Proc. Natl. Acad. Sci. USAVol. 86, pp. 3639-3643, May
1989Biophysics
Structure of activated aconitase: Formation of the [4Fe-4S]
clusterin the crystal
(Fe-S enzyme/x-ray diffraction)
A. H. ROBBINS* AND C. D. STOUTtDepartment of Molecular Biology,
Research Institute of Scripps Clinic, 10666 North Torrey Pines
Road, La Jolla, CA 92037
Communicated by Helmut Beinert, January 24, 1989 (received for
review October 12, 1988)
ABSTRACT The structure of activated pig heart
aconitase[citrate(isocitrate) hydro-lyase, EC 4.2.1.3] containing a
[4Fe-4S] cluster has been refined at 2.5-A resolution to a
crystallo-graphic residual of 18.2%. Comparison of this structure
to therecently determined 2.1-A resolution structure of the
inactiveenzyme containing a [3Fe-4S] cluster, by difference
Fourieranalysis, shows that upon activation iron is inserted into
thestructure isomorphously. The common atoms of the [3Fe-4S]and
[4Fe-4S] cores agree within 0.1 A; the three commoncysteinyl S.,
ligand atoms agree within 0.25 A. The fourthligand of the Fe
inserted into the [3Fe-4S] cluster is a water orhydroxyl from
solvent, consistent with the absence of a freecysteine ligand in
the enzyme active site cleft and the isomor-phism of the two
structures. A water molecule occupies asimilar site in the crystal
structure of the inactive enzyme.
The stereospecific dehydration/rehydration reaction cata-lyzed
by aconitase [citrate(isocitrate) hydro-lyase, EC4.2.1.3] requires
iron (1-3), which is present in the enzyme asan iron-sulfur cluster
(4, 5). In the inactive, aerobicallyisolated beef heart enzyme this
cluster is [3Fe-4S]; the clusterhas cubane-like geometry (6-8).
Upon activation of theenzyme with Fe2+ under reducing conditions, a
[4Fe-4S]cluster is formed (6-12). The fourth Fe added to form
the[4Fe-4S] cluster is directly involved in coordinating to
sub-strates (13-16). Mossbauer (13, 14) and electron nucleardouble
resonance spectroscopy in conjunction with 170 and13C labeling
experiments (15, 16) show that the fourth Fe (Feasite, refs. 13 and
14) coordinates one carboxyl of substrate(citrate, cis-aconitate,
or isocitrate) (16). When nitroisocit-rate is complexed to reduced
activated aconitase both thehydroxyl group and H70 (x = 1 or 2) are
bound simultane-ously (15). Kinetic experiments ofaconitase
turnover in 3H20suggest that the enzyme traps protons or water from
thesolvent (17); the proton abstracted from substrate is con-served
in product by the enzyme (2). Literature leading to thecurrent
mechanistic understanding of the aconitase reactionhas been
reviewed (18).
Interconversion of a [3Fe-4S] to [4Fe-4S] cluster has
beenobserved in Desulfovibrio gigas ferredoxin (19). In
addition,the [3Fe-4S] cluster in this protein can incorporate Co2+
orZn2+ into the fourth site of the cluster (20, 21). The geometryof
the [3Fe-4S] cluster in the 1.9-A resolution structure
ofAzotobacter vinelandii 7Fe ferredoxin is very similar to thatof
[4Fe-4S] cubanes (22-24), consistent with extended x-rayabsorption
fine structure results for aconitase (6) and D.gigas ferredoxin
(25).
Recently, the structure of inactive [3Fe-4S] aconitase frompig
heart has been solved and refined at 2.1-A resolution(unpublished
data). In this paper we report the refined struc-ture of the enzyme
at 2.5-A resolution following activation in
the crystal. Comparison ofthe [3Fe-4S] and [4Fe-4S]
aconitasestructures, which are isomorphous, provides
crystallographicevidence for a Fe-S cluster interconversion.
Previously, it wasshown from analysis of the anomalous difference
Pattersonmap for inactive aconitase that the Fe-Fe distances are
3.0 times or(I) (Table 2).
*Present address: Miles Research Division, West Haven, CT
06516.tTo whom reprint requests should be addressed.
3639
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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3640 Biophysics: Robbins and Stout
Table 1. Data collection and scalingNo. of No. of
No. of total independentData set crystals reflections
reflections Resolution, A Rsymm(l)* Rdiff(F)tInactive 3 350,360
47,583 2.1 0.106Activated 2 283,187 28,269 2.5 0.134 0.124
*R = Zh>li~i - II/hZhaI, where IT is the mean intensity of
the i observations of reflection h.tR = 1hj1Fph1 - 1FpII/jIFp1,
where lFphl and IFpI are the activated and inactive structure
amplitudes, respectively.
Refinement calculations were done using the programX-PLOR (28).
The structure of inactive aconitase was takenfrom the 2.1-A
refinement (R factor 0.209, 43,288 indepen-dent reflections 2 O0O.F
in the resolution range 5.0-2.1 A,6171 atoms including 322 water
molecules with B c 60 A2,and rms deviation from ideality of 0.026 A
for bonds). Themodel was modified with the program Frodo (29). The
aminoacid sequence of 755 residues of the pig heart enzyme usedin
refinement was derived from the DNA (H. Zalkin,
personalcommunication) and the protein (W. E. Brown,
personalcommunication). It should be noted that theN terminus
oftheprotein has not been identified from the DNA or
proteinsequence or in the electron density. Residue 1 in both
crystalstructures is the first residue for which there is
identifiabledensity. Coordinates of both the activated and
inactivestructures have been deposited with the Protein Data BankA
complete description of the inactive structure determina-tion will
be presented elsewhere.
RESULTS
A difference Fourier map calculated at 2.5-A resolution
withcoefficients [IFI(activated) - IFI(inactive)] and phases from
therefined, inactive aconitase structure revealed a single,
posi-tive electron density peak. This peak is shown in Fig. la
inrelation to the electron density and model for the
[3Fe-4S]cluster in the inactive protein structure. Because this
peak is2.7 A from each of the Fe atoms of the [3Fe-4S] cluster,
thenew cluster was modeled as [4Fe-4S] (Fig. lb); however, noligand
was modeled at the fourth coordination site of thecluster.
Refinement of the inactive protein structure coordi-nates against
the 2.5-A activated data reduced the R factorfrom 0.29 to 0.23.
Refinement of individual temperaturefactors further reduced R to
0.20.
Phases calculated from the partially refined activatedstructure
were used to compute an electron density map withthe activated
data. The Fourier coefficients, 21FOI - IFcI, wereweighted by the
method of Sim (30). This map confirmed the[4Fe-4S] model for the
activated cluster and also showed adistinct lobe of electron
density on the new Fe site (Fe4) (Fig.lc). The position of this
density is within 1 A of a watermolecule placed in the 2.1-A
refinement of the inactivestructure. This water molecule (W806) was
not moved from itsposition in the inactive structure; a bound SO2-
ion presentin both structures and additional water molecules
observed inthe activated structure electron density map were also
addedto the model. The model was subjected to additional
refine-ment against the 2.5-A activated data. The refined
structureincluding 226 waters and the SO2- with isotropic
temperaturefactors on all 6076 atoms has an R factor of 0.182 for
all data.O.OcF in the resolution range 5.0-2.5 A (Table 3). The
rmsdeviations from ideality of bonds and angles are 0.027 A
and4.360, respectively; 184 water molecules have B < 40 A2
andthe remaining 42 have B 55 A2; all waters have at least
onehydrogen bond to the protein and were refined with
unitoccupancy. Sections of the final 2.5-A resolution 21FOl -
IFJIelectron density map are shown in Fig. 2 a-f.
tBrookhaven Protein Data Bank (Brookhaven Natl. Lab., Upton,NY),
File 2ACN.
The position of the new, fourth Fe site in the
activatedstructure of aconitase was confirmed with a Bijvoet
differ-ence Fourier map (31). The anomalous scattering data fromthe
activated crystals to 2.5 A was combined with phasesfrom the
inactive structure to give an independent image ofthe Fe structure
(Fig. ld). Because there is no Fe at the Fe4site in the inactive
structure, this map independently con-firms the presence of a
[4Fe-4S] cluster in activated aconi-tase.The activated enzyme
structure is remarkably isomorphous
to the inactive structure. The Ca structure of inactive
aconi-tase and the [3Fe-4S] cluster is shown in Fig. 3a in a view
intothe enzyme cleft. A description of the protein structure will
bepresented elsewhere. The 755 common pairs of Ca atoms ininactive
and activated aconitase differ on average by 0.19 A inthe two
structures (maximum deviation, 0.47 A). Becausethese small
differences are randomly distributed throughoutthe molecule, and
because the unit cell parameters of the twocrystal forms are the
same, it is likely that the observeddifferences in the structures
represent no more than randomerrors in the coordinates at 2.5- to
2.1-A resolution.The isomorphism of activated and inactive
aconitase also
pertains to the side chains when the structures are examinedin
three dimensions. Fig. 3b shows the N, Ca, C, and sidechain atoms
of 14 active site residues in the two models. Bothstructures also
have in common a bound SO2- and a numberof ordered solvent
molecules modeled as waters. Four watermolecules adjacent to the
Fe-S cluster in the cleft in theactivated structure (W789, W806,
W807, W963) are observed atcommon sites in the inactive structure
(Fig. 3 c and d); theinactive structure contains a fifth water
(W892) in the imme-diate vicinity of the active site. B factors for
these watermolecules range from 10 to 25 A2 and 15 to 26 A2 in
theinactive and activated models, respectively. The positions ofthe
four common pairs of waters differ by 0.95 A, 0.06 A, 0.48A, and
0.55 A, respectively, at W806, W807, W789, and W963.The shift of
W806 is toward the Fe4 site from its startingposition in the
inactive structure and into the density asso-ciated with Fe4 (Fig.
lc). W806 was not constrained to bebonded to Fe4 in the refinement;
the resulting Fe-O distanceis 1.6 A. The 0.95-A shift of W806 is
significant in that thelargest shift at any Ca position is 0.47
A.Table 4 summarizes the bond distances and angles for the
[3Fe-4S] and [4Fe-4S] clusters in the two structures. B
factorsare 14-23 A2 for [3Fe-4S] and 13-18 A2 for [4Fe-4S].
The[3Fe-4S] moieties in the two structures appear to be indis-
Table 2. Activated aconitase dataNo. of independent
Resolution reflectionsrange, A Observed Possible % .20.0F
Average J/lo(I)
oo-4.47 5739 5783 99.2 18.24.47-3.55 5557 5565 99.8
14.63.55-3.10 5437 5481 99.2 8.93.10-2.82 4926 5469 90.1
6.32.82-2.62 4076 5449 74.8 3.92.62-2.46 2534 5433 46.6 3.2
28,269 33,180 85.2 12.7
Proc. Natl. Acad. Sci. USA 86 (1989)
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Proc. Natl. Acad. Sci. USA 86 (1989) 3641
Table 3. R factor for the activated aconitase structureNo.
of
reflections*
Resolution range,
A2.45-2.552.55-2.662.66-2.802.80-2.972.97-3.203.20-3.523.52-4.014.01-5.00
TotalF.bW-amplitude range
0.02-8.448.44-16.8616.86-25.2825.28-33.7033.70-42.1342.13-50.5550.55-58.9758.97-67.39
Total
6082,3282,6663,0893,4%3,6%3,7823,817
23,482
10,1639,0073,2368202063974
23,482
R factort
0.30370.28330.26590.21860.20140.17200.14730.14060.1820
0.34670.17260.11670.08680.06140.06990.08230.04110.1820
*Includes all observed data .O.OcF.tR = >IhlFol -
IFCLI/ZIFoI, where IFOI and IFcl are observed andcalculated
structure amplitudes, respectively.
three cysteine Sy ligand atoms differ by 0.25 A. By compar-ison,
the [3Fe-4S] clusters of inactive aconitase (2.1-Arefinement) and
7Fe ferredoxin (1.9-A refinement; ref. 24)agree within 0.09 A; in
this case the Sy atoms agree within0.25 A. The similarity of
[3Fe-4S] and [4Fe-4S] core geometryas observed in aconitase is also
observed in 7Fe ferredoxin;in this protein the seven common atoms
of the two differentclusters agree within 0.08 A. Variations in
average S-Fe-S vs.S-Fe-Sy bond angles (Table 4) are also similar to
thoseobserved in other Fe-S clusters (32).
FIG. 1. Stereo figures of electron density maps of activated
andinactive aconitase. (a) Superposition ofthe 2.5-A resolution
differenceFourier map with coefficients (IF(activated)I -
IF(inactive)I) showing thefourth Fe site (heavy lines) and the
2.1-A resolution (21FOI - IFJI)Fourier map of the inactive protein
(thin lines). The model of the[3Fe-4S](Sy)3 cluster in the inactive
protein is shown. Phases for bothmaps were calculated from the
2.1-A refinement of the inactivestructure; each map is contoured at
0.45 of the maximum density. (b)Difference Fourier map as in a
superposed on the model for [4Fe-4S]cluster in the activated
structure. Also shown are the three cysteineSy atoms attached to
the cluster and a water molecule (W8%) refinedadjacent to the
fourth Fe site (Fe4). In this view W806 is at the tip ofthe vector
attached to Fe4-i.e., above the cluster and differenceelectron
density peak. Additional crosses are atoms within a s.o-Asphere of
the center of the cluster. (c) Model for the [4Fe-4S] clusterwith
three cysteine Sy ligands and a water ligand (WN6, above clusterin
this view as in b superposed on the 2.5-A (21FO1 - IFcI)
electrondensity map of the activated protein. The map is contoured
at 0.27and 0.45 the maximum density. The cysteine ligands are
residues 359(left), 422 (right), and 425 (behind cluster in this
view). (d) Bijvoetdifference Fourier map using activated aconitase
data to 2.5-Aresolution with coefficients (IF+ - IF-I) and phases
from therefined inactive protein structure. The fourth Fe site is
at the top ofthe cluster in this view. Only the four Fe atoms are
shown (averageseparation, 2.62 A). The map is contoured at 0.45 of
the maximumdensity.
tinguishable, consistent with the overall isomorphism of
theprotein structures. The seven common atoms of the Fe-Score
structures differ on average by 0.11 A in position; the
Table 4. Iron-sulfur geometry[4Fe-4S] cluster [3Fe-4S]
cluster*
Fe...Fe distances6 Number 3
2.51-2.69 A Range 2.64-2.73 A2.62 Average 2.69
Fe...S distances12 Number 9
2.22-2.37 A Range 2.25-2.35 A2.30 Average 2.30
Fe...Sy distances3 Number 3
2.33-2.38 A Range 2.28-2.34 A2.36 Average 2.32
S...Fe...S angles12 Number 9
103-1130 Range 101-11301070 Average 1070
Sy...Fe...S angles9 Number 9
98-1210 Range 94-12301120 Average 1120
Fe...S ...Fe angles12 Number 6
66-730 Range 70-720690 Average 710
Restraints used in refinement: Fe...Fe 2.75 A; Fe ...S, Sy 2.31
A;S...Fe...S, Sy 109.40; Fe...S...Fe, 750*Coordinates taken from
inactive structure refinement.
a
b **
*
c
*
d
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Proc. Natl. Acad. Sci. USA 86 (1989) 3643
a c[3Fe-4S] Aconitase
b d[4Fe-4S) Aconitase
[3Fe-4S] Aconitase
(4Fe-4S] Aconitase
FIG. 3. Active site residues and Fe-S cluster in aconitase. (a)
Ca structure of inactive aconitase and the [3Fe-4S](Sy)3 cluster
viewed towardthe active site cleft. The three N-terminal domains
are arranged around the Fe-S cluster in a threefold manner; the
C-terminal domain is abovethe cluster in this view. (b)
Superposition of 14 residues of the activated and inactive protein
structures showing N, Ca, C, and side chain atomsand the
[3Fe-4S](Sy)3 cluster. These residues surround a cavity at the base
of a cleft leading from the surface of the protein to the Fe-S
cluster.(c) The side chains of eight active site residues in the
immediate vicinity of the [3Fe-4S](Sy)3 cluster, the bound SOJ-
ion, and five watermolecules from the 2.1-A resolution refinement.
The unlabeled side chains are H14 and S167. The SOJ- is hydrogen
bonded to Q73 and also R581,Sw, and R645 (not shown, see b). (d)
The amino acid side chains as in c, the [4Fe-4S](Sy)3 cluster, and
four water molecules in the active sitein common with the inactive
structure (c), which refined in the activated structure. A fifth
water, W892, refined to B > 55 A2 and is not included.One of the
common waters, W806 (no label), refined to a position 1.6 A from
Fe4 of the [4Fe-4S] cluster and is shown bonded to Fe4. The
sidechains of H148 and S167 are unlabeled.
sequence data prior to publication, and M. H. Emptage, J.
B.Howard, and D. Case for discussions. This work was supported
byNational Institutes of Health Grant GM-36325.
1. Villafranca, J. J. & Mildvan, A. S. (1971) J. Biol. Chem.
246, 772-779.2. Rose, I. A. & O'Connell, E. L. (1967) J. Biol.
Chem. 242, 1870-1879.3. Ruzicka, F. J. & Beinert, H. (1974)
Biochem. Biophys. Res. Commun.
58, 556-563.4. Kennedy, C., Rauner, R. & Gawron, 0. (1972)
Biochem. Biophys. Res.
Commun. 47, 740-745.5. Ruzicka, F. J. & Beinert, H. (1978)
J. Biol. Chem. 253, 2514-2517.6. Beinert, H., Emptage, M. H.,
Dreyer, J.-L., Scott, R. A., Hahn, J. E.,
Hodgson, K. 0. & Thomson, A. J. (1983) Proc. Nat!. Acad.
Sci. USA 80,393-3%.
7. Ryden, L., Ofverstedt, L.-G., Beinert, H., Emptage, M. H.
& Kennedy,M. C. (1984) J. Biol. Chem. 259, 3141-3144.
8. Beinert, H. & Thomson, A. J. (1983) Arch. Biochem.
Biophys. 222, 333-361.
9. Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H.,
Emptage,M. H., Beinert, H. & Munck, E. (1982) Proc. Nat!. Acad.
Sci. USA 79,1096-1100.
10. Kennedy, M. C., Emptage, M. H., Dreyer, J.-L. & Beinert,
H. (1983) J.Biol. Chem. 258, 11098-11105.
11. Emptage, M. H., Dreyer, J.-L., Kennedy, M. C. & Beinert,
H. (1983) J.Biol. Chem. 258, 11106-11111.
12. Kennedy, M. C., Emptage, M. H. & Beinert, H. (1984) J.
Biol. Chem.259, 3145-3151.
13. Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H.
& Munck,E. (1983) Proc. Nat!. Acad. Sci. USA 80, 4674-4678.
14. Kent, T. A., Emptage, M. H., Merkle, H., Kennedy, M. C.,
Beinert, H.& Munck, E. (1985) J. Biol. Chem. 260,
6871-6881.
15. Telser, J., Emptage, M. H., Merkle, H., Kennedy, M. C.,
Beinert, H. &Hoffman, B. M. (1986) J. Biol. Chem. 261,
4840-4846.
16. Kennedy, M. C., Werst, M., Telser, J., Emptage, M. H.,
Beinert, H. &Hoffman, B. M. (1987) Proc. Nat!. Acad. Sci. USA
84, 8854-8858.
17. Kuo, D. J. & Rose, 1. A. (1987) Biochemistry 26,
7589-75%.18. Emptage, M. H. (1988) ACS Symp. Ser. 372, 343-371.19.
Moura, J. J. G., Moura, I., Kent, T. A., Lipscomb, J. D., Huynh, B.
H.,
LeGall, J., Xavier, A. V. & Munck, E. (1982) J. Biol. Chem.
257, 6259-6267.
20. Moura, I., Moura, J. J. G., Munck, E., Papaefthymiou, V.
& LeGall, J.(1986) J. Am. Chem. Soc. 108, 349-351.
21. Surerus, K. K., Munck, E., Moura, I., Moura, J. J. G. &
LeGall, J.(1987) J. Am. Chem. Soc. 109, 3805-3807.
22. Stout, G. H., Turley, S., Sieker, L. C. & Jensen, L. H.
(1988) Proc.Nat!. Acad. Sci. USA 85, 1020-1022.
23. Stout, C. D. (1988) J. Biol. Chem. 263, 9256-9260.24. Stout,
C. D. (1989) J. Mol. Biol. 205, 545-555.25. Antonio, M. R.,
Averill, B. A., Moura, I., Moura, J. J. G., Orme-
Johnson, W. H., Teo, B.-K. & Xavier, A. V. (1982) J. Biol.
Chem. 257,6646-6649.
26. Robbins, A. H. & Stout, C. D. (1985) J. Biol Chem. 260,
2328-2333.27. Howard, A. J., Nielsen, C. & Xuong, N. H. (1985)
Methods Enzymol.
114, 452-472.28. Brunger, A. T., Kuriyan, J. & Karplus, M.
(1987) Science 235, 458-460.29. Jones, T. A. (1978) J. Appl.
Crystallogr. 11, 268-272.30. Sim, G. A. (1960) Acta Crystallogr.
13, 511-512.31. Strahs, G. & Kraut, J. (1968) J. Mol. Biol. 35,
503-512.32. Carter, C. W. (1977) in Iron-Sulfur Proteins, ed.
Lovenberg, W. (Aca-
demic, New York), Vol. 3, pp. 157-204.33. Plank, D. W. &
Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189.34. Kennedy, M.
C. & Beinert, H. (1988) J. Biol. Chem. 263, 8194-8198.35.
Schloss, J. V., Emptage, M. H. & Cleland, W. W. (1984)
Biochemistry
23, 4572-4580.36. Kennedy, M. C., Kent, T. A., Emptage, M.,
Merkle, H., Beinert, H. &
Munck, E. (1984) J. Biol. Chem. 259, 14463-14471.37. Ramsay, R.
R. & Singer, T. P. (1984) Biochem. J. 221, 489- 497.
Biophysics: Robbins and Stout
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