Iron-Sulphur Clusters With Labile Metal Ions Andrew J. Thomson, Jacques Breton, Julea N. Butt, E. Claude Hatchikian, and Fraser A. Armstrong AJT, JB. School o f Chemical Sciences, University o f East Anglia, Norwich United Kingdom.--JNB, FAA. Department o f Chemistry, University o f California at Irvine, Irvine, California, U.S.A.--ECH. CNRS-LCB, Chemin Joseph-Aiguier, Marseille, France
ABSTRACT A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd HI from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [3Fe-4S] I + ~ [3Fe-4S] °
~rM(u) [M3Fe-4S] 2 + ~ [M3Fe - 4S] l+ The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]° cluster for divalent ions lie in the sequence Cd > Zn ~ Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S] I + as well as for [3Fe-4S]°. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-/~2-sniphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least One additional ligand which may be from solvent (H20 or O H - ) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn0I), that may trigger conformational change.
Address reprint requests to: Professor A. J. Thomson, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
Journalof InorganicBiochemistry, 47, 197-207 (1992)
197 © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$5.00
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INTRODUCTION .Iron-sulphur clusters bound to proteins have a role, long recognized as electron-storage and reversible-electron transfer centers [see Ref. 1 for review]. It is now clear that the [4Fe4Sl cubane core cluster also can function at the catalytic center of dehydratase enzymes such as aconitase [see Ref. 21. Evidence is now emerging of a possible third role, namely, control of protein biosynthesis at the level of mRNA [see Refs. 3, 4, and Kiihn and Hentze in this issue]. In this review we focus upon the cubane cluster [4Fe4S] and the three-iron cluster [3Fe4S] which undergo a set of interconversion and other redox linked processes involving loss or uptake of metal ions or protons. The cubane cluster [4Fe4S], has a remarkably wide range of chemistry [see, Ref. 51. It can adopt, in a protein environment, at least three oxidation states [4Fe4S]i+/2+/3+, ahh ough for a given protein only the single electron redox processes 1 + /2 + or 2 + /3 + are permitted. In this role the cubane core is liganded by four cysteinyl ligands so that each iron atom is tetrahedrally coordinated. However, [4Fe4S]‘+ in aconitase [see Ref. 61 catalyzes the non-&ox reaction of citrate-isocitrate interconversion which entails sequential dehydratase-hydratase reactions [see Ref. 71. The active form of aconitase contains the [4Fe-4S12+ cluster liganded by three thiolate groups of cysteine. One iron site coordinates substrate and H,O or OH-. Aconitase is apparently a member of a growing family of dehydratase enzyme which utilize a [4Fe-4S] 2+ cluster in non-redox catalysis. The list includes L-serine dehydratase ( Peptostreptococcus asaccharalyticus) [see Ref 81, 6-phosphogluconate dehydratase (Zymonas mobilis) [see Ref. 91, tartrate dehydratase [see Ref. lo], and maleic acid hydratase [see Ref. 111. One curious feature of the chemistry of the [4Fe4S] cluster in some proteins is that on oxidation one iron atom is lost to generate the [3Fe4S]‘+ core [see Ref. 121. Conversely, on reduction of the latter core to [3Fe-4Sl” the divalent ion Fe(B) is taken up to restore the cubane framework [see Ref. 131. Redox linked metal-ion uptake and loss processes have been identified in a number of bacterial ferredoxins (Fd) such as Fd (Ciostridium pasteurianum) [see Ref. 121, Fd II (Desulphovibrio gigas) [see Ref. 141, Fd III (Desufphovibrio africanus) [see Ref. 151, and Fd (Pyrococcus furiosus) [see Ref. 161. It has been shown that metal ions besides iron can be taken up by the vacant corner position of the [3Fe-M cluster]. These include cobalt(II) [see Ref. 171, zinc@) [see Ref. 181, nickel(B) [see Ref. 191, and cadmium(II) [see Ref. 201. Iron loss also occurs from the active site [4Fe4S] cluster of aconitase under oxidizing conditions [see Ref. 21, and this leads to loss of enzymic activity. Under reducing conditions in the presence of Fe(B), activity is restored as the cluster is reconstituted. A similar process appears to be taking place in L-serine dehydratase (Peptostreptococcus asaccharolyticus) [see Ref. 81. The capability is thus afforded for corresponding in vivo metabolic activities to be modulated by the local Fe@) concentration and electrochemical potential. This role for Fe(B) in controlling the activity of an enzyme used in energy generation in mitochondria and in certain bacteria has been given added significance by reports of the sequence of an iron-regulated mRNA-binding protein, the so-called iron-responsive element binding protein (IRE-BP) [see Refs. 3,4]. The sequence of this protein shows regions of high homology with that of mitochondrial aconitase, especially in the vicinity of the iron-sulphur cluster binding domain. This has led to the proposal that the IRE-BP is
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an iron-sulphur protein containing a [3Fe4S] cluster which will bind Fe(n) under reducing conditions [see Refs. 3-41. Cellular iron metabolism is controlled by regulation of ferritin and transferrin receptor synthesis at the level of mRNA [Ref. 211. This is effected by the IRE-BP which binds to similar sequences (IREs) that exist within the 5’ untranslated region of ferritin mRNA and the 3’ untranslated region of the transferrin receptor mRNA. The IREs from both mRNAs interact with a cytoplasmic protein the IRE-BP, whose binding affinity is dependent upon the iron status of the cell. The clear implication is that [4Fe4S] * [3Fe4S] transformation is involved in control of IRE-BP binding to the IRE and hence governs protein expression. The rich redox and metal ion uptake chemistry exhibited by the [4Fe4S] cluster in proteins has not yet been fully modeled with synthetic analogues of these clusters [see Ref. 51. Our approach is to explore this chemistry through studies of appropriate low-molecular weight ferredoxins that provide a tailor-made site for metal ion and ligand binding. We have developed a sensitive, electrochemically-based strategy that enables us to control and monitor these redox-linked processes, to resolve convoluted reactivities, and to isolate samples for spectroscopic characterization [see Ref. 221. The bacterial ferredoxins we have used to study these processes are ferredoxin III, Desuiphovibrio crfticanus (Da) [see Ref. 231 and ferredoxin I, Azotobacter chroococcum (AC) [see Ref. 241. Both proteins contain, as aerobically isolated, one [3Fe-4S] and one [4Fe4S] cluster. Fd III, Da, has a molecular weight of 6000 Da, and a known sequence showing only seven cysteine residues. Between the cysteine residues 11 and 17 lies an aspartate group, at position 14, instead of cysteine as would be found in a typical 2[4Fe4S] Fd. The [3Fe-4S]’ cluster incorporates Fe@) rapidly to form a [4Fe4S]*+ cluster which can be reduced further to generate a [4Fe4S]‘+ solution with unusual magnetic properties [see Ref. 151. Spectroscopic and electrochemical evidence shows that A. chroococuum Fd I is similar if not identical to that of Azotobacter vinelandii Fd I [see Ref. 251 whose structure has recently been redetermined [see Refs. 6, 261. This Fd contains nine cysteines. The ligation of the cysteine thiol groups to the two clusters is compared in Figure 1 with that of the typical 8Fe ferredoxin from Clostridium pasteurianum [see Ref. 271. The ligation of one of the [4Fe-4S] clusters is highly conserved in all three proteins as judged by sequence data. The x-ray structures of Av Fd I and Pa Fd also show that the cluster binding regions is similar. However, variation in cysteine spacing within the first 16 or 17 residues and the replacement of one of the cysteine residues by an aspartic acid residue leads to marked changes in the reactivity of the second cluster. 8Fe Fd from Ciostridium pasteurianum does not readily lose iron from one of its clusters except under extreme oxidizing conditions such as treatment with hexacyanoferrate ion [see Ref. 121. Here we describe combined spectroscopic and electrochemical studies of two bacterial Fds both with [3Fe4S][4Fe4S] core structures, one of which undergoes facile and reversible redox coupled metal ion uptake, Da Fd III, and the other Av Fd I, which is resistant to metal-ion uptake. EXPJIRIMENTAL
The EPR diagnostic including illustrates
METHODS and MCD spectra under conditions of low-temperature provide excellent signatures of the cluster type and of the ground state magnetic pammeters, electron spin, g-values, and zero-field splitting parameters. Figure 2 the EPR, W-visible absorption, and MCD spectra of the oxidized form of
200 A. J. Thomson et al.
8 (N)
11
14
CYS-X-X-CYS-X-X-CYS
PRO-C%
0
18
. . . . . . . . . . . . . . . . . . . . . .CYS-PRO
. . . . . , . . . . . . . . . . .CYS-x-x-CYS-x-x-CYS 41
45
38
35
1 8 (N)
11
16
20
24
CYS-X-X-CYS-X-X-X-X-CYS
. . . . . . . . . . . . . . . . . .CYS-PRO . . . . . . CYS
3Fc
4Fe
/I\
PRO-CYS . . . . . . . . . . . . . , .CYS-x-x-CYS-x-x-CYS
(‘3
45
49
11
14
39
21
17
(N) CYS-X-X-ASP-X-X-CYS
42
. . . . . . . . . . . . . . . . . CYS-PRO
4Fe
3Fe
/I\
(C) OLN-GLU-CYS . . . . . . . , . . . . . . . .CYS-x-x-CYS-x-x-CYS 51
47
44
41
FIGURE
1. The sequences of the 2[Fe_4S] Fd, Closfridium Pusteurianum, the [3Fe4S][4Fe4S] Fd, Azotobacter vinelandii, and the [3Fe_4S][4Fe_4S]Fd III, Desulphovibrio qfricanus, showingthe ligation mode of the clusters.
Da Fd III measured at liquid helium temperature. In this state the four-iron cluster is in the diamagnetic oxidation level [4Fe_QS]*+. The MCD spectra of paramagnetic species increase in intensity as the temperature is lowered whereas the intensity of diamagnetic species are independent of temperature and hence weak. The [3Fe_4S]‘+ core has a spin S = l/2 which gives rise to a characteristic EPR signal at g = 2.01. The lower panel shows the absorption spectra measured at 1.5 K and also the MCD spectra at several temperatures between 1.57 and 22 K and at 5 Tesla magnetic field.
IRON-SULPHUR CLUSTERS WITH LABILE METAL IONS
201
g-v0 I UQ 2. 15
I '
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2. 05
2. DO
1.
1.95
1. 80
1.85
90
1
Oesulphovibrio africanus Fd El, native 300
AEIMhi 200
-
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K
.._..... 4.215 ,( _........._ 11.95K
---
V
2.01 K
-._._22.0K -300 L,
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I
I
300
400
500
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,
6;O A;nm 7;O
900
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FIGURE
2. The EPR spectrum (upper), absorption spectrum (lower, A) and MCD spectrum at 5 Tesla (lower) of Fd III, Lksufphovibrio clfricunus in the oxidized state. The EPR and MCD signals arise only from the [3Fe4]‘+ cluster.
The detailed assignment of this spectrum has not been achieved but similar spectra have been recorded from [3Fe4S] cores in a variety of different protein environments [see Ref. 281. It can be difficult to manipulate proteins under conditions of low potential and well-defined metal ion concentration. Cluster transformations are more readiiy identified and studied by a voltammetric method that, in practice uses a nanomole of protein for each experiment but permits equilibrium constants and kinetic parameters to be measured [see Ref. 231. The discovery arises from the observation that Fds co-absorb with amino sugars at a pyrolytic graphite edge (PGE) electrode giving a stable electroactive fihn, provided that a promoter molecule of amino-sugar is present in solution [see Ref. 131. The FGE surface is rich in acidic oxides which interact with proteins bearing negatively-charged surface regions in the presence of poly-cations such as the naturally occurring amino-sugar, neomycin. Fast, direct
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electron transfer takes place between the protein and the electrode surface allowing cyclic voltammograms to be recorded. The preparation of electroactive films has been described [see Ref. 201. RESULTS AND DISCUSSION Fd III, D. africanus The direct, unmediated cyclic voltammogram (CV) of an adsorbed film of Da Fd IIl in the presence of the chelator EGTA at pH 7 shows three well-defined waves due to three redox couples (Fig. 3). One of these corresponds to the stable [4Fe-4S]‘+/‘+ cluster and the other two are associated with the [3Fe4S] cluster and are assigned respectively to the normal 1 + /O couple and a chemically-reversible two-electron process of uncertain identity. By comparative integration and from the effect of pH upon the wave-position the latter appears to correspond to a three-proton uptake. If the coated electrode is transferred to a stirred solution devoid of EGTA but containing a low concentration of Fe(B), reductive passage through the couple [3Fe-4S]‘+” initiates rapid changes. Subsequent cycles over the course of several seconds causes the waves due to [3Fe4S] I+” to disappear together and be replaced by a new couple corresponding to the newly formed cluster [4Fe4S]*+/‘+. The position of the new couple corresponds closely with CV’s of Fd III undergoing transformation in the solution phase. The height of each CV peak is proportional to the concentration of the redox active species present. Hence a study of these peaks as a function of Fe(B) concentration enables cluster-metal dissociation constants to be determined [see Ref. 201. The redox linked uptake of a divalent metal ion is not restricted to Fe@). Zn(II) and Cd(B) also bind strongly and the affinity order Cd*+> Zn*% Fe*+ has been established [see Ref. 201. This is the order expected for a sulphide rich site [see Ref.
I
5CLA
I
I
-800
I
I
-600
I
1
-400
I
I
-200
1
I
1
0
E/mV vs. SHE FIGURE 3. Steady-state cylclic voltatnmogram of a film of 7Fe Fd, Desulphovibrio qfricanus III. The redox couple corresponding to each wave is indicated.
IRON-SULPHUR CLUSTERS WITH LABILE METAL IONS
203
291. It has also been shown that Tl(I) has a high afIinity for the reduced [3Fe4S]’ site but also shows a measurable atlinity for the oxidized state of the cluster [3Fe4S]‘+ [see Ref. 301. The [3Fe4S]’ cluster is paramagnetic with a spin S = 2 subject to a negative, predominantly axial zero-field splitting [see Ref. 121. The [4Fe4S]‘+ cluster is diamagnetic at low temperatures. On reduction of this cluster by one electron the resulting state [4Fe4S]’ + has an unusual spin-state S = 3/2 subject to an axial zero-field splitting. This state has g values of 5.27, (2.34), (1.62). The MCD spectra are changed little in form by this spin-state change from S = l/2. The coordination geometry of the newly formed cluster is not ‘yet established. Three of the ligands are cysteine whereas the labile iron may be ligated either by the carboxylate group of asp 11, or by H,O or OH -. There is no pH dependence to the redox potential over the range 6-8. FdI, A. chrwococcum By means of direct electrochemistry at a PGE electrode in the presence of the promoter neomycin the redox potentials of the two iron-sulphur clusters in Fd I( AC) were measured [see Ref. 311 and the magnetic properties were determined (see Ref. 24). The relevant equilibria are [ 3Fe4S] i + + e + [ 3Fe4S] ’
E,,,=
[3Fe4S]‘+H++[3Fe4S]‘*-*H+
-46Ok
1OmV
pK,=7.8
and [4Fe4S]2++e-+[4Fe4S]1+
E,,2=
-645klOmVatpH8.3.
The pH dependence of the three-iron cluster corresponding to the uptake of a proton in the [3Fe4S]’ state has been discovered only in Azotobucter Fd. A further reduction of the three-iron cluster can be observed at much lower potentials. This process has been detected electrochemically in all [3Fe-4S] cluster-containing ferredoxins so far examined The [3Fe4S]’ cluster in Fd I, AC, is peculiarly resistant to take up divalent metal ions when in a purified state. Two reports have appeared of the formation of a [4Fe4S] core. In one case the tertiary structure of the protein Fd I, Av, is disrupted by guanadium hydrochloride to generate a 2[4Fe4S] Fd [see Ref. 321. In the other case, spontaneous uptake of Fe(B) was reported for a sample at pH 8.3 in the presence of nucleic acid contamination [see Ref. 241. It has been proposed that binding between Fd I AC and DNA may take place and facilitate the three-to-four iron conversion [see Ref. 331. CONCLUSIONS The [3Fe4S] cluster is a redox dependent ligand for a variety of cations. The one-electron reduced state [3Fe4S]‘, has a higher affinity for divalent metal ions than the oxidixed state. The only example to date of metal ion binding to the [3Fe4S]+ cluster is Tl(I) in Da Fd III. However, the oxidized form of the four-iron
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cluster, [4Fe-4S13+, is stable in the redox proteins called high potential iron proteins (HiPiP). Since this core is a complex of Fe@) with [3Fe4S]’ + the protein fold and the presence of a fourth cysteine ligand has stabilized the oxidized four-iron core. The K, values determined for Da Fd III show that the order of stability of the clusters [M3Fe_4S]* accords with the Irving-Williams series for binding of high-spin M(II) ions to a sulphide rich ligand. We have discovered examples of both the [3Fe4S]* and [3Fe-4S]‘- states reacting with protons. The location of the bound protons is not established. However, it is likely that they become bound to the cluster itself possibly by protonating the bridging sulphide in the open cluster face. The possibility that H30+, a pyramidal cation, could cap the open corner of the [3Fe4S] by hydrogen-bonding to the three j+ulphide atoms is an intriguing one. The reactivity of the [3Fe-4S]* core towards cations is not surprising given the stereochemical disposition of the three sulphide ions. However, the noteworthy features are, first, the much reduced affinity of the [3Fe-4S]‘+ core towards cations, and second, the total lack of reactivity of both l+ and 0 oxidation states of the [3Fe4S] core in some proteins. The addition of a metal ion to the comer of the three-iron core to complete the cube gives (Fig. 4) an intermediate which has a metal ion with a coordination number of only three. This center will therefore have a high affinity for a further ligation. This could be either H,O or OH- from solvent or a protein side-chain ligand possibly cysteine (thiol) or aspartic acid (carboxylate). This poses a number of interesting questions. The first, how is it that some [3Fe4S] cores are unreactive in proteins even when reduced in the presence of excess Fe(U)? The open sulphide-face could be blocked by the close approach of other residues. The h-sulphide atoms may have a reduced metal ion affinity due to H-bonding. The H-bonds could either stabilize the triangular face or cause the sulphide ions to flex so that the triangular edges of the face are lengthened. The presence or otherwise of a fourth ligand from the protein which is correctly positioned to provide a tetrahedral coordination site for an incoming metal ion will also affect reactivity of the triangular sulphide face. Hence we must expect to encounter proteins with [3Fe4S] centers with a wide range of different stabilities and reactivities towards additional metal ions. The other important feature of the redox-linked metal ion uptake process is that it can be an engine to drive protein conformational change. For example, if the fourth ligand acquired by the added metal ion is a protein side-chain which must move into position this may trigger movement of the polypeptide backbone and hence conformational change. The [3Fe4S] core can be a redox-linked sensor of divalent metal ion concentrations within a cell that may lead to protein conformational change scheme. At high potentials the umeactive [3Fe4S]‘+ state is formed. As the potential drops the reduced state increases in concentration and provided that an appropriate divalent metal ion is available a cubane cluster will be formed. This may trigger conformational change. The potential which senses the internal potential of the cell is the [3Fe_4S]‘+‘* redox couple. The concentration range over which a divalent metal ion can be sensed is governed both by the potential of the resulting cluster and the affinity of the [3Fe4S]* core for the metal ion. The evidence from sequence data that the IRE-BP has high homology with mitochondrial aconitase suggests that a [3Fe4S] cluster may be present in this
206 A. J. Thomson et al.
anaerobic respiration by sensing oxygen levels. They may be activated by Fe@). It remains to be determined whether the [3Fe-4S] core cluster plays any role in these control processes. This work has been supported by Molecular Recognition Initiative of the S.E.R.C., by the University of California, by grants from N.A.T.O. (No. CRC 900302), by an EXXON Education Foundation Award (FAA), and by C.N.R.S., France.
REFERENCES 1. A. J. Thomson, in Metalioproteins, P. M. Harrison, Ed. Verlag Chemie, Weinheim, F.R.G., 1985, Part 1, pp. 79-120. 2. H. Beinert, FASEB J. 4, 2483 (1990). 3. T. A. Rouault, C. D. Stout, S. Kaptain, J. B. Harford, and R. D. Klausner, Cell 64, 881 (1991). 4. M. W. Hentze and P. Argos, Nucleic Acid Res. 19, 1739 (1991). 5. R. H. Hahn. S. Ciurli, and J. A. Weigel, Prog. Znorg. Chem. 38, 1, (1990). 6. D. J. Robbins and C. D. Stout, Proc. Natl. Acad. Sci. 86, 3639 (1989). 7. H. Beinert and M. C. Kennedy, Eur. J. Biochem. 186, (1989). 8. R. Grahowski and W. Buckel, Eur. J. Biochem. 199, 89 (1991). 9. R. K. Scopes and K. Griffiths-Smith, Anal. Biochem. 136, 530 (1984). 10. H. Rode and F. Giflham, J. Bacterioi. 151, 1602 (1982). 11. J.-L. Dreyer, Eur. J. Biochem., 150, 145 (1985). 12. A. J. Thomson, A. E. Robinson, M. K. Johnson, R. Cammack, K. K. Rao, and D. 0. Hall, Biochim. Biophys. Acta, 637, 423 (1981). 13. F. A. Armstrong, J. N. Butt, S. J. George, E. C. Hatehikian, and A. J. Thomson, FEBS Lett., 125, 15 (1989). 14. B. H. Huynh, J. J. G. Moura, I. Moura, T. A. Kent, J. L&all, A. V. Xavier, and E. Munck, J. Bioi. Chem. 255, 3242 (1980). 15. S. J. George, F. A. Armstrong, E. C. Hatchikian, and A. J. Thomson, Biochem. J., 264, 275 (1989). 16. R. C. Conover, A. T. Kowal, W. Fu, J.-B. Park, S. Aano, M. W. W. Adams, and M. K. Johnson, J. Biol. Chem. 265, 8533 (1990). 17. I. Moura, J. J. G. Moura, E. Miinck, V. Papaefthymou, and J. LeGall, J. Amer. Chem. Sot., 108, 349 (1986).
18. K. K. Surerus, E. Miinck, I. Moura, J. J. G. Moura, and J. LeGall, J. Amer. Chem. Sot., 109, 3805 (1987). 19. R. C. Conover, J.-B. Park, M. W. W. Adams, and M. K. Johnson, J. Amer. Chem. Sot., 112, 4562 (1990). 20. J. N. Butt, F. A. Armstrong, J. Breton, S. J. George, A. J. Thomson, and E. C. Hatchikian, J. Amer. Chem. Sot., 113, 6663 (1991). 21. E. C. Theil, J. Bioi. Chem., 265, 4771 (1990). 22. F. A. Armstrong, Structure and Bonding, 72, 137 (1990). 23. F. A. Armstrong, S. J. George, R. Cammack, E. C. Hatchikian, and A. J. Thomson, Biochem. J. 264, 265 (1989). 24. S. J. George, A. J. M. Richards, A. J. Thomson, and M. G. Yates, Biochem. J., 224, 247 (1984). 25. S. J. George, Ph.D. Thesis, University of East Anglia, 1986. 26. G. H. Stout, S. Turley, L. C. Sieker, and L. H. Jensen, Ptoc. Natl. Acad. Sci., 85, 1020 (1988).
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27. M. Tanaka,
28. 29. 30. 31. 32. 33. 34.
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T. Nakashima, A. M. Benson, H. F. Mower, and K. T. Yasunobu, Biochemistry, 5, 1666 (196f$. H. Beinert and A. J. Thomson, Arch. B&hem. Biophys., 222, 333 (1983). J. J. R. da Silva, and R. J. P. Williams, The Biological Chemistry of the Elements, OUP, Oxford, 1991. J. N. Butt, A. Sucheta, F. A. Armstrong, J. Breton, A. J. Thomson, and E. C. Hatchikian, .I. Amer. Chem. Sot., 113, 8948 (1991). F. A. Armstrong, S. J. George, A. J. Thomson, and M. G. Yates, FEBS Lett., 234, 107 (1988). T. V. Morgan, P. J. Stephens, B. K. Burgess, and C. D. Stout, FEBS Lett., 167, 137 (1984). A. J. Thomson, FEBS Lett., 285, 230 (1991). S. Spiro and J. R. Guest, TZBS, 16, 310 (1991).
Received January 27, 1992; accepted February 3, 1992
ABSTRACT A study has been carried out of the redox-linked metal ion uptake processes of the iron-sulphur cluster [3Fe-4S] in the bacterial ferredoxin, Fd HI from Desulphovibrio africanus using a combination of electron paramagnetic resonance (EPR) and low-temperature magnetic circular dichroism (MCD) spectroscopy and direct, unmediated electrochemistry of the Fd in a film deposited at a pyrolytic graphite electrode. Reduction of the three-iron cluster is required before a divalent metal ion becomes bound as in the reaction sequence [3Fe-4S] I + ~ [3Fe-4S] °
~rM(u) [M3Fe-4S] 2 + ~ [M3Fe - 4S] l+ The redox potentials of these processes and the metal binding constants have been determined. The affinities of the [3Fe-4S]° cluster for divalent ions lie in the sequence Cd > Zn ~ Fe. In addition, specific binding of a monovalent ion, Thallium(I), is detected for [3Fe-4S] I + as well as for [3Fe-4S]°. The results provide a clear and quantitative demonstration of the capability of the open triangular tri-/~2-sniphido face of a [3Fe-4S] cluster to bind a variety of metal ions if the protein environment permits. In each case the entering metal ion is coordinated by at least One additional ligand which may be from solvent (H20 or O H - ) or from a protein side chain (e.g., carboxylate from aspartic acid). Hence the [3Fe-4S] core can be a redox-linked sensor of divalent metal ions, Fe(II) or Zn0I), that may trigger conformational change.
Address reprint requests to: Professor A. J. Thomson, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
Journalof InorganicBiochemistry, 47, 197-207 (1992)
197 © 1992 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/92/$5.00
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INTRODUCTION .Iron-sulphur clusters bound to proteins have a role, long recognized as electron-storage and reversible-electron transfer centers [see Ref. 1 for review]. It is now clear that the [4Fe4Sl cubane core cluster also can function at the catalytic center of dehydratase enzymes such as aconitase [see Ref. 21. Evidence is now emerging of a possible third role, namely, control of protein biosynthesis at the level of mRNA [see Refs. 3, 4, and Kiihn and Hentze in this issue]. In this review we focus upon the cubane cluster [4Fe4S] and the three-iron cluster [3Fe4S] which undergo a set of interconversion and other redox linked processes involving loss or uptake of metal ions or protons. The cubane cluster [4Fe4S], has a remarkably wide range of chemistry [see, Ref. 51. It can adopt, in a protein environment, at least three oxidation states [4Fe4S]i+/2+/3+, ahh ough for a given protein only the single electron redox processes 1 + /2 + or 2 + /3 + are permitted. In this role the cubane core is liganded by four cysteinyl ligands so that each iron atom is tetrahedrally coordinated. However, [4Fe4S]‘+ in aconitase [see Ref. 61 catalyzes the non-&ox reaction of citrate-isocitrate interconversion which entails sequential dehydratase-hydratase reactions [see Ref. 71. The active form of aconitase contains the [4Fe-4S12+ cluster liganded by three thiolate groups of cysteine. One iron site coordinates substrate and H,O or OH-. Aconitase is apparently a member of a growing family of dehydratase enzyme which utilize a [4Fe-4S] 2+ cluster in non-redox catalysis. The list includes L-serine dehydratase ( Peptostreptococcus asaccharalyticus) [see Ref 81, 6-phosphogluconate dehydratase (Zymonas mobilis) [see Ref. 91, tartrate dehydratase [see Ref. lo], and maleic acid hydratase [see Ref. 111. One curious feature of the chemistry of the [4Fe4S] cluster in some proteins is that on oxidation one iron atom is lost to generate the [3Fe4S]‘+ core [see Ref. 121. Conversely, on reduction of the latter core to [3Fe-4Sl” the divalent ion Fe(B) is taken up to restore the cubane framework [see Ref. 131. Redox linked metal-ion uptake and loss processes have been identified in a number of bacterial ferredoxins (Fd) such as Fd (Ciostridium pasteurianum) [see Ref. 121, Fd II (Desulphovibrio gigas) [see Ref. 141, Fd III (Desufphovibrio africanus) [see Ref. 151, and Fd (Pyrococcus furiosus) [see Ref. 161. It has been shown that metal ions besides iron can be taken up by the vacant corner position of the [3Fe-M cluster]. These include cobalt(II) [see Ref. 171, zinc@) [see Ref. 181, nickel(B) [see Ref. 191, and cadmium(II) [see Ref. 201. Iron loss also occurs from the active site [4Fe4S] cluster of aconitase under oxidizing conditions [see Ref. 21, and this leads to loss of enzymic activity. Under reducing conditions in the presence of Fe(B), activity is restored as the cluster is reconstituted. A similar process appears to be taking place in L-serine dehydratase (Peptostreptococcus asaccharolyticus) [see Ref. 81. The capability is thus afforded for corresponding in vivo metabolic activities to be modulated by the local Fe@) concentration and electrochemical potential. This role for Fe(B) in controlling the activity of an enzyme used in energy generation in mitochondria and in certain bacteria has been given added significance by reports of the sequence of an iron-regulated mRNA-binding protein, the so-called iron-responsive element binding protein (IRE-BP) [see Refs. 3,4]. The sequence of this protein shows regions of high homology with that of mitochondrial aconitase, especially in the vicinity of the iron-sulphur cluster binding domain. This has led to the proposal that the IRE-BP is
IRON-SULPHUR CLUSTERS WITH LABILE METAL IONS
199
an iron-sulphur protein containing a [3Fe4S] cluster which will bind Fe(n) under reducing conditions [see Refs. 3-41. Cellular iron metabolism is controlled by regulation of ferritin and transferrin receptor synthesis at the level of mRNA [Ref. 211. This is effected by the IRE-BP which binds to similar sequences (IREs) that exist within the 5’ untranslated region of ferritin mRNA and the 3’ untranslated region of the transferrin receptor mRNA. The IREs from both mRNAs interact with a cytoplasmic protein the IRE-BP, whose binding affinity is dependent upon the iron status of the cell. The clear implication is that [4Fe4S] * [3Fe4S] transformation is involved in control of IRE-BP binding to the IRE and hence governs protein expression. The rich redox and metal ion uptake chemistry exhibited by the [4Fe4S] cluster in proteins has not yet been fully modeled with synthetic analogues of these clusters [see Ref. 51. Our approach is to explore this chemistry through studies of appropriate low-molecular weight ferredoxins that provide a tailor-made site for metal ion and ligand binding. We have developed a sensitive, electrochemically-based strategy that enables us to control and monitor these redox-linked processes, to resolve convoluted reactivities, and to isolate samples for spectroscopic characterization [see Ref. 221. The bacterial ferredoxins we have used to study these processes are ferredoxin III, Desuiphovibrio crfticanus (Da) [see Ref. 231 and ferredoxin I, Azotobacter chroococcum (AC) [see Ref. 241. Both proteins contain, as aerobically isolated, one [3Fe-4S] and one [4Fe4S] cluster. Fd III, Da, has a molecular weight of 6000 Da, and a known sequence showing only seven cysteine residues. Between the cysteine residues 11 and 17 lies an aspartate group, at position 14, instead of cysteine as would be found in a typical 2[4Fe4S] Fd. The [3Fe-4S]’ cluster incorporates Fe@) rapidly to form a [4Fe4S]*+ cluster which can be reduced further to generate a [4Fe4S]‘+ solution with unusual magnetic properties [see Ref. 151. Spectroscopic and electrochemical evidence shows that A. chroococuum Fd I is similar if not identical to that of Azotobacter vinelandii Fd I [see Ref. 251 whose structure has recently been redetermined [see Refs. 6, 261. This Fd contains nine cysteines. The ligation of the cysteine thiol groups to the two clusters is compared in Figure 1 with that of the typical 8Fe ferredoxin from Clostridium pasteurianum [see Ref. 271. The ligation of one of the [4Fe-4S] clusters is highly conserved in all three proteins as judged by sequence data. The x-ray structures of Av Fd I and Pa Fd also show that the cluster binding regions is similar. However, variation in cysteine spacing within the first 16 or 17 residues and the replacement of one of the cysteine residues by an aspartic acid residue leads to marked changes in the reactivity of the second cluster. 8Fe Fd from Ciostridium pasteurianum does not readily lose iron from one of its clusters except under extreme oxidizing conditions such as treatment with hexacyanoferrate ion [see Ref. 121. Here we describe combined spectroscopic and electrochemical studies of two bacterial Fds both with [3Fe4S][4Fe4S] core structures, one of which undergoes facile and reversible redox coupled metal ion uptake, Da Fd III, and the other Av Fd I, which is resistant to metal-ion uptake. EXPJIRIMENTAL
The EPR diagnostic including illustrates
METHODS and MCD spectra under conditions of low-temperature provide excellent signatures of the cluster type and of the ground state magnetic pammeters, electron spin, g-values, and zero-field splitting parameters. Figure 2 the EPR, W-visible absorption, and MCD spectra of the oxidized form of
200 A. J. Thomson et al.
8 (N)
11
14
CYS-X-X-CYS-X-X-CYS
PRO-C%
0
18
. . . . . . . . . . . . . . . . . . . . . .CYS-PRO
. . . . . , . . . . . . . . . . .CYS-x-x-CYS-x-x-CYS 41
45
38
35
1 8 (N)
11
16
20
24
CYS-X-X-CYS-X-X-X-X-CYS
. . . . . . . . . . . . . . . . . .CYS-PRO . . . . . . CYS
3Fc
4Fe
/I\
PRO-CYS . . . . . . . . . . . . . , .CYS-x-x-CYS-x-x-CYS
(‘3
45
49
11
14
39
21
17
(N) CYS-X-X-ASP-X-X-CYS
42
. . . . . . . . . . . . . . . . . CYS-PRO
4Fe
3Fe
/I\
(C) OLN-GLU-CYS . . . . . . . , . . . . . . . .CYS-x-x-CYS-x-x-CYS 51
47
44
41
FIGURE
1. The sequences of the 2[Fe_4S] Fd, Closfridium Pusteurianum, the [3Fe4S][4Fe4S] Fd, Azotobacter vinelandii, and the [3Fe_4S][4Fe_4S]Fd III, Desulphovibrio qfricanus, showingthe ligation mode of the clusters.
Da Fd III measured at liquid helium temperature. In this state the four-iron cluster is in the diamagnetic oxidation level [4Fe_QS]*+. The MCD spectra of paramagnetic species increase in intensity as the temperature is lowered whereas the intensity of diamagnetic species are independent of temperature and hence weak. The [3Fe_4S]‘+ core has a spin S = l/2 which gives rise to a characteristic EPR signal at g = 2.01. The lower panel shows the absorption spectra measured at 1.5 K and also the MCD spectra at several temperatures between 1.57 and 22 K and at 5 Tesla magnetic field.
IRON-SULPHUR CLUSTERS WITH LABILE METAL IONS
201
g-v0 I UQ 2. 15
I '
2. 10
2. 05
2. DO
1.
1.95
1. 80
1.85
90
1
Oesulphovibrio africanus Fd El, native 300
AEIMhi 200
-
I
157
K
.._..... 4.215 ,( _........._ 11.95K
---
V
2.01 K
-._._22.0K -300 L,
I
I
I
300
400
500
-
,
6;O A;nm 7;O
900
_I
FIGURE
2. The EPR spectrum (upper), absorption spectrum (lower, A) and MCD spectrum at 5 Tesla (lower) of Fd III, Lksufphovibrio clfricunus in the oxidized state. The EPR and MCD signals arise only from the [3Fe4]‘+ cluster.
The detailed assignment of this spectrum has not been achieved but similar spectra have been recorded from [3Fe4S] cores in a variety of different protein environments [see Ref. 281. It can be difficult to manipulate proteins under conditions of low potential and well-defined metal ion concentration. Cluster transformations are more readiiy identified and studied by a voltammetric method that, in practice uses a nanomole of protein for each experiment but permits equilibrium constants and kinetic parameters to be measured [see Ref. 231. The discovery arises from the observation that Fds co-absorb with amino sugars at a pyrolytic graphite edge (PGE) electrode giving a stable electroactive fihn, provided that a promoter molecule of amino-sugar is present in solution [see Ref. 131. The FGE surface is rich in acidic oxides which interact with proteins bearing negatively-charged surface regions in the presence of poly-cations such as the naturally occurring amino-sugar, neomycin. Fast, direct
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electron transfer takes place between the protein and the electrode surface allowing cyclic voltammograms to be recorded. The preparation of electroactive films has been described [see Ref. 201. RESULTS AND DISCUSSION Fd III, D. africanus The direct, unmediated cyclic voltammogram (CV) of an adsorbed film of Da Fd IIl in the presence of the chelator EGTA at pH 7 shows three well-defined waves due to three redox couples (Fig. 3). One of these corresponds to the stable [4Fe-4S]‘+/‘+ cluster and the other two are associated with the [3Fe4S] cluster and are assigned respectively to the normal 1 + /O couple and a chemically-reversible two-electron process of uncertain identity. By comparative integration and from the effect of pH upon the wave-position the latter appears to correspond to a three-proton uptake. If the coated electrode is transferred to a stirred solution devoid of EGTA but containing a low concentration of Fe(B), reductive passage through the couple [3Fe-4S]‘+” initiates rapid changes. Subsequent cycles over the course of several seconds causes the waves due to [3Fe4S] I+” to disappear together and be replaced by a new couple corresponding to the newly formed cluster [4Fe4S]*+/‘+. The position of the new couple corresponds closely with CV’s of Fd III undergoing transformation in the solution phase. The height of each CV peak is proportional to the concentration of the redox active species present. Hence a study of these peaks as a function of Fe(B) concentration enables cluster-metal dissociation constants to be determined [see Ref. 201. The redox linked uptake of a divalent metal ion is not restricted to Fe@). Zn(II) and Cd(B) also bind strongly and the affinity order Cd*+> Zn*% Fe*+ has been established [see Ref. 201. This is the order expected for a sulphide rich site [see Ref.
I
5CLA
I
I
-800
I
I
-600
I
1
-400
I
I
-200
1
I
1
0
E/mV vs. SHE FIGURE 3. Steady-state cylclic voltatnmogram of a film of 7Fe Fd, Desulphovibrio qfricanus III. The redox couple corresponding to each wave is indicated.
IRON-SULPHUR CLUSTERS WITH LABILE METAL IONS
203
291. It has also been shown that Tl(I) has a high afIinity for the reduced [3Fe4S]’ site but also shows a measurable atlinity for the oxidized state of the cluster [3Fe4S]‘+ [see Ref. 301. The [3Fe4S]’ cluster is paramagnetic with a spin S = 2 subject to a negative, predominantly axial zero-field splitting [see Ref. 121. The [4Fe4S]‘+ cluster is diamagnetic at low temperatures. On reduction of this cluster by one electron the resulting state [4Fe4S]’ + has an unusual spin-state S = 3/2 subject to an axial zero-field splitting. This state has g values of 5.27, (2.34), (1.62). The MCD spectra are changed little in form by this spin-state change from S = l/2. The coordination geometry of the newly formed cluster is not ‘yet established. Three of the ligands are cysteine whereas the labile iron may be ligated either by the carboxylate group of asp 11, or by H,O or OH -. There is no pH dependence to the redox potential over the range 6-8. FdI, A. chrwococcum By means of direct electrochemistry at a PGE electrode in the presence of the promoter neomycin the redox potentials of the two iron-sulphur clusters in Fd I( AC) were measured [see Ref. 311 and the magnetic properties were determined (see Ref. 24). The relevant equilibria are [ 3Fe4S] i + + e + [ 3Fe4S] ’
E,,,=
[3Fe4S]‘+H++[3Fe4S]‘*-*H+
-46Ok
1OmV
pK,=7.8
and [4Fe4S]2++e-+[4Fe4S]1+
E,,2=
-645klOmVatpH8.3.
The pH dependence of the three-iron cluster corresponding to the uptake of a proton in the [3Fe4S]’ state has been discovered only in Azotobucter Fd. A further reduction of the three-iron cluster can be observed at much lower potentials. This process has been detected electrochemically in all [3Fe-4S] cluster-containing ferredoxins so far examined The [3Fe4S]’ cluster in Fd I, AC, is peculiarly resistant to take up divalent metal ions when in a purified state. Two reports have appeared of the formation of a [4Fe4S] core. In one case the tertiary structure of the protein Fd I, Av, is disrupted by guanadium hydrochloride to generate a 2[4Fe4S] Fd [see Ref. 321. In the other case, spontaneous uptake of Fe(B) was reported for a sample at pH 8.3 in the presence of nucleic acid contamination [see Ref. 241. It has been proposed that binding between Fd I AC and DNA may take place and facilitate the three-to-four iron conversion [see Ref. 331. CONCLUSIONS The [3Fe4S] cluster is a redox dependent ligand for a variety of cations. The one-electron reduced state [3Fe4S]‘, has a higher affinity for divalent metal ions than the oxidixed state. The only example to date of metal ion binding to the [3Fe4S]+ cluster is Tl(I) in Da Fd III. However, the oxidized form of the four-iron
204
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cluster, [4Fe-4S13+, is stable in the redox proteins called high potential iron proteins (HiPiP). Since this core is a complex of Fe@) with [3Fe4S]’ + the protein fold and the presence of a fourth cysteine ligand has stabilized the oxidized four-iron core. The K, values determined for Da Fd III show that the order of stability of the clusters [M3Fe_4S]* accords with the Irving-Williams series for binding of high-spin M(II) ions to a sulphide rich ligand. We have discovered examples of both the [3Fe4S]* and [3Fe-4S]‘- states reacting with protons. The location of the bound protons is not established. However, it is likely that they become bound to the cluster itself possibly by protonating the bridging sulphide in the open cluster face. The possibility that H30+, a pyramidal cation, could cap the open corner of the [3Fe4S] by hydrogen-bonding to the three j+ulphide atoms is an intriguing one. The reactivity of the [3Fe-4S]* core towards cations is not surprising given the stereochemical disposition of the three sulphide ions. However, the noteworthy features are, first, the much reduced affinity of the [3Fe-4S]‘+ core towards cations, and second, the total lack of reactivity of both l+ and 0 oxidation states of the [3Fe4S] core in some proteins. The addition of a metal ion to the comer of the three-iron core to complete the cube gives (Fig. 4) an intermediate which has a metal ion with a coordination number of only three. This center will therefore have a high affinity for a further ligation. This could be either H,O or OH- from solvent or a protein side-chain ligand possibly cysteine (thiol) or aspartic acid (carboxylate). This poses a number of interesting questions. The first, how is it that some [3Fe4S] cores are unreactive in proteins even when reduced in the presence of excess Fe(U)? The open sulphide-face could be blocked by the close approach of other residues. The h-sulphide atoms may have a reduced metal ion affinity due to H-bonding. The H-bonds could either stabilize the triangular face or cause the sulphide ions to flex so that the triangular edges of the face are lengthened. The presence or otherwise of a fourth ligand from the protein which is correctly positioned to provide a tetrahedral coordination site for an incoming metal ion will also affect reactivity of the triangular sulphide face. Hence we must expect to encounter proteins with [3Fe4S] centers with a wide range of different stabilities and reactivities towards additional metal ions. The other important feature of the redox-linked metal ion uptake process is that it can be an engine to drive protein conformational change. For example, if the fourth ligand acquired by the added metal ion is a protein side-chain which must move into position this may trigger movement of the polypeptide backbone and hence conformational change. The [3Fe4S] core can be a redox-linked sensor of divalent metal ion concentrations within a cell that may lead to protein conformational change scheme. At high potentials the umeactive [3Fe4S]‘+ state is formed. As the potential drops the reduced state increases in concentration and provided that an appropriate divalent metal ion is available a cubane cluster will be formed. This may trigger conformational change. The potential which senses the internal potential of the cell is the [3Fe_4S]‘+‘* redox couple. The concentration range over which a divalent metal ion can be sensed is governed both by the potential of the resulting cluster and the affinity of the [3Fe4S]* core for the metal ion. The evidence from sequence data that the IRE-BP has high homology with mitochondrial aconitase suggests that a [3Fe4S] cluster may be present in this
206 A. J. Thomson et al.
anaerobic respiration by sensing oxygen levels. They may be activated by Fe@). It remains to be determined whether the [3Fe-4S] core cluster plays any role in these control processes. This work has been supported by Molecular Recognition Initiative of the S.E.R.C., by the University of California, by grants from N.A.T.O. (No. CRC 900302), by an EXXON Education Foundation Award (FAA), and by C.N.R.S., France.
REFERENCES 1. A. J. Thomson, in Metalioproteins, P. M. Harrison, Ed. Verlag Chemie, Weinheim, F.R.G., 1985, Part 1, pp. 79-120. 2. H. Beinert, FASEB J. 4, 2483 (1990). 3. T. A. Rouault, C. D. Stout, S. Kaptain, J. B. Harford, and R. D. Klausner, Cell 64, 881 (1991). 4. M. W. Hentze and P. Argos, Nucleic Acid Res. 19, 1739 (1991). 5. R. H. Hahn. S. Ciurli, and J. A. Weigel, Prog. Znorg. Chem. 38, 1, (1990). 6. D. J. Robbins and C. D. Stout, Proc. Natl. Acad. Sci. 86, 3639 (1989). 7. H. Beinert and M. C. Kennedy, Eur. J. Biochem. 186, (1989). 8. R. Grahowski and W. Buckel, Eur. J. Biochem. 199, 89 (1991). 9. R. K. Scopes and K. Griffiths-Smith, Anal. Biochem. 136, 530 (1984). 10. H. Rode and F. Giflham, J. Bacterioi. 151, 1602 (1982). 11. J.-L. Dreyer, Eur. J. Biochem., 150, 145 (1985). 12. A. J. Thomson, A. E. Robinson, M. K. Johnson, R. Cammack, K. K. Rao, and D. 0. Hall, Biochim. Biophys. Acta, 637, 423 (1981). 13. F. A. Armstrong, J. N. Butt, S. J. George, E. C. Hatehikian, and A. J. Thomson, FEBS Lett., 125, 15 (1989). 14. B. H. Huynh, J. J. G. Moura, I. Moura, T. A. Kent, J. L&all, A. V. Xavier, and E. Munck, J. Bioi. Chem. 255, 3242 (1980). 15. S. J. George, F. A. Armstrong, E. C. Hatchikian, and A. J. Thomson, Biochem. J., 264, 275 (1989). 16. R. C. Conover, A. T. Kowal, W. Fu, J.-B. Park, S. Aano, M. W. W. Adams, and M. K. Johnson, J. Biol. Chem. 265, 8533 (1990). 17. I. Moura, J. J. G. Moura, E. Miinck, V. Papaefthymou, and J. LeGall, J. Amer. Chem. Sot., 108, 349 (1986).
18. K. K. Surerus, E. Miinck, I. Moura, J. J. G. Moura, and J. LeGall, J. Amer. Chem. Sot., 109, 3805 (1987). 19. R. C. Conover, J.-B. Park, M. W. W. Adams, and M. K. Johnson, J. Amer. Chem. Sot., 112, 4562 (1990). 20. J. N. Butt, F. A. Armstrong, J. Breton, S. J. George, A. J. Thomson, and E. C. Hatchikian, J. Amer. Chem. Sot., 113, 6663 (1991). 21. E. C. Theil, J. Bioi. Chem., 265, 4771 (1990). 22. F. A. Armstrong, Structure and Bonding, 72, 137 (1990). 23. F. A. Armstrong, S. J. George, R. Cammack, E. C. Hatchikian, and A. J. Thomson, Biochem. J. 264, 265 (1989). 24. S. J. George, A. J. M. Richards, A. J. Thomson, and M. G. Yates, Biochem. J., 224, 247 (1984). 25. S. J. George, Ph.D. Thesis, University of East Anglia, 1986. 26. G. H. Stout, S. Turley, L. C. Sieker, and L. H. Jensen, Ptoc. Natl. Acad. Sci., 85, 1020 (1988).
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27. M. Tanaka,
28. 29. 30. 31. 32. 33. 34.
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T. Nakashima, A. M. Benson, H. F. Mower, and K. T. Yasunobu, Biochemistry, 5, 1666 (196f$. H. Beinert and A. J. Thomson, Arch. B&hem. Biophys., 222, 333 (1983). J. J. R. da Silva, and R. J. P. Williams, The Biological Chemistry of the Elements, OUP, Oxford, 1991. J. N. Butt, A. Sucheta, F. A. Armstrong, J. Breton, A. J. Thomson, and E. C. Hatchikian, .I. Amer. Chem. Sot., 113, 8948 (1991). F. A. Armstrong, S. J. George, A. J. Thomson, and M. G. Yates, FEBS Lett., 234, 107 (1988). T. V. Morgan, P. J. Stephens, B. K. Burgess, and C. D. Stout, FEBS Lett., 167, 137 (1984). A. J. Thomson, FEBS Lett., 285, 230 (1991). S. Spiro and J. R. Guest, TZBS, 16, 310 (1991).
Received January 27, 1992; accepted February 3, 1992
