Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli

Eur. J. Biochem. 233, 317-326 (1995) 0 FEBS 1995

Spectroscopic characterisation of an aconitase (AcnA) of Escherichia coli Brian BENNETT', Megan J. GRUER2, John R. GUEST' and Andrew J. THOMSON' ' School of Chemical Sciences, University of East Anglia, Norwich, UK ' Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank.

Sheffield, UK

(Received 23 May/l7 July 1995) - EJB 95 0822/3

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53 % identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S] +-containing, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal from c-aconitase. This is perhaps related to the greater similarity of the AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but not identical to that of m-aconitase. Whilst the 13Fe-4SJ clusters from both of these enzymes display some features in their MCD spectra common to [3Fe-4S] clusters in general, their spectra overall are unique and indicate that the Fe, atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters of aconitases. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S]' cluster which is indistinguishable from the signals from the [4Fe-4S] ' cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [4Fe-4SI2+-containing AcnA is fivefold higher than that of m-aconitase. The mecha- nistic implications of these data are discussed. A novel S = 1/2 EPR signal with g=2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction.

Keywords: aconitase; Escherichia coli; EPR; magnetic circular dichroism.

The dehydratase-hydratase aconitase [citrate (isocitrate) hydro-lyase] catalyses the isomerisation of citrate and isocitrate via the dehydration product cis-aconitate. The application of spectroscopic techniques to the enzyme, including EPR (Emptage et al., 1983a), EXAFS (Beinert et al., 1983), Mossbauer (Kent et al., 1982; Emptage et al., 1983b) and magnetic circular dichroism (MCD; Johnson et al., 1984), revealed the presence of an iron-sulphur cluster interconvertible between the [4Fe-4S] and the [3Fe-4S] form. The oxidative loss of a specific iron atom, denoted Fe,, leads to deactivation of the enzyme. However, this process is reversible: under reducing conditions iron is re-incor- porated into the Fe, site in a two-stage process and the [4Fe-4S] cluster reformed with concomitant appearance of enzyme activity (Kennedy et al., 1983; Faridoon et al., 1991). X-ray crystallography (Robbins and Stout, 1989) confirmed that the [4Fe-4S] cluster has a cubane structure and demonstrated that removal of Fe,, does not lead to any significant change in the positions of the remaining [3Fe-4S] cluster atoms. Crystallography has also shown that Fe, is not ligated by an amino acid side chain of the

Correspondence to A. J. Thomson, School of Chemical Sciences, University of East Anglia. University Plain, Norwich, England NR4 7TJ

Fax: +44 1603 259396. Abbreviations. c-aconitase, mammalian cytosolic aconitase ; EN-

DOR, electron-nuclear double resonance; EXAFS, extended X-ray absorption edge fine structure; IRE, iron-responsive element; IRF (IRE- BP), iron regulatory factor (iron-responsive-element-binding protein); m-aconitase, mammalian mitochondrial aconitase; MCD, magnetic circular dichroism.

Enzyme. Citrate (isocitrate) hydro-lyase (EC 4.2.1.3).

protein. Mossbauer (Kent et al., 1982; Emptage et al., 1983b) and electron-nuclear double resonance (ENDOR; Telser et al., 1986; Kennedy et al., 1987; Werst et al., 1990a) spectroscopic studies have revealed that Fe;, has, in addition to three inorganic sulphur bridging ligands, a hydroxyl anion ligand which persists but becomes protonated upon binding of the C2 carboxylate anion of cis-aconitate to Fe,. The inequivalence of the iron sites in the cluster has been highlighted by 57Fe-ENDOR and "S- ENDOR and it has been further shown that the nuclei of two of the inorganic sulphide bridging ligands are considerably more strongly coupled to the paramagnetic electron density in the reduced [4Fe-4S] ' cluster than are those of the other two (Werst e t al., 1990b).

The [4Fe-4SI2 ' cluster in mammalian mitochondrial aconitase can be reduced to yield a [4Fe-4S]-', which retains G30% activity and displays a rhoinbic S = 1/2 EPR spectrum. The pre- cise form of the signal is to some extent buffer dependent but upon the addition of substrate a much larger effect, marked by a 30% increase in the anisotropy (i.e. gl -gl) of the signal, was observed (Emptage et al., 1983 a). Mossbauer spectroscopy also indicated that the ligand environment of Fe, is significantly al- tered upon the addition of substrate (Emptage et al., 1983b). These findings are consistent with the indication from "0- ENDOR studies (Werst et al., 1990a) that substrate binds directly to the cluster, through Fe,, in this partially active form of the enzyme. Crystallographic studies have demonstrated that upon the addition of substrate (citrate, cis-aconitate or isocitrate) to the oxidised [4Fe-4SI2+ form Fe, forms an isocitrate complex

31 8 Bennett et al. (Eur. J . Biochern. 233)

with the substrate molecule binding via the Ca-hydroxyl oxygen atom and a Ca-carboxylate oxygen (denoted OHA and OA2, respectively; Lauble et al., 1992). The OA2-Fe,-OHA angle of 68" is indicative of the distortion required by the bidentate substrate chelate. No modification of the [3Fe-4SJt S = 112 EPR signal from oxidised (inactive) enzyme is observed upon addition of substrate although this signal has particularly narrow linewidths and therefore any interaction of substrate would be expected to result in a detectable change in the spectrum. Treat- ment of aconitase with excess EDTA and cyanide removes all of the iron and inorganic sulphide, yielding an inactive u p - aconitase which can be reactivated to the [4Fe-4S]-containing form by incubating with Fez+, S2- and a thiol-containing reductant (Werst et al., 1990b).

By far the most extensively characterised aconitase is the mammalian mitochondrial enzyme (m-aconitase) but a cytosolic aconitase (c-aconitase) has been purified from bovine liver (Kennedy et al., 1992). Both these enzymes can be isolated in the active, [4Fe4S]-containing state and their EPR signals, both in the presence and absence of substrate, suggest that both contain very similar clusters. c-Aconitase also undergoes reversible Fe, loss upon oxidation but the resulting [3Fe-4S] clusters of the two aconitases differ in that the axial EPR signal from c-aconitase has g ! > g,, whereas that from m-aconitase has gll > g , . Both signals are distinct, however, from those from the electron- transferring clusters of other L3Fe-4SI-containing proteins. Ken- nedy et al. (1992) noted striking similarities between c-aconitase and the iron regulatory factor (IRF; also known as iron-responsive-element-binding protein, IRE-BP). I R F is a cytoplasmic mRNA-binding protein which post-transcriptionally regulates the expression of a number of proteins involved in iron metabolism (Leibold and Munro, 1988; Casey et al., 1988; Roualt et al., 1988; Mullner et al., 1989; Walden et al., 1989). IRF binds to specific stem-loop structures known as iron-responsive ele- ments (IRE; Klausner and Harford, 1989). IRF and c-aconitase are now known to be identical proteins in which the presence or otherwise of iron reciprocally regulates aconitase and IRE-binding activity (Haile et al., 1992a, b). However, whilst the loss of only Fe, from the cluster is sufficient to abolish aconitase activity, complete disassembly of the cluster is required for IRE-binding activity. Evidence has been presented suggesting that in vivo interconversion of apo-c-aconitase (IRF) and the active [4Fe- 4S]-containing form in response to iron levels is a physiologi- cally significant regulatory mechanism (Mullner et al., 1992; Tang et al., 1992: Gray et al., 1993: Emery-Goodman et al., 1993; Klausner et al., 1993).

Escherichiu coli contains two genetically distinct aconitases, AcnA and AcnB, encoded by the respective acnA and acnB genes (Gruer and Guest, 1994). AcnA has been purified to homogeneity but not subjected to a detailed biochemical and spectroscopic characterisation (Prodromou et al., 1991). AcnB has only recently been amplified by overexpressing the cloned acnB gene (Gruer, M. J., Bradbury, A. J. and Guest, J. R., unpublished results). The amino acid sequence of AcnA has been deduced from the nucleotide sequence of ucnA ; it is 27-29% identical to the mitochondria1 aconitases, but 53 % identical to human IRF (c-aconitase). It retains 19 of the 20 active-site residues identified by crystallographic studies with porcine m-aconitase, including three cysteine residues (Prodromou et al., 1992). This study presents the results of a spectroscopic study of the AcnA enzyme purified from E. coli. The study is aimed at a comparison between the spectroscopic signatures of AcnA and those of the mammalian m-aconitases and c-aconitases. The structural implications are discussed.

MATERIALS AND METHODS

Purification of aconitase A (AcnA). The source of AcnA was E. coli K12 strain JRG2387, a transformant of DH1 (thi-I hsdR17 supE44 recA I gyrA96 relA1) containing the multicopy phagemid pGS447 (Prodromou et al., 1991). The phagemid is a derivative of pUC119 in which the acnA coding region is expressed from its own promoter to give 20-200-fold amplifica- tion of AcnA activity in the transformed strain (Prodromou et al., 1991).

AcnA was purified from cultures of JRG2387 grown for 7.5 h at 37 "C with vigorous aeration in 15 2-1 flasks containing double-strength Luria-Bertani medium plus ampicillin (0.2 mg/ ml) to obtain a high level of AcnA expression (Prodromou et al., 1991). A 16-h culture of bacteria grown in the same medium served as the inoculum (1%). The bacteria were sedimented (14000Xg for 15 min at 4°C) and either stored at -70°C or immediately resuspended in Mes/MgCl buffer (10 mM K' -Mes, pH 6.0, containing 5 mM MgCl,; 1 ml/g wet bacteria) then dis- rupted by two passes through a French pressure cell at 133 MPa. The extract was clarified (20000Xg for 30 min at 4°C followed by 150000Xg for 2 h at 4°C) and subsequently was either stored at -20°C until required or used immediately. For large-scale preparations (e.g., batch 4, see Table 1) the clarified extract (30 ml) was loaded on a Procion Red (H-3B, I. C. I.) dye-affinity column (14 cmX5 cm), previously equilibrated with Mes/MgCI buffer, then washed with 800 ml Mes/MgCl buffer. Bound AcnA was eluted with a 1.6- 1 linear gradient of 0 to 100 mM sodium citrate in Mes/MgCI buffer and fractions containing 250% of the activity of the most active fraction were pooled and concentrated to a volume of less than 40 ml using Centricon-30 concentrators (Amicon) prior to dialysis against 2 x 3 1 Mes/MgCI buffer at 4°C. The sample was then loaded onto a Procion Green (HE-4BD, I. C. I.) dye-affinity column (34.5 cmX2 cm) equilibrated with Mes/MgCI buffer and washed with 300 ml of Mes/ MgCl buffer. AcnA was eluted with an 800-ml linear gradient of 0 to 100 mM citrate in Mes/MgC1 buffer. The preparation of the Procion Red matrix is described in detail elsewhere (Prodro- mou et al., 1991); the Procion Green matrix was prepared using an entirely analogous procedure.

Steps were sometimes taken to minimise the oxidative loss of Fe, from the AcnA [4Fe-4S] cluster (see Results and Table 1). This involved saturating all buffers with N, by exten- sive bubbling with high purity N, gas (B. 0. C.) and maintaining a stream of N2 during chromatography and dialysis.

Enzyme purity was estimated by densitometric analysis of Coomassie-brilliant-blue-stained gels obtained by PAGE performed under denaturing conditions (SDS/PAGE; 15 % acrylam- ide, 0.1 % SDS in all buffers) using the discontinuous buffer system of Laemmli (1970).

Enzyme assays. The citrate (isocitrate) hydro-lyase activity of AcnA was assayed spectrophotometrically at 240 nm and at 20-25 "C, following the formation of cis-aconitate from isocitrate, using a molar absorption coefficient, &;", of 3600 M ~ I .

Assays were carried out in 50 mM TrisIHC1, pH 8.0, without any additives except where explicitly stated. Activities measured at temperatures (roc) other than 25°C were normalised for 25°C by multiplying by the factor l.08(25-'' (Bennett, 1994). Protein was estimated by the dye-binding method of Bradford (1976) using the Bio-Rad protein assay kit with BSA as standard. The concentration of AcnA molecules was also estimated from the electronic absorption of the [Fe-S] cluster at 400 nm using an absorption coefficient, c i g , calculated from the spectra of [3Fe- 4.51 +-, (3Fe-4SI"- and (4Fe-4S]*+-containing m-aconitase presented by Emptage et al., (1983). A value of c&" of 8300 M-' was used for samples suspected to contain mixtures of the three

D ---- 16 ,.* " 1 IC.... . J . Biochcnz. 233) 319

forms of AcnA. At this wavelength the absorption coefficients are within 10%. The presence of [4Fe-4S]'-containing AcnA would cause a large error in this estimate since it has an ck' ' of 6020 M- ' . However, the [4Fe-4S] ' content could be easily determined by quantification of its EPR signal. Integration of the g,, -2.01 EPR signal from freshly prepared [3Fe-4S] '-containing AcnA yielded an estimate of AcnA concentration indistinguishable from that estimated from A,,,,,.

The incorporation of Fe;, into [3Fe-4S]-containing AcnA for either kinetic or spectroscopic analysis was effected by the addition of 1 mM Na,S20,, 2.5 mM (NH,),Fe(S0,)2 and 10 mM DL- isocitrate to the enzyme. Enzyme thus treated is referred to as activated. For activity assays these reagents were not removed because 1-5 p1 activated enzyme was added to 3 ml assay mix- ture and the reagents had no effect on activity at their final con- centrations. Stock solutions of 10-50 mM (NH,),Fe(SO,), were freshly prepared anaerobically in 10 mM HCI.

Enzyme samples. Enzyme samples for spectroscopy were concentrated to 50 pM- 1 mM for spectroscopy using Centri- prep and Centricon-30 concentrators (Amicon). Anaerobic samples were prepared either under argon (B. 0. C.) on a manifold or under nitrogen (<5 ppm 0,) in an anaerobic glovebox (Faircrest). Dithionite solutions were freshly prepared in either argon-saturated buffer under argon or in vacuum-degassed buffer in a nitrogen atmosphere. Where applicable, enzyme samples for spectroscopy were rapidly frozen under anaerobic conditions as described in detail elsewhere (Bennett et al., 1994a). Buffer ex- change into 'H20 (D,O; 99.9% mol 'H/mol, Fluorochem Ltd.) was achieved by repeated ultrafiltration (Centricon-30) or dialysis.

EPR and MCD spectroscopy. X-band EPR spectra were recorded under non-saturating conditions at approximately 9.4 GHz on an updated Bruker ER 200-D SRC spectrometer equipped with a microwave counter (Marconi model 2440) and an NMR gaussmeter (Bruker ER 032 M). Cryogenic operating temperatures were maintained using a helium flow cryostat (Ox- ford Instruments ESR-900). Integrations were performed relative to a 2 mM Cu-EDTA solution according to Aasa and Vanngird (1975). K,-band EPR was performed on a Bruker ESP-300 spectrometer equipped with a Bruker 5102 QTH/8715 cavity operating at its maximum modulation frequency of 12.5 kHn and at a microwave frequency of approximately 34 GHz. Temperature was maintained using a helium flow cryostat (CF-935, Oxford Instruments). Where applicable, difference spectroscopy techniques were as in earlier work (Bennett et al., 1994b).

Low-temperature MCD spectra were recorded on a JASCO J-500D spectropolarimeter interfaced to an IBM PC-AT. Glyc- erol (50% by vol.; spectroscopic grade, Aldrich) was used as the glassing agent. The sample was mounted in a split-coil su- perconducting magnet (SM4, Oxford Instruments) capable of generating a magnetic field of 5T and permitted sample temperature variation from 1.6-150 K. The magnet was fitted with optical windows to allow access of the light beam to the sample.

RESULTS

Enzyme preparations. A typical preparation from 35 g wet cell paste yielded approximately 150 mg AcnA, judged to be 2 98 % pure by SDS/PAGE. The specific activities of four batches of as-prepared, purified AcnA are given in Table 1. As can be seen from the Table 1, enzyme prepared using Nz-saturated buffers (batch 4) was significantly more active than that prepared without any anaerobic precautions. Activity measurements on this batch of AcnA (= 80% pure) after the first stage of affinity chromatography indicated that it had a specific activity of 167 pmol

Table 1. Activity of AcnA before and after addition of ferrous ion. The specific activities of preparations of AcnA after partial purification on the Procion-Red dye-affinity column, after final purification on the Procion-Green dye-affinity column and after activation with Fe' are presented along with fractional intensities of [3Fe-4S]' EPR signals of the as-purified material. Activities after the Procion Red and Green col- umns were determined aerobically in the absence of added iron on the pooled eluent from the column prior to further sample handling. The [3Fe-4S] + EPR intensities were recorded on samples analogous to those assayed as shown in column 3. EPR integrations are estimated to 10%.

Activity [3Fe-4S] ' Enzyme Activity Activity preparation after Procion after Procion after Fe" EPR (yield in mg) Red column Green column activation intensity

U/mg e -/mol AcnA

1.' (8) loh 0.38'

3" (50) 50 8.0 59' 4' (150) 167 48" 200' 0.75 4R 11 'I 81 0.90

2.' (20) 0.1 45 1.07 18'

it No anaerobic precautions were taken during enzyme preparation. " Activities were determined on the pooled eluent from the column

' The EPR signal observed was unusual. (' Activities were determined on the pooled eluent from the column

following aerobic dialysis into citrate-free buffer, (e.g. K+/Mes, pH 6.0; K'IHepes, pH 7.0; Tris/HCI, pH 8.0; K'nr ic ine, pH 8.5), and aerobic concentration.

Activities were determined aerobically in the presence of 0.5- 2.5 pM Fe' ' following anaerobic treatment of the as-prepared enzyme (after dialysis into 50 mM K'/Hepes, pH 7.0, and aerobic Concentration) with 2.5 mM (NH,)2Fe(S0,), and 1 mM Na,S,O,.

' The activity was determined after incubation of the enzyme anaerobically with a mild excess of solid NaBH, until hydrogen ceased to be evolved followed by the addition of 2.5 mM (NHJ2Fe(SO&.

Buffers used for chromatography and dialysis were saturated with N, during the preparation.

Enzyme originally from preparation 4 was recovered by aerobic dialysis and concentration after use in a number of experiments which involved exposure of aliyuots of the enzyme to up to 1 mM Na&O,, 35 mM citrate, 20 mM rxL-isocitrate or 2.5 mM (NHJ)IFe(SOJ)Z. Insolu- ble protein-derived matter, which caused some turbidity of the recovered batch, was removed by centrifugation prior to quantification and assay of the material.

prior to further sample handling.

"

cis-aconitate formed . min-' . mg-' protein. The specific activity of this material after final purification was lower (48 pmol cis- aconitate formed . min-' . mg-' protein) but treatment of the sample with 2.5 mM Fe" and 1 mM Na,S,O, in the presence of 10 mM DLisocitrate (see Materials and Methods) increased the specific activity to 200 pmol cis-aconitate formed . min-' . mg-l protein (Table 1). EPR analysis of this as-prepared material, which exhibited 25% of the maximum activity obtained by activation, showed that 75 % of the protein was in the inactive 13Fe- 4S]+ form. Enzyme prepared without any attempts to exclude oxygen was not only less active but was also less activatible with Fe2+/Na2S,0, (compare batches 2 and 3 with batch 4, Table 1 ) . Aerobic concentration of the purified enzyme, followed by dialysis into citrate-free buffers (e.g. K+/Mes, pH 6.0; K '/Hepes, pH 7.0; Tris/HCI, pH 8.0; K'Rricine, pH 8.5) led to a decrease in the specific activity (compare batches 1 and 3 with batch 2, Table 1), but did not appear to have a great effect on the activatibility of the enzyme (compare batch 2 with batch 3, Table 1). Attempts to reactivate the enzyme using NaBH, as the reductant were only partially successful (batch 2, Table 1).

320 Bennett et al. (EUK J. Biochem. 233)

The [3Fe-4S]-containing form of AcnA. Anaerobic treatment of as-prepared AcnA with a stoichiometric amount of EDTA for 1 h at 20-25°C followed by brief (5 min) exposure to air was found to yield inactive enzyme which exhibited a g = 2.01 EPR signal (Fig. l a ) due to the [3Fe-4S]+ cluster. The signal integ- rated to a spin density of 1 .O t 0.1 spins/molecule. Longer, aerobic treatment of the enzyme with EDTA produced more complex changes in the EPR of AcnA. The EPR signal of the [3Fe-4S]+ cluster (Fig. 1 a) appears to differ from that of m-aconitase in that g,, >g, and the signal much more closely resembles that from c-aconitase (Kennedy et al., 1992). Although the signal recorded at X-band appears axial, examination of the spectrum at K,-band (Fig. 1 b) clearly reveals that the species is rhombic. To measure the g values accurately (the microwave frequency at K,-band could not be measured with great precision) the second- (62x/dB2, where B = magnetic field; Fig. 1 c) and third-de- rivatives (d3x/6B3; Fig. 1 d) of X-band EPR absorption spectra were generated and the arrows on Fig. 1 c and d mark the positions of the principal g values at 2.024, 2.008 and 2.000. The [3Fe-4S]+ EPR signal from AcnA was found to be invariant upon dialysis into a number of different buffers (K'Nes, pH 6.0; K+/Hepes, pH 7.0; Tris/HCI, pH 8.0; K'mricine, pH 8.5) and to the presence of citrate. The presence of SO% (by vol.) glycerol, used as a glassing agent in MCD samples, sharp- ens the linewidths somewhat and shifts the gz value to 2.004 (Fig. l e ) . No change in the EPR spectrum was observed upon transferring a sample into buffered *H,O.

Variable-temperature ultravioleu'visible MCD spectra of [3Fe-4S]+-containing AcnA are presented in Fig. 2. The temperature dependence of the intensity of the spectrum indicates that it is largely due to the paramagnetic [3Fe-4S]+ cluster. The spectrum is similar to that from m-aconitase (Johnson et al., 1984; there is no published data on MCD of c-aconitase), exhibiting features observed in the m-aconitase spectrum such as the positive doublet with peaks at 370 nm and 400 nm, a triplet centred on 450 nm (in AcnA, clearly resolved as three bands but less so in ni-aconitase), a weak, positive feature just above 600 nm, and two fairly broad, equally intense positive bands at 700 nm and 800 nm (both doublets, the former resolved, the latter exhibiting a shoulder at 820nm). The spectra from AcnA and m-aconitase are not identical but the most prominent features appear to be present in both spectra and the spectra clearly resemble each other more than they do the spectra of the [3Fe- 4S1' clusters of e.g. Fd 1 of Azotobacter chvoococcum and Fd I1 of Desulfovibrio gigas, where the similarities break down particularly at longer wavelengths (Johnson et al., 1984).

The [3Fe-4SIo-containing form of AcnA was generated by reduction of [3Fe-4S]+-containing AcnA with 3 mM NaS,O, for 1-5 min in 50 mM Tris/HCl, pH 8.0, without added iron (John- son et al., 1984). This species gave rise to a broad, featureless EPR signal (Fig. 3) which extends into zero-field, indicative of an integral spin system (Hagen, 1992; Hendrich and Debrunner, 1989). From Fig. 3, it can be clearly seen that as the microwave power is increased from 0.2 mW to 2 mW then to 10 mW (Fig. 3a, b and c, respectively), the EPR signal moves upfield, out of zero-field. The low intensity of the signal recorded at 10 mW is indicative of saturation at this microwave power and the signal was barely observable at 50 mW (data not shown).

Variable temperature MCD spectra of [3Fe-4SIo-containing aconitase are shown in Fig. 4. These exhibit a number of features common to the spectra of [3Fe-4S]"-containing proteins (George, 1986; Johnson et al., 1984), particularly the bands with maxima at 375, 450 and 480 nm. However, an intense band at 700 nm which is exhibited by a number of [3Fe-4SI0-containing ferredoxins but which is less prominent in the spectrum of c- aconitase is not observed in the spectrum of [3Fe-4SIo AcnA.

\ /-------= 2

v g-value

I I I I I I 2.041 2 . 0 0 7 1 . 9 7 4

Fig. 1. EPR spectra of the [3Fe-4S]+ cluster in AcnA. (a) and (b) show first derivative (J,,,,,) EPR spectra of AcnA in 50 mM Tris/HCI buffer, pH8.0, recorded at (a) X-band ( ~ 9 . 3 5 GHz) and (b) K,,-band (= 34 GHz). The second-derivative (6&J and third-derivative (b~,sUi) spectra corresponding to the X-band SX,,, spectrum, (a), are presented in (c) and (d), respectively. The arrows show the positions at which the principal g values of 2.024, 2.008 and 2.000 were determined. Spectrum (e) is of a sample of [3Fe-4S]'-containing AcnA in TrislHCI, pH 8.0, containing 50% (by vol.) glycerol. X-band spectra were recorded at 15 K, 2 mW microwave power and 0.2 I T modulation amplitude. The K,-band spectrum was recorded at 25 K, 0.9 mW microwave power and 0.5 mT modulation amplitude.

Fig. S shows the magnetisation curve for the [3Fe-4S]" cluster. Whilst full analysis of these data is complex, the similar, corresponding data for m-aconitase and D. gigas FdII has been inter- preted (Johnson et al., 1985; Thomson et al., 1981) as describing an S = 2 ground state with a predominantly axial distortion resulting in an M , = 2 2 lowest energy doublet. The similarity of

Bennett et al. ( E M J . Biochern. 233) 321

Fig. 2. hriable temperature CD spectra of [3Fe-4S]+-containing AcnA. Traces (a), (b) and (c) show MCD spectra of [3Fe-4SIi-containing A&A in 50 mM TrisiHCl containing 50% (by vol.) glycerol, pH 8.0, recorded at a magnetic field strength of 5T and at temperatures of (a) 1.6 K, (b) 4.2 K and (c) 8.5 K.

9 1 6 9-12 t 1

a

C

I I I I -1__1__ I I I I 60 80 40 BImT 20

Fig. 3. EPR spectra of [3Fe-4S]u-containing AcnA. Fig. 5 shows X-band EPR spectra of [SFe-4S]-containing AcnA in 50 mM TrisIHCl, containing 50% (by vol.) glycerol, pH 8.0, which had been anaerobically incubated with 3 mM Na,S,O, at 2 0 ~ 2 5 ° C for 5 min. The data were recorded at 4.5 K, with a modulation amplitude of 1.0 mT, a microwave frequency of approximately 9.66 GHz and using microwave powers of (a) 0.2, (b) 2.0 and (c) 10 mW. The horizontal straight lines indicate the baseline for each spectrum and labelled arrows mark the g e l 6 and g=12 positions. The intensities of the spectra are as recorded, i.e. they have not been normalised or corrected for (microwave power)"'.

the data presented in Fig. 5 to the earlier data is good evidence that the [3Fe-4S]" cluster of aconitase is also an S = 2 system.

Both the oxidised and reduced forms of [3Fe-4S]-containing AcnA also exhibited intense circular dichroism (CD) in the absence of an applied magnetic field. The spectra, presented in Fig. 6, were found to be temperature independent between 2.8 and 50 K.

The [4Fe-4S]-containing form of AcnA. The active, [4Fe- 4SI2' -containing form of AcnA could be generated by treatment of the enzyme with dithionite and Fez+ (see above).

In the presence of Na,S,O,, the [4Fe-4S]-containing form of AcnA was seen to exhibit rhombic EPR signals in the g = 2 region. The development of the signal with dithionite was slow and the maximum content of approximately 10% of 14Fe-4SJ'

322 Bennett et al. (Eur: J . Biochem. 233)

-100 ' I I 400 son h,nm 600 700 aon

Fig. 4. Variable temperature MCD spectra of [3Fe-4S]-containing AcnA after reduction with dithionite. Traces (a), (b) and (c) show MCD spectra of [3Fe-4S]-containing AcnA in SO mM Tris/HCI, containing SO% (by vol.) glycerol, pH 8.0, which had been anaerobically incubated with 3 mM Na,S,O, at 20-25°C for 5 min. Spectra were recorded at a magnetic field strength of 5T and temperatures of (a) 1.6 K, (b) 4.2 K and (c) 8.5 K.

"1 17.5X . , "

1 FiPU

0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 .2 PBIZkT

Fig. 5. MCD magnetisation data for [3Fe-4S]-containing AcnA after reduction with dithionite. Magnetisation curves showing the field dependence and temperature dependence of the MCD absorption of dithionite-treated AcnA at 450 nm are presented. The sample used was that of Fig. 4. The data were recorded using a swept magnetic field (0-5T) and at fixed temperatures of 1.63, 4.2 and 17.5 K as indicated on the figure.

was observed after approximately 40 min. Fig. 7 a shows the signal from AcnA generated by treatment with 2 mM Na,S,O, for 30 min at pH 8.0. The signal was found to be temperature dependent: saturation studies of this signal at 17 K indicated a PI,, value of approximately 40 mW. At 5 mW microwave power, the maximum signal intensity was at 25 K. Signal intensity fell to half-maximum at temperatures of 17 K and 30 K. The form of the signal clearly suggests that the spectrum includes contribu- tions from more than one species and difference spectroscopy techniques applied to data recorded at 13 K (Fig. 7a) and 9.6 K

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J I L .

400 500 600 700 800 hlnm

Fig. 6. Low-temperature CD spectra of [3Fe-4S]-containing AcnA before and after reduction with dithionite. Fig. 6A shows the CD spectrum of [3Fe-4S]'-containing AcnA, recorded at 1.6 K in the absence of an applied static magnetic field. The sample used was that of Fig. 4. Fig. 6B shows the CD spectrum of [3Fe-4S]-containing AcnA after reduction with dithionite, recorded at 1.6 K in the absence of an applied static magnetic field. The sample used was that of Fig. 4.

(Fig. 7 b) allowed the separation of three proposed contributory single species, one shown as Fig. 7 c and the other two shown in Fig. 7d and indicated by the stick-spectra A and B. Similarly complex spectra were seen during preliminary work on samples at pH 6.0 (Breton, 1994).

The addition of a 100-fold excess of citrate to AcnA at pH 8.5 had little effect on the [4Fe-4S]+ EPR signal. As can be seen from Fig. 8 b the spectrum i s simplified somewhat and is largely due to a single species with gl,2,3 values 2.049, 1.918, 1.846. This small effect contrasts sharply with that seen in m- aconitases and c-aconitases in which the g values are shifted considerably to gl,*, of 2.040, 1.855, 1.780 (Emptage et al., 1983; Kennedy et al., 1992). The same signal (Fig. 8c) was generated by the addition of citrate to AcnA in 10 mM Meskitrate, pH 6.0. Addition of K'flricine, pH 8.5, to a final concentration of 200 mM had no effect on the spectrum (Fig. 8d).

Bennett et al. ( E M J . Rzochern. 233) 323

I I I3

---1 I I--.. J 1

3 0 0 370 340 160 :i ti 0 I h i r

Fig. 7. EPR spectra of dithionite-reduced [4Fe-4S]+-containing AcnA. The X-band ( V = 9.4 GHz) EPR spectra of AcnA, anaerobically incubated in 50 mM K+/Tricine, containing 2 mM Na&O,, pH 8.0, for 30 min, were recorded with a modulation amplitude of 0.5 mT, a microwave power of 5 mW and at temperatures of (a) 13.0 K and (b) 9.6 K. The difference spectrum (c) was isolated by subtraction of (b) from (a) and the difference spectrum (d) was generated by back-subtraction of (c) from (b). The stick spectra A and B correspond to the two rhombic species which contribute unequally to (d): from both integration of the g , features and measurement of the peak-to-trough intensities of the gz features of the two signals it is clew that the signal corresponding to A accounts for 55 % of the overall spin density. The g , regions of the stick spectra are shown as dashed to highlight the uncertainty with which the positions of the individual g1 resonances can be determined from the data.

300 320 340 360 300 HimT

Fig. 8. The effect of citrate on the EPR signal of dithionite-reduced [4Fe-4S]-containing AcnA. Spectrum (a) is of dithionite-reduced [4Fe- 4S]-containing AcnA in 50 mM K'/Tricine, pH 8.0; the sample and spectrum correspond to Fig. 7(a). Spectrum (h) is of the same sample after anaerobic addition of 0.1 vol 300 mM citrate in 500 mM K+/Tric- ine, pH 8.0 (final [citrate] = 30 mM). Spectrum (c) is of dithionite-reduced [4Fe-4S]-containing AcnA in 10 mM K+/Mes, containing 30 mM citrate, pH 6.0, and spectrum (d) is of the same sample after addition of 0.5 vol of 400 mM K'/Tricine, pH 8.5 (final [Tricine] = 200 mM). Spectra were recorded at approximately 9.4 GHz, 5 mW microwave power, 0.5 mT modulation amplitude and at a temperature of 13 K.

Dialysis of a freshly prepared sample of AcnA against 0.1 mM EDTA at 4°C for 16 h was carried out with the intention of removing residual Fe., from a heterogenous sample (activity 48 pmol cis-aconitate formed . min-' . mg-' protein) which con- tained both [3Fe-4S] and [4Fe-4S] forms of the enzyme. The appearance of a large EPR signal at gz4.3, characteristic of

1 1 ~ 1 ' 1 : 1 1 1 325 3 0 334 3 2 542 346

BimT

Fig.9. The novel EPR signal from AcnA. Fig. 9 shows the X-band ( v = 9.4496 GHz) EPR spectrum obtained upon aerobically dialysing 45 pM [3Fe-4S1' -containing AcnA against a volumetric excess of 0.1 mM Na,EDTA in 50 mM K'nricine, pH 8.0, for 16 h at 4°C. The spectrum was recorded at 2 mW microwave power at a temperature of 20 K and with a modulation amplitude of 0.2 mT.

Fe' I , and partial loss of electronic absorption in the visible region indicated that the EDTA treatment had liberated much of the iron from the enzyme. After exhaustive dialysis against Tric- ine, pH 8.0, a small g z 4 . 3 signal remained (data not shown) which also exhibited a feature at p 9 . 6 , characteristic (Kennedy et a]., 1984) of the [Fe(pS),Fe(pS),Fe] cluster of purple aconitase. Some caution should be exercised, however, in taking the p 9 . 6 feature as being diagnostic of this species. Signals at g = 9 due to the S = i- 1/2 and S = 2 5/2 doublets are often seen in association with g z 4 . 3 signals due to the S = t 312 doublet of a rhombic Fe? ' species.

An EPR signal (data not shown) which was very similar to that from [Fe( pS),Fe( pS),Fe]-containing aconitase was observed during the course of the present work upon air oxidation of a buffer solution to which Fez ' and dithiothreitol had been added.

A novel EPR signal observed in AcnA. Another EPR signal, a novel, complex EPR signal at g=2, was also observed in EDTA- treated AcnA. Integration of this signal revealed that less than 10% of the aconitase molecules in the sample contributed to it. This signal (termed here the novel signal) could be reproducibly obtained and is presented in Fig. 9. The signal was often observed at 20 K along with the p 2 . 0 1 signal due to [3Fe-4S]+, but unlike the g = 2.01 signal it persisted at higher temperatures (2 35 K). Removal of EDTA by aerobic dialysis of samples resulted in an increase in the intensity of the novel signal and a diminution and eventual disappearance of the [3Fe-4S] ' signal. Spectra recorded at 1.5 K on samples in which the [3Fe-4SIi signal had disappeared were indistinguishable from those recorded on the heterogeneous samples at 35 K. The signal appears to be due either to two separate, similar species or a single, misotropically I = 1/2-split species with g,,,,,,,,, ,=2.040, 2.029, 2.007 and A:,;,:$,,,=1.45, 1.70, 0.70 mT. Although it is very unusual for iron-containing species to exhibit resolvable hyper- fine splittings, the invariance of the form of the signal from different enzyme samples in either K'mricine, pH 8.0, or Mes/ citrate, pH 6.0, and when recorded over a range of temperatures (1.5-3.5 K) and microwave powers (2-100 mW) suggests that the signal may well be due to a single 'H-split species. Transfer of the enzyme into *H,O did not, however, result in a loss of the

324 Bennett et al. ( E m J . Biochem. 233)

splitting and hence the effect cannot, be due to solvent-derived protonation of the cluster.

The novel signal from AcnA was found to be stable in air at 20-25°C for at least 16 h. The addition of 2 mM Na2S,0, abol- ished the EPR signal, though it was recoverable upon reoxida- tion with air. Anaerobic incubation of AcnA exhibiting the novel signal with 1 mM Na2S201 and 2.5 mM Fe' for 20 min resulted in a loss of the novel signal and reappearance of the [3Fe-4SIi g52.01 signal upon removal of reductant by aerobic dialysis. Prolonged incubation of AcnA with EDTA eventually resulted in an EPR-silent form of the enzyme which exhibited negligible electronic absorbance and MCD at wavelengths above approximately 300 nm. This form of the enzyme could not re-incorpo- rate iron upon treatment with Fez+ and Na2S2O4 in the abcence of sulphide and presumably corresponds to apo-protein from which all the iron and inorganic sulphide has been lost.

DISCUSSION

As-prepared AcnA. As-purified AcnA was found to be largely in the inactive, [3Fe-4S]+-containing state. However, after the first stage of dye-affinity chromatography, using N,-sparged buffers, the enzyme exhibited 80% of the maximum activity at- tainable by Na,S20,/Fe2 + treatment. It therefore seems likely that the active form is present in the harvested bacteria despite the high level of overexpression. EPR spectroscopy of pelleted AcnA-overexpressing cells failed to detect any signals due to the [3Fe-4S]+ cluster (Bennett, B., unpublished results). Purifi- cation of the enzyme in the presence of air was found not only to diminish the specific activity of the as-prepared enzyme but also its ability to be reactivated with Fe' '/Na,S,O, whereas aerobic dialysis into citrate-free buffers affected the specific activity but not the activatibility. This observation can be rationalised by postulating that whilst 0, merely effects reversible iron loss from the cluster, the liberated iron (as Fe'+ in the precence of 0,) can inflict irreversible, oxidative damage on the enzyme. Systematic studies are needed to address this question further.

Inactive forms of AcnA. EPR spectroscopy of [3Fe-4S] +-containing AcnA indicates that whilst the cluster is similar to that of m-aconitase, it is even more closely related to that in c-aconitase. The high degree of similarity between the amino acid sequences and the structure of the active site of human c-aconitase and E. coli AcnAis surprising considering the evolutionary dis- tance between the organisms. Attempts to identify IRE-like sequences in E. coli have so far proved fruitless (Hentze, M. w., personal communication). At least one other aconitase, AcnB, is expressed by E. coli and this enzyme can seemingly perform all the essential functions of AcnA in an ucnA mutant (Gruer and Guest, 1994). AcnA expression is regulated, directly or indi- rectly, by ArcA, FNR, Fur, SoxRS and CRP whereas AcnB expression has only been shown to be partially repressed by anaerobic conditions, indicating lesser involvement of ArcA and FNR. AcnB is repressed in contrast to AcnA which is induced by oxidative stress. Several citric acid cycle reactions are cata- lysed by genetically distinct and differentially regulated enzymes in E. coli. Further studies with AcnB are needed to estab- lish whether or not i t performs an IRF-like r61e in iron regulation.

From the MCD spectrum of the AcnA [3Fe-4S]+ cluster it can be concluded that the electronic structure of the AcnA [3Fe- 4S]+ cluster is very similar to that in m-aconitase but the EPR spectra suggest that the electronic structures of the clusters are slightly different. Whether the differences between the clusters are due to specific amino acid residues or to more global differ-

ences in protein conformation remains to be seen. Detailed com- parisons of the MCD spectrum of [3Fe-4S] '-containing aconitases with those of [3Fe-4S]+ clusters in the bacterial ferredoxins A. croococcum Fd I and D. gigas Fd I1 (Johnson et al., 1984) and with the [3Fe-4SIt cluster in the oxidised molybdenum- containing and [Fe-S]-containing enzyme E. coli nitrate reductase-A (Johnson et al., 1985) indicate that the electronic structure of the aconitase cluster is not as conserved as the others. Subtle differences between the EPR signal from the aconitase [3Fe-4S]+ cluster and those from the [3Fe-4S]+ clusters from E. coli nitrate reductase (Vincent and Bray, 1978), Azotobucter vinelundii Fd I (Emptage et al., 1980), D. gigas Fd I1 (Huynh et al., 1980) and Thermus therrnophilus Fd I (Hille et al., 1983) also highlight the uniqueness of the aconitase cluster. Moreover, these data indicate that the unusual nature of Fe, is not the only atypical feature of the aconitase cluster which differentiates it from the electron-transferring clusters in other systems.

The MCD magnetisation data obtained from [3Fe-4Sj-containing AcnA reduced for a short time in the absence of iron indicates, by analogy with earlier work (e.g., Thomson et al., 1981 ; Johnson et al., 1985), that the paramagnetic chromophore is a S = 2 system, i.e. a [3Fe-4SJ0 cluster. Such S = 2 species do not give rise to easily detected EPR signals, though broad EPR signals extending into zero-field are occasionally seen, such as that from the [3Fe-4S]" cluster of the 7: thermophilus 7Fe ferredoxin (Hagen et al., 1985). Similar signals, with g.,pp.l,rn,=16r have also been claimed (Hendrich, M. P., unpublished results; see Hendrich and Debrunner, 1985) to have been observed from m-aconitase. Such signals are due to the ldml = 0 transition within the second ( I -+ 2 >) non-Kramer's doublet of the S = 2 spin system (more usually referred to as the ldml = 4 transition; Hagen, 1992, and references therein) and both the form and the microwave-power-dependent field shift of the signals from AcnA are consistent with their being due to an S = 2 [ 3Fe-4S 1" cluster.

The novel EPR signal from AcnA. EPR and Mossbauer analy- ses (Kennedy et al., 1984) of inactive m-aconitase at pH>9.5 have suggested, by comparison with data for synthetic clusters (Hagen et al., 1983), that linearisation of the [3Fe-4S] cluster can occur with the formation of a [Fe(pS),Fe(pS),Fej+ cluster. Kinetic studies (Zhuang and Sykes, 1994) have shown that this process behaves uniphasically and exhibits a dependence. The resulting linear [3Fe-4S] cluster exhibits an S = 5/2 EPR signal when oxidised. Upon addition of dithionite to this species, one of two EPR spectra were observed, depending on the reaction conditions. Each spectrum appears to contain contri- butions from at least two S = 1/2 rhombic species, and Kennedy et al. (1984) have argued that one of the signals is probably due to generation of a [2Fe-2S]+ cluster. Prolonged incubation with dithionite regenerated the cubane [4Fe-4S] centre, suggesting that the putative [2Fe-2S] species may be an obligatory intermediate in the reconstruction of the [4Fe-4Sj cluster from the linear [3Fe-4S] cluster. Perhaps related to this is the proposal that [3Fe- 4S]"-containing m-aconitase can be superreduced to a labile [3Fe-4SJ2- form, decomposition of which in a proportion of enzyme molecules provides Fez+ for incorporation into the remaining molecules to yield [4Fe-4S]-containing enzyme (Tong and Feinberg, 1994).

The novel signal observed in AcnA is similar to the signal observed (Kennedy et al., 1984) in m-aconitase at pH 10.5 upon treatment with excess dithionite which was presumed to be due to a reduced (2Fe-2SI centre. In AcnA, however, the signal was due to an oxidised rather than a reduced species. Unlike the linear species in m-aconitase the novel species of AcnA could be generated at physiological pH. Furthermore, generation of

Bennett et al. (Eus J. Biochem. 233) 325

this species was found to be reversible upon addition of Fe" to the reduced, EPR-silent species (see Results). It is therefore entirely possible that the novel species is an obhgdtory intermediate in cluster disassembly in AcnA. Though we tentatively as- sign the novel EPR signal as being due to a 2Fe species because of the similarity with the reduced purple aconitase signal from m-aconitase, further work is clearly necessary.

The active form of AcnA. The fully active form of aconitase is the [4Fe-4S l 2 +-containing form. Unfortunately the [4Fe-4SI2 +

cluster is the least accessible to spectroscopic methods : the cluster is diamagnetic and therefore EPR silent and the MCD intensity is very weak ( d e < I O M - ' c m - ' between 300nm and 800 nm at magnetic fields of up to 4.9T and temperatures in the range 1.7-95 K). Such weak MCD intensity can result in dominance of the spectrum by, e.g., contaminating haemopro- teins present at < 0.1 % by-iron or, above 450 nm, possibly even by free Fe" (Johnson et al., 1984). Furthermore, the enzyme was seen to exhibit intense natural CD signals which could in- terfere; those for the [3Fe-4S] +-containing and [3Fe-4S]"-containing enzyme are shown in Fig. 6. In view of these potential problems, no attempt was made to record the MCD of the [4Fe- 4S]*' cluster.

For reasons similar to those discussed above for the [4Fe- 4Sj2+ form, MCD spectroscopy of the [4Fe-4S] ' form of AcnA was not attempted, but the paramagnetic nature of the latter allowed EPR analysis. In the absence of substrate, the form of AcnA containing [4Fe-4S]+ displays an EPR signal due to two or three species, each of which must be electronically similar. Structurally and thermodynamically similar conformers of [4Fe- 4S] clusters are known to co-exist in other systems e.g., Desul- phovibrio qfricanus Fd I (Hatchikian et al., 1984), E. coli mem- brane-bound respiratory nitrate reductases A and Z (Guigliarelli et al., 1992) and the corrinoid/(Fe-S] protein from Clostridiurn therrnoaceticuin (Ragsdale et al., 1987). The EPR parameters of the [4Fe-4S]+ signals from AcnA are also very similar to those of the signals from m-aconitases and c-aconitases, indicating that the clusters in these three aconitases are alike. The existence of three species which contribute to the EPR of AcnA in Tricine, pH 8.0, where only two are observed in Mes, pH 6.0, may indicate that the pK, for the protonation of the presumed hydroxylf water ligand of Fe,i in AcnA lies in the region 6.0-8.0.

The [4Fe-4S]+ cluster of AcnA does, however, differ mark- edly from those in m-aconitases and c-aconitases in its reaction to the addition of substrate. The large increase observed in the anisotropy of the [4Fe-4S]+ EPR signals from the mammalian enzymes upon adding citrate suggests that the binding of substrate through two carboxylates introduces a significant degree of perturbation of the electronic structure of the cluster. The EPR parameters of AcnA, however, are barely perturbed upon the addition of citrate, though that it drives the heterogeneous pop- ulation of [4Fe-4S] clusters into adopting a single, most thermodynamically favourable conformation clearly indicates interaction between the substrate and enzyme molecule. This much reduced effect of substrate on the electronic structure of the 14Fe-4SI.' cluster of AcnA compared to that on m-aconitases and c-aconitases is perhaps indicative of a different method of substrate binding. The much higher maximum catalytic activity of AcnA relative to those of m-aconitases and c-aconitases may also be an indication of the operation of a different catalytic mechanism of AcnA. Clearly, the use of other spectroscopic techniques such as ENDOR, Mossbauer and resonance Raman and of X-ray crystallography will be required to address this question further.

The authors wish to thank Dr J. Breton, University of East Anglia for assistance with some of the MCD spectroscopy. B. B. and M. J. G.

were supported by a grant from the Wellcome Trust to A. J. T. and J. R. G. The authors acknowledge the use of spectroscopic facilities of the Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia.

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Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli