fe for activation we have studied withMossbauer spectroscopy the beef heart enzyme in the presenceof citrate. Our studies show that the environment of one iron site(Fea) of the [4Fe-4S] cluster is drastically altered in the presenceof citrate. Fea is the iron acquired during activation of aconitase.In the oxidized [4Fe-4S]2+ state two species with enzyme-boundsubstrate are observed, whereas only one is observed in the re-duced [4Fe-4S]+ state. The Mossbauer parameters of Fea revealthat the site has acquired substantial high-spin ferrous character.This is most pronounced in the 1+ state where at Fea the clusterexhibits a localized valence state. The dramatic increase of theisomer shift upon substrate binding strongly suggests that the li-gand environment of Fea has become at least five-coordinate andthat the cluster may function as a Lewis acid. In the absence ofcitrate the EPR spectra of the active [4Fe-4S]+ enzyme (g1,2,3 =2.06, 1.93, 1.86) show no hyperfine broadening in the presenceof H2'70. However, in the presence of citrate (g1,2,3 = 2.04, 1.85,1.78) sizable transferred hyperfine interactions are observed; un-der the experimental conditions the hydroxyl groups of citrate andisocitrate as well as water are labeled with 170. We did not detectbroadening by '70-labeled carboxyl groups of citrate in H2160.Implications for the mechanism of aconitase are discussed.

Aconitase catalyzes the interconversion of citrate, cis-aconitate,and isocitrate in a dehydration-hydration reaction. The enzymehas been known for many years to require iron for activity orto contain iron (1). A decade ago Kennedy and co-workers (2)showed that it also contained labile sulfide but the potentialimplications of this discovery remained unknown and unex-plored. Ruzicka and Beinert (3) then established a relationshipbetween the oxidation state of the iron-sulfur (Fe-S) cluster andenzymatic activity, which seemed to suggest a regulatory func-tion for the cluster in this enzyme. However, there remaineduncertainties about the nature of the Fe-S cluster of aconitase(3-5). The almost isotropic EPR signal of the enzyme (g = 2.01;S = 1/2) as obtained on purification was first interpreted (6)as that of a high potential type [4Fe-4S] ferredoxin (Fd) but wasthen shown by M6ssbauer spectroscopy to originate from a 3Fecluster (7). It was also shown by Mossbauer spectroscopy thaton activation of aconitase with iron and reductant this 3Fe clus-ter is converted into a [4Fe-4S] cluster in its (diamagnetic) 2+oxidation state (8). Similar cluster interconversions have beenobserved for the Fds of Desulfovibrio gigas (9). The Fe sites of

the [4Fe-4S] clusters in these Fds as well as in aconitase arerepresented in the Mossbauer spectra by two distinct quad-rupole doublets a and b (8, 9), one (a, in aconitase: quadrupolesplitting, AEQ = 0.83 mm/s; isomer shift, 6 = 0.46 mm/s)originating from a single Fe site and the other (b, AEQ = 1.30mm/s; 6 = 0.46 mm/s) from the remaining three.

M6ssbauer spectroscopy requires the isotope 57Fe, which oc-curs at only 2.2% natural abundance. By using 57Fe enrichedto >90% for the activation of aconitase we can follow specifi-cally the newly incorporated Fe. By using the non-M6ssbauerisotope 56Fe for activation we can study the spectra of the otherthree sites, albeit with a much poorer signal-to-noise ratio. Bythis approach we have shown that doublet a represents the Feacquired during activation (8). This Fe, designated here as Fea,is particularly labile and is lost, concomitant with enzyme ac-tivity, on oxidation by, e.g., oxygen or ferricyanide (10). Chem-ical analyses for Fe and labile S2- on the 3Fe and 4Fe enzymestogether with results of extended x-ray absorption fine struc-ture spectroscopy have provided strong evidence that the clus-ter remaining after loss of the labile Fe does not have the [3Fe-3S] structure, as originally assumed (7, 8), but retains its S2-in a [3Fe-4S] structure (11).

The diamagnetic [4Fe-4S]2+ cluster of active aconitase canbe reduced by one electron to the [4Fe-4S]+ state, which showsthe typical g 1.94 (S = 1/2) EPR signal (8, 12). In this statethe enzyme is less active (c30%). Although the addition of ci-trate to the inactive [3Fe-4S]+ enzyme has only a minor effecton the EPR signal of its cluster (g = 2.01)-not more than thatof a number of unrelated anions-there is a dramatic and spe-cific effect of substrate on the EPR signal of the reduced stateof the active enzyme; the g values (12) shift from 2.06, 1.93,and 1.86 to 2.04, 1.85, and 1.78.

Here we have studied the effect of substrate on the [4Fe-4S]cluster of activated aconitase in its diamagnetic (2+) state byM6ssbauer spectroscopy and in its reduced (1+) paramagneticstate by EPR and Mossbauer spectroscopy. The Mossbauer datashow that the inequivalence of cluster sites is greatly enhancedby the presence of substrate, with the single Fea site acquiringdistinct ferrous character as compared to the remaining threeFeb sites, where little change is observed. The EPR spectra ofaconitase in the presence of [3-hydroxyl-'70]citrate, and H217oshow pronounced line broadening because of transferred hy-perfine interactions. This implies direct binding of citrate orwater to the Fe-S cluster.

Inasmuch as we see in these observations potential evidencefor involvement of the Fe-S cluster in the reaction catalyzed byaconitase, we report here the relevant experiments and discusstheir implications.

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MATERIALS AND METHODSAconitase was isolated in its inactive form from beef heart mi-tochondria and activated anaerobically with iron and dithio-threitol as in ref. 10. Enzyme assays, sample preparation, andEPR spectroscopy followed procedures and yielded results asin refs. 10 and 12. Reduction of samples was accomplished eitherby light in the presence of deazaflavin (EPR samples) (12, 13)or by excess dithionite in the presence of methylviologen(Mbssbauer samples).

H2170 was purchased from MSD Isotopes (St. Louis, MO).The 170 enrichment was determined from isotope-ratio massspectrometry analysis of CO2 after equilibration with a H217osample (14). [3-hydroxyl-170]Citrate was produced in situ byincubating an activated aconitase sample with cis-aconitate in38% enriched H217O (15). Citrate containing 170-labeled car-boxylic acid groups was prepared by incubating citric acid in46% enriched H217O at pH -1 and 70'C for 11 days (16).Mossbauer and EPR spectroscopies were carried out as de-scribed (8, 9, 12). Isomer shifts, 8, are quoted relative to Femetal at room temperature. The quoted uncertainties are es-timates based on visual comparisons of simulations with thespectra.

RESULTSEPR Spectroscopy. The addition of citrate to reduced active

aconitase yields a species with g values at g, = 2.04, g2 = 1.85,and g3 = 1.78 (12). To test whether H20 or the 3-OH group ofcitrate is coordinated to the cluster we have prepared samplesin H2170 (38% enriched in 170).§ Fig. 1A shows that the low-field resonance at gi = 2.04 is broadened, by about 0.5 mT,when H2170 rather than H2160 is present. The broadening is

§ It must be considered here that in all instances when one of the threesubstrates is added to active aconitase, an equilibrium mixture is rap-idly established with 88% citrate, 8% isocitrate, and 4% cis-aconitatefree in solution (1) and with enzyme-bound substrates of unknown con-centrations. Because of this and without additional and more specificinformation, it is not vet possible to establish the effect on the clusterof each substrate separately. With this in mind we will often refer tocitrate with the implication that the same can apply to isocitrate. Fur-thermore, we use the term "substrate" when any of the three sub-strates, or unknown bound intermediates, may be involved.

FIG. 1. Superposition of the low-field resonances of the EPR spec-

tra (normalized to equal amounts ofunpaired spin) ofreduced activatedaconitase incubated with 2 mM cis-aconitate (A) or 10mM trans-aconi-tate (B). The narrow resonances were observed in H2160 and the broaderones in 38% enriched H2170. Each sample contained 13 mg of aconitaseper ml, 25 mM Hepes (pH 7.5), 16 mM potassium oxalate, 8 ,uM de-azaflavin, and substrate or analog as stated above. Samples were pho-toreduced for 20 min in vacuo and then were quickly frozen in liquidN2. Spectrometer conditions: microwave frequency, 9.24 GHz; micro-wave power, 1 mW; modulation amplitude, 0.4 mT; scanning rate, 16mT/min; time constant, 0.064 s; temperature, 12.5 K.

attributable to transferred hyperfine interactions, demonstrat-ing that '7O derived from H2"7O is coordinated to the iron-sul-fur cluster. The bound oxygen belongs either to a water ligandor to the 3-OH group of citrate. Broadening is also observedat g2 and g3. Citrate enriched to 41% in its carboxylic groupsexhibited no hyperfine broadening of the EPR signal in H2160.We have also examined the effects of two competitive in-

hibitors. Tricarballylate (1,2,3-propanetricarboxylic acid) at 50-fold excess over enzyme has no observable effect on the EPRsignal of reduced active aconitase (or on the Mbssbauer spec-trum of oxidized active aconitase) nor is there any broadeningin H2170. This demonstrates that occupation of the active sitealone does not promote water coordination to the cluster. Theaddition of the competitive inhibitor trans-aconitate to reducedaconitase produces a species with g values at 2.01, 1.87, and1.80. As seen in Fig. 1B, on substitution of H2160 by waterenriched in 170, a 0.3-mT broadening of the g1 = 2.01 line isobserved. Because trans-aconitate is not a substrate of aconi-tase and because the carboxylate oxygens do not exchange un-der the conditions employed, this broadening can only be causedby H2170 coordination to the cluster.

Mossbauer Spectroscopy. Fig. 2A shows a 4.2 K Mossbauerspectrum of 57Fe activated aconitase. It consists essentially of

VELOCITY (mm/s)

FIG. 2. M6ssbauer spectra of activated aconitase recorded at4.2 K. Spectra inA andB were recorded in zero field; in C a magneticfield of 60 mT was applied parallel to the observed y-radiation. (A) En-zyme (110 mg/ml) activated with 57Fe. The solid line traces the doubletof Fe.. (B) 57Fe-Activated enzyme-i.e., Fe. enriched-in the presenceof a 5-fold excess of citrate. The solid line was generated by superim-posing three quadrupole doublets with relative intensities 20%, 40%(for S1), and 40% (for S2). We used a width of 0.30 mm/s for each of thesix absorption lines. By allowing different widths for S1 and S2 the datacan also be represented by choosing 45% and 35% for the intensities ofS1 and S2, respectively. InA andB we have removed from the raw datathe small contribution (7%) of the Feb sites that have 57Fe in naturalabundance. (C) 56Fe-Activated enzyme (250 mg/ml) in the presence ofcitrate. The solid line is the sum of a quadrupole doublet (AEQ = 1.15mm/s; 8 = 0.47 mm/s and 0.38 mm/s linewidth) and the experimentalspectrum (data of figure 2A of ref. 8) of the oxidized 3Fe centers thatare not converted during activation.

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one quadrupole doublet with AEQ = (0.83 + 0.02) mm/s and8 = (0.46 ± 0.01) mm/s. About 10% of the total 57Fe belongsto a second species, of unknown nature, discernible as a shoul-der around +1.5 mm/s Doppler velocity. By comparing theobserved absorption with a known standard (sample FdR of ref.9) we found that the 'Fe concentration of the AEQ = 0.83 mm/s doublet corresponds to within 10% to the [4Fe-4SJ concen-tration as determined by EPR, suggesting that the5 Fe is in-corporated only into one subsite (site Fe,). This is also indicatedby the shape of the spectrum: as shown earlier (figure 2A of ref.8) the other three sites (Feb) have a larger splitting, AEQ = 1.30mm/s. There is no evidence for such a component in Fig. 2A.

Fig. 2B shows a 4.2 K spectrum obtained after a 5-fold excessof citrate had been added to the 'Fe-activated enzyme. It con-sists of three quadrupole doublets, two of which were not pres-ent before the addition of substrate. The new doublets, labeledSI and S2, have parameters$ AEQ(Si) = (1.26 + 0.04) mm/s and8(S1) = (0.84 ± 0.02) mm/s, and AEQ(S2) = (1.83 ± 0.04) mm/s and 8(S2) = (0.89 + 0.02) mm/s. AE0(S1) is independent oftemperature, whereas AEQ(S2) has a slight temperature de-pendence [AEQ(S2) = 1.75 mm/s at 150 K]. We will argue be-low that the dramatically increased isomer shifts reflect a changeof coordination upon binding of substrate. Fig. 2C shows aspectrum that gives information complementary to that of Fig.2B. It was obtained for aconitase activated withOFe-i.e., weobserve the spectra of the three Feb sites. The spectrum ex-hibits two components. A broad component stretching from -3mm/s to +3 mm/s represents 3Fe clusters not converted to4Fe clusters during the activation. (These clusters are not ob-served in the spectra of Figs. 2 A and B because the conversionyield was much higher and because the unconverted clustersare not enriched in 'Fe.) An EPR quantitation of the g = 2.01signal of the 3Fe cluster revealed that 25% of the clusters hadnot been converted. This corresponds well to the 3Fe clusterconcentration determined by Mossbauer spectroscopy. Super-imposed on the spectrum of the 3Fe cluster in Fig. 2C is adoublet representing the contribution of the three Feb sites.First, it can be seen that the three sites yield the same spectra.Second, the value for AEQ = 1.15 mm/s is just slightly lessthan the value observed for Feb of the uncomplexed enzyme,AEQ = 1.30 mm/s. Thus, the effect of substrate is felt essen-tially only at the Fea site.

The data of Fig. 2C lead to the conclusion that SI and S2 be-long to one subsite-i.e., substrate binding results in two spec-troscopically distinct species at that site. The intensities of Siand S2 are about the same. We have studied whether their rel-ative intensities can be changed by lowering the substrate con-centration. When the citrate concentration was only 30% of thecluster concentration, the relative intensities of SI and S2 werestill the same.

As judged by EPR and optical studies (12) the dissociationconstant of citrate is of the order 1 tkM.11 Thus, the sample of

The assignment of the four absorption lines to the two doublets is sug-gested by the spectra observed in the presence of 2-hydroxy-3-nitro-1,2-propanedicarboxylic acid. The 4.2 K spectrum with this compet-itive inhibitor is the same as that shown in Fig. 2B except that doubletS1 is absent.

1I This value is two orders of magnitude less than the Km of citrate (200fuM). When the measured values of Km and Vmx of all three substrates(1, 17) are considered in the Briggs-Haldane formulation of steady-statekinetics, it would appear that the values for Km and Kd of citrate shouldbe much closer than that observed. At this time the cause of this dis-crepancy between spectroscopic and enzymatic measured binding ofcitrate is not known. However, considering the complexity of havingthree substrates in equilibrium and unknown concentrations of boundstates, the differences observed may not be implausible.

-4 -2 0 2 4

VELOCITY (miM/s)

FIG. 3. M6ssbauer spectra of 57Fe-activatedreduced aconitase (100mg/ml) in the presence of citrate. (A) Spectrum recorded at 150 K. Thesolid line traces a contribution (30%) of adventitious Fe2". The arrows

mark a minority species (10%). The prominent doublet (SR) representsthe Fe. site of the [4Fe-4S] cluster. (B) Spectrum recorded at 4.2 K ina magnetic field of 60 mT applied parallel to the observed ytradiation.(C) Spectrum recorded at 4.2 K ofSR obtained by subtracting the Fe2+impurity and the minority species from the spectrum ofB. See text. (D)Difference spectrum obtained by subtracting the 4.2 K spectrum ob-

tained in transverse field from that shown in B. Contributions of quad-rupole doublets-i.e., the Fe2+ impurity and the minority species-arecanceled in this procedure. The solid lines in C andD are theoreticalspectra computed from Eq. 1 with the parameters quoted in the text.

Fig. 2B should have all clusters complexed with substrate. Yetabout 20% of the absorption in Fig. 2B belongs to a doublet likethat of uncomplexed enzyme. (These results were reproduciblefor three preparations, after longer incubation times, and on

addition of 2-fold more citrate.) Some clusters in our concen-

trated samples may be unable to bind substrate. Alternatively,the component resembling the uncomplexed enzyme may rep-resent a state in which citrate, cis-aconitate, or isocitrate is boundin such a way that the spectral properties of the cluster are littleaffected.To date the values of 8 reported for [4Fe-4S]2+ clusters have

all been smaller than 0.50 mm/s. Thus, the large shifts of SIand S2, which suggest sites with substantial high-spin ferrouscharacter, raise the question of whether these sites are still anintegral part of the cluster. We have studied the sample in ap-plied magnetic fields up to 6.0 T. The data showed that all threespectral components reside in environments with electronic spinS = 0. These observations strongly suggest that SI and S2 resultfrom a subsite of an intact [4Fe-4S] cluster. In the following wewill further substantiate this assertion.

Reduction of the [4Fe-4S] cluster leads to the appearance ofan EPR signal of the g = 1.94 type (see above). Fig. 3A showsa 150 K Mossbauer spectrum of the reduced enzyme-substratecomplex. At 150 K the electron spin relaxation rate is fast andno magnetic hyperfine interactions are observed. Fig. 3B showsa spectrum of the same sample recorded at 4.2 K in a magneticfield of 60 mT. The spectra consist of two major and one minorcomponent. One, accounting for 30% of the total 57Fe, is in-dicated by the solid line in Fig. 3A. Its Mossbauer parameters

A .~!.\fE* ~~~~~1.0010

'.^ SR~~~ O.5-'I

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AEQ = 3.2 mm/s and 8 = 1.15 mm/s identify it as high-spinferrous material, almost certainly due to adventitiously boundiron originating from a decomposition of the [4Fe-4S] clusterinto a 3Fe fragment and ferrous ion during the lengthy reduc-tion procedure.** We have observed a similar phenomenon witha [4Fe-4S] ferredoxin from D. gigas (9). The spectrum of theFe2+ impurity is almost independent of temperature and isprominent in Fig. 3B. The data of Fig. 3 A and B contain alsoa minority species (10% of total 57Fe) with AEQ 0.8 mm/sand 0.45 mm/s (see arrows). An Fe3+ impurity, oxidized

uncomplexed enzyme, or enzyme-substrate may contribute toit.The remaining component in Fig. 3A, accounting for 60% of

57Fe, is a quadrupole doublet representing the reduced en-

zyme-substrate complex (SR). It is noteworthy that only one

doublet is observed in contrast to the two doublets Si and S2of the oxidized complex. AEQ of doublet SR has a pronouncedtemperature dependence; AEQ = 2.40 mm/s, 2.23 mm/s, 1.80mm/s, and 1.48 mm/s at 77 K, 100 K, 150 K, and 195 K, re-

spectively. Together with the value of 8 = 0.96 mm/s at 150K these parameters reveal a site that is distinctly high-spin fer-rous in character. tt At temperatures below 70 K the lines of SRbroaden because magnetic hyperfine interactions begin to ap-pear. At 4.2 K the species SR has features as shown in Fig. 3C.This spectrum was prepared by removing the SR doublet froma 77 K spectrum (data not shown) and then subtracting the re-

sulting spectrum from the spectrum of Fig. 3B. This procedurecauses the shape of the central region of the spectrum to besomewhat uncertain. Hence, we have displayed in Fig. 3D a

difference spectrum obtained by subtracting a spectrum re-

corded at 4.2 K in 60-mT transverse field from that of Fig. 3B.In such a difference spectrum the contributions of the impur-ities are canceled. An important feature of the spectrum of Fig.3D is the fact that the intensities of the absorption bands de-pend on the orientation of the applied field relative to the ybeam. This field dependence proves (19) that SR belongs to a

complex that must exhibit an EPR signal at 4.2 K. After com-

pletion of the Mbssbauer studies the material was transferredinto an EPR tube, while either being kept in liquid N2 or iso-pentane at 130 K. The EPR studies revealed only one species-namely, the g = 1.94 type signal of the enzyme-substratecomplex. This proves that SR results from a subsite of the[4Fe-4S]+ cluster.The solid lines in Fig. 3 C and D are theoretical spectra gen-

erated with a computer program from the S = 1/2 spin ham-iltonian

H= ,BSgH + S'A-

+ [3I2 15/4 + (I2 2]12zx Y [1]

**The sample of Fig. 3 is from an earlier batch than that of Figs. 2 Aand B. Prior to reduction it contained ca. 10% of adventitiously boundFe3+. Thus, about 20% of the [4Fe-4S] cluster appears to have frag-mented during the reduction. (The slow kinetics of reduction re-

quires incubation times of 2-3 hr.)t When the [4Fe-4S] cluster of aconitase is reduced in the absence ofcitrate an entirely different doublet, with AEQ 0.75 mm/s and

0.53 mm/s, is observed at 150 K. We have discussed elsewhere

that some proteins with [4Fe-4S] -i.e., reduced-clusters exhibittwo doublets, each representing two equivalent iron sites (18). Whenthe 3Fe cluster of D. gigas ferredoxin II is converted into a [4Fe-4S]cluster, the 57Fe is incorporated into the site that is more ferrous incharacter (9). In aconitase, on the other hand, the 57Fe resides in asite with parameters characteristic of the ferric-type site. We havepointed out earlier (9) that different sites are occupied as well whenthe clusters of the two proteins are in the 2+ state.

where all symbols have their conventional meanings. The g val-ues are known from EPR. Because the g values are quite isotro-pic and because the Mdssbauer spectra are averages over ran-domly oriented molecules the spectra contain little informationabout the spatial relation of the g tensor relative to the A tensorand the electric field gradient tensor. For the calculations weusedAx = Ay = 34 MHz and Az = 26MHz together with AEQ=+2.50 mm/s and 8 = 0.99 mm/s. Considering our limited datawe restricted these calculations to axial symmetry with Ax = Ayand q = 0. The presence of the impurities leaves some un-certainties regarding the true shape of the spectra. With thiscaveat the theoretical curves represent the data quite well.

DISCUSSIONActive aconitase contains a [4Fe-4S] cluster. We have studiedhere the cluster in the oxidized (2+) state with Mossbauer spec-troscopy and in the reduced (1+) state with both Mossbauerand EPR spectroscopies.

H2170 Broadens EPR Linewidths. The EPR spectrum of the[4Fe-4S]+ cluster changes drastically on addition of citrate. Inthe presence of substrate and H2170 the spectrum is noticeablybroadened because of transferred hyperfine interactions. Thebroadening is of similar magnitude, about half, as that reportedfor metmyoglobin (20). Because the 3-OH group of citrate islabeled with 170 when the enzyme-substrate mixture is frozenin the steady state (cf. 16), this broadening implies that the 170belongs either to bound H20 (or OH-) or to the 3-OH groupof citrate. No broadening is observed in the absence of citrateor when only the citrate carboxyl groups are labeled with 170.The latter observation suggests either that the carboxyl groupsof substrate are not bound to the cluster or that the bond is tooweak for the development of observable transferred hyperfineinteractions. On the other hand, the broadening observed withH2170 and trans-aconitate clearly suggests water coordinationto the cluster because this competitive inhibitor does not con-tain a functional group that can be labeled with 170 under theexperimental conditions. The trans-aconitate experiment thenraises the question as to whether the citrate is actually boundto the cluster or whether its. binding to aconitase might allowthe binding of water in an indirect way. In attempting to answerthis question we may consider the following arguments: the EPRspectra of the 1+ state depend quite sensitively on the sub-strate or substrate analog present. Further, in the catalyticallymost active 2+ state we have observed with M6ssbauer spec-troscopy two species, S1 and S2, which are only observed in thepresence of substrate. Two species, both similar but clearly dif-ferent from Si and S2, are also observed in the presence of trans-aconitate (M6ssbauer spectra are not shown here). It may bethat some of the species indicate water coordination to the clus-ter, with the substrate or analog conditioning such binding. Inour view, however, it is more plausible that some of the speciesrepresent substrate or analog directly bound to the cluster. Be-cause water is a reactant in the aconitase reaction the 170 thatis coordinated to the cluster in the presence of citrate most likelybelongs to the 3-OH group of citrate, either dissociated fromthe substrate or still attached to it. (We should point out thatthe doublets S1 and S2 are observed in the 2+ state, whereasEPR is performed in the 1+ state. Thus, the observation ofcoordinated 170 by EPR does not necessarily imply that the sameligand is bound in the catalytically more active 2+ state.)

Mossbauer Spectroscopy Shows Localized Valence. TheMossbauer spectra show that substrate is bound to one iron atom(Fea) of the [4Fe-4S] cluster. Fea is the iron acquired duringthe activation of the enzyme. The Mbssbauer data also showthat the electronic structures of the Feb iron atoms are little

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affected by binding of substrate. As witnessed by the appear-ance of the doublets SI and S2, two species with enzyme-boundsubstrate are present when the cluster is in the 2+ state. Incontrast, in the [4Fe-4S]+ state, only one species, SR, is ob-served. EPR also detects only one component assignable to acluster-substrate complex.To appreciate the dramatic effects observed upon substrate

binding it is useful to focus on the isomer shift data. The foursites of [4Fe-4S]2+ clusters have in general about the same shiftsand these lie around 8 = (0 44 + 0.03) mm/s, at 4.2 K, for pro-teins and suitable model complexes. The isomer shift reflectsessentially the electronic state of the cluster core (and is there-fore a good indicator of the oxidation state) and to a minor ex-tent the nature of the ligand (in general Cys sulfur) which com-pletes the environment of tetrahedral stereochemistry. Recentstudies of model complexes in collaboration with B. A. Averillhave shown that 8 increases by only about 0.04 mm/s when thefour thiolate ligands are replaced by phenolate. Further, a largebody of data has established that the average 8 increases by about0.10-0.12 mm/s upon reduction of the cluster.The shifts 8(S1) = 0.84 mm/s and 8(S2) = 0.89 mm/s are

incompatible with the maintenance of a tetrahedral environ-ment and they suggest strongly that the ligand environment hasexpanded to at least five-coordination. 14 The observed shifts ofSI and S2 show that the iron of the substrate binding site hasacquired substantial high-spin ferrous character. This is evenmore apparent in the [4Fe-4S]+ state. The parameters AEQ(SR)= 2.50 mm/s and S(SR) = 0.99 mm/s, at 4.2 K, are typical ofthose observed for high-spin ferrous compounds. For example,myoglobin (21) has AEQ = 2.20 mm/s and 8 = 0.93 mm/s at4.2 K. (Fe3+ compounds have 8 smaller than 0.5 mm/s.) Whereasmyoglobin has a magnetically isolated high-spin ferrous (S = 2)spectral component, SR belongs to a subsite of the [4Fe-4S]+unit, which has cluster spin S = 1/2. Therefore, we have usedthe phrase "high-spin ferrous in character." (The cluster spinS = 1/2 is the result of spin coupling and therefore the localspin of SR is not observable.) It is noteworthy that SR (and Siand S2 of the 2+ state) is a localized valence state and the clus-ter with bound substrate is thus a mixed-valence species (22).Such a valence state is observed for reduced [2Fe-2S] clusters(23-25). A localized valence state (labeled component Fe2+) hasalso been observed (26) for the P clusters of nitrogenase, a Fe-S cluster type containing four Fe atoms in an as yet not fullyunderstood arrangement.

Implications for the Mechanism of Aconitase. If we assumethat either SI or S2 in the 2+ state and SR in the 1+ state rep-resent complexes where citrate or isocitrate is coordinated tothe cluster with its OH group, we can speculate on the functionof the cluster. The increased values of 8 upon coordination ofsubstrate show that the d-electron density at Fea is increased.Because the isomer shifts at the Feb sites are the same as in theabsence of citrate, the data suggest that the cluster acts as a

t Recent unpublished x-ray and Mtssbauer studies of model complexessupport this interpretation. D. Coucouvanis and co-workers (personalcommunication) have synthesized the complex Fe4S4L2L'2 (L =thiolato, L' = dithiocarbamato), which has two four-coordinate andtwo five-coordinate iron sites. Compared to Fe4S4L4 the shift of twoiron sites has increased by 0.3 mm/s. R. H. Holm and co-workers(personal communication) have studied the Fe4S4L"4 anion, where L"= 2-hydroxy-thiophenolate. X-ray diffraction studies reveal that threesites are tetrahedral, whereas the fourth site is pentacoordinate withan additional (weakly bound) phenol oxygen. Preliminary analysis ofthe Mossbauer spectra of polycrystalline material suggests three com-ponents with a 2:1:1 site ratio; relative to Fe4S4L4 the isomer shiftof one site has increased by 0.15 mm/s.

Lewis acid withdrawing electron density from the substrate andthus facilitating loss of the hydroxyl group from citrate or iso-citrate. For the reverse reaction-namely, hydration of cis-aconitate-binding of this substrate would lead to a confor-mational change allowing H20 coordination to the cluster. Wedid indeed observe H20 binding for the analog trans-aconitate.

We are indebted to Dr. W. W. Cleland for useful discussions, to Dr.J. V. Schloss for kindly providing us with substrate analogs, to Mr. J.D. Hermes for isotope-ratio analyses and advice on labeling, to Mr. R.E. Hansen for assistance with EPR spectroscopy, and to Mrs. E. Ruz-icka for assistance with preparation of aconitase. This work was sup-ported by National Institutes of Health Grant GM-12394, Research Ca-reer Award 5-K06-GM-18442, and Training Grant T-32-AM-07049 andNational Science Foundation Grant PCM 80-05610.

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8. Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H.,Emptage, M. H., Beinert, H. & Mfinck, E. (1982) Proc. Nati Acad.Sci. USA 79, 1096-1100.

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Proc. Natl..Acad. S6. USA 80 (1983)

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