- Email: [email protected]
Fe-S proteins in sensing and regulatory functions
152
Fe-S proteins
in sensing and regulatory
functions
Helmut Beinert* and Patricia J Kileyt In the past five to ten years, it has become apparent that the function of Fe-S clusters
increasingly is not limited
electron
discovery.
transfer,
a function
implicit
in their
know that the vulnerability of these structures destruction is used by nature in sensing O,, also
nitric
clusters
oxide.
Changes
can also
serve
in the oxidation as a reversible
We
to now
to oxidative iron, and possibly
state
of Fe-S
switch.
Addresses *Institute for Enzyme Research, Graduate School, and the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, 1710 University Avenue, Madison, WI 53705, USA; e-mail: [email protected] Department of Biomolecular Chemistry, Medical School, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA; e-mail: [email protected]
Current
Opinion
in Chemical
Biology
1999,
3:152-l
57
http://biomednet.com/elecref/1367593100300152 0
Elsevier
Science
Ltd
ISSN
1367-5931
Abbreviations C
EPR FNR
IRE IRPl m NO
PRPP
cytoplasmic electron paramagnetic resonance fumarate nitrate reduction iron-responsive element iron regulatory protein 1 mitochondrial nitric oxide phosphoribosylpyrophosphate
Introduction It should barely come as a surprise that FeS clusters modular structures made up from two of the most versatile and abundant elements -should have found numerous and divergent uses during the billenia of our planet’s existence. It is only in the past few years, however, that their implication in sensing, signaling and regulation of gene expression has attracted attention and is now being explored. Because of their structure and chemical properties [ 1**,2**J5 Fe-S clusters are ideal agents for electron uptake and donation, intramolecular electron shifts and polarization. These are also the properties that can make them sensors of molecules in their immediate environment. Because the redox potentials of the couples formed by the majority of Fe-S clusters are in the range of -500-150 mV, one would expect that in biological systems Fe-S clusters are most responsive to available agents that could serve as oxidants under these low-potential conditions. Thus, one might expect that Fe-S clusters could have been recruited by nature to act as sensors of cellular oxidants; in vitro, they may also respond to strong reducing agents or ligands for iron that are stronger than their sulfur ligands. Oxidation of Fe-S clusters may have the following consequences: first, cluster rearrangement or total disassembly, which is generally irreversible and requires auxiliary agents or enzymes for reversal (category 1); second,
one-electron oxidation, which is readily reversible, depending on the prevalent redox potential or the specific oxidant present (category 2). The effects of this sensing of environmental influences by oxidation (and its eventual reversal) according to either mechanism would then have to be communicated to the surrounding protein structure for the regulatory effect to occur. The regulatory response may involve facilitating changes in the oligomeric state or may destabilize structures required for binding to other molecules such as nucleotides or proteins, which may in turn alter the resistance to proteolysis. In this review, we will describe examples for all these possibilities. We do not consider here the many simple electron transfer functions of Fe-S clusters in proteins that have been explored and described since their discovery (for recent reviews, see [ l”J”.3,4]). Rather, we will deal largely with those cases where specific oxidation (or reductive reversal) reactions are involved and a regulatory circuit is directly influenced. We also consider some examples where oxidation/reduction or destruction of an Fe-S cluster influences the regulation of a metabolic pathway. While we know for most examples described below that Fe-S clusters are involved in regulatory e\-ents, critical details of the kind of involvement are still missing at this time. Such cases will be discussed briefly, after we have covered in detail several more thoroughly studied examples (e.g. FNR [fumarate nitrate reduction] of Escbidia roji [SO], aconitaseliron regulatory protein 1 [IRPl] [6-S], glutamine phosphoribosylpyrophosphate [PRPP] amidotransferase of Bad/us suhfil’ic [9,10], endonuclease III of E. CO/~[ll] and SoxR of fi:. co/i [12’.13]). It may be useful here to remind the reader that the terms ‘oxidized’ or ‘reduced’, generally used to describe the state of a particular cluster, can be ambiguous because in many cases more than two possible oxidation states are involved. Ambiguity can be avoided by stating the charge of the cluster core (ignoring the ligands). Then, we recognize that even the most oxidized naturally occurring 4Fe cluster (e.g. in the high-potential Fe-S protein [HiPIP]), [4Fe--4S]“+, formally still contains one Fez+ iron, and the most reduced 4Fe cluster ([4FellS]+ ; see ‘the Fe-protein of nitrogenase’ section below, however), as seen in reduced ferredoxin, contains one Fe”+ ion. While the electrons (or the respective ‘holes’ in the more oxidized forms) are largely delocalized over the whole or parts of the structure, on reaction with any suitable outside molecule, these electrons (or holes) are available for reaction at any position in the cluster structure.
Sensing
by Fe-S
cluster
destruction
The FNR protein of E. co/i, an oxygen sensor This is the simplest case that falls into category 1 because, at least with respect to the sensing function, there does not
Fe-S
seem to be any compound or protein involved other than the sensor and oxygen. This is not likely to be so for the recovery phase (i.e. when FNR returns to its active [4Fe-4S] state) of the sensor in YEW, however. FNR is a global regulator controlling the synthesis of proteins required for anaerobic respiration with nitrate, fumarate, trimethylamine oxide and similar electron acceptors replacing oxygen (reviewed in 114,151). This -30 kDa protein is present in E. co/i at a fairly constant level, irrespective of the oxygen concentration. Only the anaerobically purified protein contains a [4Fe--4S]Z+ cluster (4Fe-FNR) and this cluster is required for FNR function. Also, FNR is functional only in its dimeric form, binding DNA with high affinity and thus controlling gene expression [16,17]. It seems, therefore, that the [4FeAS]z+ cluster stabilizes the dimeric form. Initially, it was considered that the oxidation state of iron in a mononuclear chelate (FeCysJ might control FNR activity (reviewed in [14]); however, when it was shown that the iron was actually part of an Fe-S cluster, namely [4Fe-4S], it was then found that the Fe-S cluster in the active form of FNR is actually present in E. coli in its 2+ form [l&19]), and not in what is generally considered the ‘reduced’, +l form (see discussion above on oxidation states and core charges). The redox potential of the 2+/l+ couple in FNR has been estimated to be 1-6.50 mV, which would rule out any function of the l+ form in V&XI [S’]. The [4Fe--4S]2+ cluster is very sensitive to oxygen, which rapidly converts it to the more 02-stable [ZFe-ZS]z+ form (the 2Fe form) [18]; this occurs with extrusion of two Fez+ ions and two sulfides, which may then become further oxidized. In the 2Fe form, FNR is inactive and appears to be monomeric. Upon addition of the reductant dithionite to the [‘ZFe-2S]2+ form, one does not obtain the [ZFe-ZS]+ form; rather, the [4Fe-4S]Z+ form is reconstituted in good yield [18]. It is not known so far, however, whether this reconstitution proceeds directly from the 2Fe form or via smaller fragments derived from it. Under these reaction conditions, there is no evidence that any other agents are involved in these processes; they are spontaneous under the conditions mentioned [18], but it seems likely that in viva the reconstitution will require additional proteins such as enzyme(s) and carriers of iron and sulfide [ZO]. In this regard, reconstitution of 4Fe-FNR can also be achieved from apoprotein [21&Z] and from the 2Fe form in the presence of iron and sulfide [18]. It is of particular interest that the sensitivity of FNR to oxygen depends dramatically on the environment of the cluster. The LeuZ8-+His FNR mupant (CysZ9 is one of the iron ligands) is stable toward oxygen for many hours. Experimental results similar to those with purified FNR, demonstrating conversion of the 4Fe to the 2Fe form and its reversal, have also been obtained with whole cells of 8. co/i, in which overexpressed FNR in either form was readily detected by s7Fe Miissbauer spectroscopy [19]. There are a number of FNR homologs found throughout the prokaryotic world [14,23”], and it is almost certain that those containing a cysteine cluster similar to that of FNR function by ligating
proteins
in sensing
and regulatory
functions
Beinert
and Kiley
153
an Fe-S cluster, perhaps with an oxygen sensitivity somewhat modulated by the detailed protein structure. It has yet to be demonstrated for the majority of the FNR homologs that are involved in regulating anaerobic processes which molecules are being sensed, however. Aconitaseliron
regulatory
protein,
an iron sensor
An intact [4Fe--4S] cluster is required for the enzymatic function of mitochondrial (m-) aconitase in the tricarboxylic acid cycle [6]. Thus, destruction or (re)synthesis of this cluster will determine the activity of aconitase and hence control the citric acid cycle if aconitase should become the limiting enzyme in the cycle. Cytoplasmic (c-) aconitase, a close relative of the mitochondrial enzyme (-30% amino acid identity), is converted to IRPl through loss of its Fe-S cluster [6,24,25]. Despite its similarity to IRPl, the closely related IRP?. has not been found to incorporate an Fe-S cluster [26,27]. IRPs regulate translation of ferritin mRNA (ferritin is an iron storage protein) and transferrin receptor mRNA (transferrin receptor transfers iron into the cell) in an opposing sensevia binding to iron-responsive elements (IREs), which are specific stemloop RNA structures present in untranslated regions of selected mRNAs. Recently, alternative splicing of the IRE has been observed in ferritin mRNA from Drosop&/n such that some of the spliced mRNAs lack the IRE and escape iron regulation [28]. IRPl has also been found to bind to IREs present in the mRNAs of m-aconitase. erythroid &aminolevulinic acid synthase, succinate dehydrogenase subunit b (known as ‘iron protein’) of DrosopUz [7] and a mammalian proton-coupled metal-ion transporter [29]. IRPl is therefore able to regulate the translation of the corresponding mRNAs. The Fe-S clustersof the aconitasesare alsoreadily attacked by oxidants, particularly by O,- and H,O,, and converted to [3Fe-4S]+ clusters [6,7,30,31]. One could therefore. in a broader sense,consider the aconitasesassensorsfor oxygen and its derivatives, which destroy the 4Fe cluster, and also assensorsfor iron, asiron is a prerequisite for resynthesisof the cluster. In its form asIRPl, the cluster-free cytoplasmic aconitaseclearly is an iron sensor;however, it owesthis sensing ability to the very fact that it is no longer an Fe-S protein. Thus, we have the opposing examples: FNR sensing oxygen via its Fe-S cluster and IRPl regulating translation and sensingiron becauseof its absence. The Fe-S cluster of m-aconitase - and by analogy presumably alsothat of c-aconitase- is located (in the crystal structure) in a deep cleft and is surrounded by cluster ligandsor substrate-binding residuesfrom all four domainsof the molecule [6]. Some of these residues (e.g. arginines that bind the tricarboxylic acid substrates)have also been identified as ligands to IREs [6,32]. Moreover, it has been shown that human IRPl cannot bind to IREs when Cys437 is encumbered through disulfide bond formation or reaction with AT-ethylmaleimide. As Cys437 is an obligatory Fe-S cluster ligand in c-aconitase, it is obvious that
154
Bio-inorganic
chemistry
the functions of the protein as aconitase and as IRPl are mutually exclusive. The conversion of c-aconitase to IRP can proceed spontaneously in the presence of oxidants, and the resynthesis of aconitase from IRPl is readily accomplished in the presence of reducing agents and appropriate reagents such as sulfide and iron. However, the rate of these processes is probably too slow to be useful in nature; additional reactants, such as nitric oxide (NO) [7,33] in the disassembly of the cluster, and enzymes and carriers of Fe or sulfide in the resynthesis [20,34] are likely to play a role. It has also been shown that phosphorylation of serine residues within the cleft of the aconitase influences the ease with which the conversion between the two forms, aconitase and IRP, can proceed [35]. Thus, for instance. IRPl is four to five times more efficiently phosphorylated by protein kinase C in vitro than is c-aconitase. Moreover, the phosphorylation state of IRPl may have an influence on cluster stability. Furthermore, there is evidence for additional pathways of iron-independent modulation of IRP function [36,37”]. Of particular interest has been a cross-relation between oxidative stressand iron-response systems involving the IRP/c-aconitase interconversion [36]. H,O, has been found to stimulate formation of active IRPl; however, this does not occur via direct oxidative attack and conversion of c-aconitase to IRP, but proceeds via a separate signaling pathway involving at least one membrane-bound component [36]. A consequence of this is that extracellular H,O, acts differently compared with intracellularly generated H,O, and, once the stimulus has occurred, the presence of H,O, is no longer required for the effect to persist. This is in contrast to the effects of NO or iron chelators in generating IRP from c-aconitase; these agents are only effective for the duration of their presence [7,36]. In addition, it must be remembered that (iso)citrate is fairly effective in chelating iron and at the same time in binding and protecting aconitase from loss of its Fe-S cluster and may thus play a role. These observations and the fact that IREs are also able to control the translation of components of the energy-producing system and of heme synthesis (see above), provide a fascinating -while still blurred - vista of the delicate cross-connections that seem to exist between various control systems in living organisms. Recent results have shown that regulation of pathogenicity factor production was altered in mutants of the cruciferous plant pathogen Xanthomonas campestris lacking rpfA, which encodes for a homologue of aconitase, indicating that an aconitase may also be involved in regulation in prokaryotes [38]. Phosphoribosylpyrophosphate-amidotransferase B. subtilis and endonuclease III of E. co/i
of
PRPP-amidotransferase and endonuclease III can also be considered as belonging to category 1 (described above). In both proteins, a [4Fe-4S]Z+ cluster stabilizes structures
required for maintaining activity. This is strongly suggested by the X-ray structures of these proteins: in PRPP-amidotransferase, the Fe-S cluster ligands are located in different domains of the structure, which is held together through the Fe-S center [lo]. Similarly, in endonuclease III the cluster stabilizes a fold that positions several positively charged residues for DNA binding [ 111.In PRPP-amidotransferase, the Fe-S cluster is oxygen-sensitive and it was observed that cluster oxidation occurs in parallel to enzyme inactivation [9]. Thus, through destruction of the Fe-S cluster, the protein becomes vulnerable towards proteolytic degradation. In the context of regulatory functions, it is of interest that in glucose-starved cells, the Fe-S cluster decays at a much higher rate than in growing cells. The Fe-S cluster of endonuclease III, however, has not been found to be particularly unstable toward oxygen. Endonuclease III has many similarities to another protein involved in DNA repair in E. coli, the glycosylase MutY protein [39]. MutY also contains an Fe-S cluster with the same pattern of cysteine ligation, but the two enzymes recognize different substrates. As with endonuclease III, the Fe-S cluster of MutY is required for activity [40]. Human endonuclease III also contains an Fe-S cluster [41].
Sensing by reversible oxidation/reduction of an Fe-S cluster: the SoxR protein of E. co/i, a sensor of oxidative stress This is a prime example for category 2 (described above), that is, sensing via a reversible one-electron redox system [ 12’,13]. SoxR is activated on exposure of cells specifically to 02-and NO, but not to H,O, or OH’. Active SoxR activates transcription of only a single gene, SOXS,leading to the formation of the SoxS protein. SoxS regulates expression of a number of genes whose products function in the defense against 02--generating agents such as paraquat, as well as against NO. In addition, activating SoxR/SoxS provides generalized antibiotic resistance. The SoxR protein is a homodimer of relative molecular mass(M,) 34,000 that contains one [2Fe-ZS] cluster per monomer. Under physiological conditions, the cluster is in the l+ state (redox potential at PH 7.6, E”7.6 - -28.5 mV). Both the l+ and 2+ form, and, even the apoprotein, binds to the soxS promoter DNA. Thus, the Fe-S cluster is not required for this function; however, only SoxR containing the cluster in the 2+ form can lead to transcription initiation by RNA polymerase [12’]. The response of the Fe-S cluster to oxidation has been demonstrated using electron paramagnetic resonance (EPR) on the purified SoxR protein [ 12’,42], as well as on whole cells, by the disappearance of the EPR signal that is typical of the [ZFe-ZS]+ form [12’]. It was also ascertained that the EPR signal of the l+ form could be restored on addition of dithionite to the sample that had been exposed to O,-, showing that the SoxR protein had not been destroyed on oxidation. The notion that the redox state of the Fe-S cluster is critical to SoxR function is further supported by experiments using
Fe-S
constitutive mutations that activate SoxR/SoxS-dependent genes in the absence of O,- generating agents. While -40-95s of the Fe-S clusters of wild type SoxR are in the l+ form, in the mutants less than 4% were in this state [43]. In one such mutant protein, the midpoint redox potential was shifted to --350 mV, which would make the Fe-S cluster more readily oxidizable, thus increasing the sensitivity of the sensor. Thus far, there is no clear indication that any more specific mechanism is at work in the reduction of the Fe-S cluster of SoxR after the oxidative stress has been removed; however, such a possibility must certainly be kept in mind. It has also been suggested that a ferredoxin or a flavodoxin might be required to keep SoxR in its reduced state [44]. If that is indeed the case, then SoxR activity might respond in GUO both to the level of specific oxidants that can directly oxidize the SoxR [ZFe-ZS]+ form, as well as to agents that could affect the oxidation state of a ferredoxin.
Other
proteins
The ‘Fe-protein’
to be considered of nitrogenase
In the context of what is often called a ‘structural role’ for Fe-S clusters, the ‘Fe-protein’ of the nitrogen-fixation enzyme nitrogenase should be mentioned. (The principle catalysts in nitrogen fixation are the so-called ‘Fe-protein’ and the ‘MO-Fe’ protein.) Fe-protein is a homodimer of M, 60,000, which contains a single [4Fe-4S] cluster located at the interface between two subunits, with two pairs of identical cysteine residues from each monomer furnishing the ligands [45]. The cluster can be removed from the dimeric protein, however, without dissociation of the subunits. One cannot, therefore, unambiguously attribute a primary role to the cluster in the formation of the dimer. The Fe-S cluster is, however, required for electron transfer to the ‘MoFeprotein’ of nitrogenase, which ultimately reduces dinitrogen [46,47]. In its reduced state, the Fe-protein binds two molecules of MgATP in the same intersubunit channel, in which the Fe-S cluster is located some 20 A away. Significant structural changes occur in the Fe-protein during binding. The ensuing conformation is not, however, able to hydrolyze ATP This can only occur after complex formation between the Fe- and the MoFeprotein. Then follows formation of MgADP and electron transfer to the P-clusters of the MoFe-protein, the protein complex dissociates and the Fe-protein can then be reduced for the next cycle. Recent results indicate that the Fe-S cluster of the Fe-protein can be reduced to the fully reduced, all ferrous [4Fe-4S]O state [48,49], which is not observed with other Fe-S proteins, but may be significant in the context of the multielectron process of dinitrogen reduction. The Fe-S cluster is essential for the reaction steps involved to occur; however, details of the involvement of the various components in these reaction sequences are at best only partially known and are the subject of intensive current investigations [SO-521. The Fe-protein is also required for the biosynthesis of FeMoco, the complex Fe-S cluster of the
proteins
in sensing
and regulatory
functions
Beinert
and
Kiley
155
MoFe-protein that also contains MO, and for the incorporation of preformed FeMoco into the apo-MoFeprotein [45]. There are a number of Fe-S proteins involved in the complicated synthesis of the MoFe-protein that have unusual functions which could be considered as regulatory [45]. Mammalian
ferrochelatase
This protein is the last enzyme involved in heme biosynthesis. It catalyzes the insertion of ferrous iron into protoporphyrin IX. Mammalian ferrochelatase (M, -50,000) has been found to contain a [2Fe-2S] cluster [53,54]. This cluster has not been found in ferrochelatases from bacteria, yeast or plants, whereas the human and mouse enzymes are inactive unlessan intact Fe-S cluster is present. Apparently, the Fe-S cluster has no function in the reduction of ferric iron in the Fe insertion reaction. It has been observed, however, that ferrochelatase is strongly inhibited by NO and that it is destruction of the Fe-S cluster by NO that causesenzyme inactivation [55,56]. It therefore seemspossible that the cluster is used as a sensor for NO, which would establish a link to other intracellular signaling pathways that depend on NO as messenger(see the aconitase/IRP section). It is suggested that inactivation of ferrochelatase, which would bring heme synthesis to a halt, may be a defense mechanism against bacterial infections [55]. Other
unexpected
functions
of Fe-S
proteins
In recent years, a number of enzymes have been encountered that require Fe-S clusters, either within the same molecule or in an associated protein (i.e. the ‘activating enzyme’), to carry out reactions in which free radicals are obligatory intermediates [2”]. Fe-S clusters are well suited to supply single electrons, as required in homolytic reactions. Examples of these enzymes include anaerobic ribonucieotide reductase [57], pyruvate-formate lyase [58] and biotin synthase [59] in E. cdi. As these enzymes have been discussedat some length in [2’*], we refer the reader to this article for detail. This area of Fe-S protein involvement will probably receive much attention in the near future.
Conclusions Fe-S proteins, ubiquitous in living matter, do not only have a versatile, modular structure that can be adapted to various conditions and requirements, they are also unexpectedly flexible in the reactivities that they display: from agents of electron storage,transfer and polarization, to signaling and regulating agents, via their tunable sensitivity to various oxidants or reductants.
Acknowledgements We acknowledge with gratitude the comments on our manuscript by BK Bnrsxss. B Demnle. RE Eisenstein, Y Hat&, MK Johnson, MC Ken&& TA Rouault~a~d RL Switzer. This work was supported by the United States Public Health Service and by a National Institutes for Health grant GM45844 (to PJ Kiley), who was also a recipient of a Young Investigator Award from the National Science Foundation and the Shaw Scientist Award from the Milwaukee Foundation.
156
Bio-inorganic
References Papers of particular have been highlighted l
**of
chemistry
and recommended interest, as:
published
within
reading the annual
IQ.
period
of review,
of special interest outstanding interest
20.
1.
Beinert H, Holm RH, Mijnck E: Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 1997, 277:653-659. &s paper and [2*] represent the most recent concise surreys of the Fe-S field, with emphasis on new insights concerning chemical, physical and biological aspects. 2. .. See
Johnson MK: iron-sulfur Opin Chem Bio/1998, annotation [l “I.
3.
Johnson MK: Iron-sulfur proteins. In Encyclopedia Chemistry. Edited by King RB. Chichester: John 1994:1896-1913.
4.
proteins: 2:173-181.
Cammack R, Sykes AG (Eds): Advances 38. San Diego: Academic Press; 1992.
5. .
Kiley PJ, Beinert H: Oxygen sensing the role of the iron-sulfur cluster. 22:341-352. This work summarizes the present status tion of Fe-S clusters in FNR of E. co/i. 6.
new
Beinert H, Kennedy MC, Stout DC: protein, enzyme, and iron-regulatory 96:2335-2373.
roles
for old
Wiley
in inorganic
Aconitase protein.
Curr
of inorganic & Sons; vol
regulator, FNR: Rev 1999, concerning
the func-
as iron-sulfur Chem Rev
1996,
7.
Hentze MW, Kuhn LC: Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Nat/ Acad Sci USA 1996, 93:8175-8182.
8.
Klausner RD, Rouault the control of cellular
9.
Grandoni JA, Switzer RL, Makaroff CA, Zalkin H: Evidence that the iron-sulfur cluster of Bacillus subfilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in viva. J Biol Chem 1989, 264:6058-6064.
IO.
11.
Thayer MM, Ahern H, Xing D, Cunntngham binding motiis in the DNA repair enzyme structure. EM60 J 1995,14:4108-4120.
H, Satow
Khoroshilova N, Beinert H, Kiley PJ: Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding. Proc Nat/ Acad Sci USA 1995, 92:2499-2503.
22.
Green J, Bennett B, Jordan P, Ralph E, Thomson A, Guest J: Reconstitution of the [4Fe-4Sl cluster in FNR and demonstration of the aerobic-anaerobic switch in vitro. Biochem 11996, 316:887-892.
Van Spanning Stouthamer denifrificans transcriptional adaptation 23:893-907. an This contams . .. of transcnptronar
RP, Tainer JA: Novel DNA endonuclease III crystal
Hidalgo E, Ding H, Demple B: Reclox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem Sci 1997,22:207-210. _. This contams a concrse presentatron and dtscussron ot knowledge ot me SoxRlSoxS system up to early 1997. 13.
Gaudu P, Weiss B: SoxR, only in its oxidized form. 93:10094-l 0098.
a [2Fe-2Sl transcription Proc Nat/ Acad Sci USA
factor, 1996,
14.
Guest JR, Green J, Irvine AS, Spiro S: The FNR modulon and FNRregulated gene expression. In Regulation of Gene Expression in Escherichia co/i Edited by Lin ECC, Lynch AS. Austin, Texas: RG Landes Co; 1996:317-342.
15.
Unden G, Bongaerts J: Alternative fscherichia co/k energetics and response to electron acceptors. 1320:217-234.
17.
Lazauera BA, Beinert H, Khoroshilova N, Kennedy MC, Kiley PJ: DNA binding and dimerization of the Fe-S containing FNR protein from Escherichia co/i are regulated by oxygen, J B/o/ Chem 1996, 271:2762-2768. Khoroshilova N, Popescu C, Mijnck E, Beinert H, Kiley P: Iron-sulfur cluster disassembly in the FNR protein of Escherichia co/i by 0,: [4Fe-4S1 to [2Fe-2Sl conversion with loss of biological activity. Proc Nat/ Acad Sci USA 1997, 94:6087-6092.
RJ, De Boer APN, Reijnders WNM, Westerhoff HV, AH, Van Der Oost J: FnrP and NNR of Paracoccus are both members of the FNR family of activators but have distinct roles in respiratory in response to oxygen limitation. MO/ Microbial 1997, excellent ., acttvators.
survey
and
discussion
of the FNR
protein
family
25.
Haile DJ, Rouault TA, Harford JB, Kennedy MC, Blondin GA, Beinert H, Klausner RD: Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc Nat/ Acad Sci USA 1992,89:11735-l 1739.
26.
Guo B, Brown FM, Phillips JD, Yu Y, Leibold and expression of iron regulatory protein 1995, 270:16529-l 6535.
27.
lwai K, Klausner RD, Rouault TA: Requirements degradation of the RNA binding protein, iron EM80 J 1995, 14~5350-5357.
for iron-regulated regulatory protein
29.
Gunshin H, MacKenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA: Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997, 388:482-488.
30.
Gardner PR, Fridovich co/i aconitase. J Biol
31.
Flint D, Tuminello J, Emptage M: The inactivation containing hydro-lyases by superoxide. J Biol 268:22369-22376.
32.
Philpott CC, Klausner RD, Rouault TA: The bifunctional ironresponsive element binding proteinkytosolic aconitase: the role of active-site residues in ligand binding and regulation. froc Nafl Acad Sci USA 1994,91:7321-7325.
33.
Drapier J-C, Hirling H, Wietzerbin J, Kaldy P, Kuhn LC: Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMEO J 1993,12:3643-3649.
34.
Land T, Rouault TA: Targeting of a human iron-sulfur cluster assembly enzyme, nifS, to different subcellular compartments regulated through alternative AUG utilization. MO/ Cell 1998, 2:807-815.
36.
I: Superoxide Chem 1991,
K: Drosophile the presence 436:476-482.
2.
Lind MI, Ekengren mRNA: alternative iron-responsive
35.
S, Melefors 0, Soderhall RNA splicing regulates element. FEB.9 Leff 1998,
EA: Characterization 2 (IRP2). J Biol Chem
28.
respiratory pathways of transcriptional regulation in BBA - Bioenergetics 1997,
Lazazzera BA, Bates DM, Kiley PJ: The activity of the Escherichia co/i transcription factor FNR is regulated by a change in oligomeric state. Genes Dev 1993, 7:1993-2005.
from
Kennedy MC, Mende-Mueller L, Blondin GA, Beinert H: Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Nat/ Acad Sci USA 1992,89:11730-11734.
is active
16.
froc
24.
Y:
12. .
18.
21.
TA, Harford JB: Regulating the fate of mRNA: iron metabolism. Cell 1993, 72:19-28.
Smith JL, Zaluzec EJ, Wery J-P, Niu L, Switzer RL, Zalkin Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 1994, 264:1427-l 433.
Zheng L, Cash VL, Flint DH, Dean DR: Assembly of iron-sulfur clusters: identification of an iscSUA-hscBA-fdx gene cluster Azotobacter vinelandii. J Biol Chem 1998, 273:13264-l 3272.
23. ..
Chemistry,
by the global FEMS Microbial of knowledge
clusters.
Popescu C, Bates D, Mijnck E, Beinert H: Mijssbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia co/i. Nat/ Acad Sci USA 1998, 9613431-l 3435.
sensitivity of the 266:i 9328-19333.
ferritin of the
Escherichia
of Fe-S cluster Chem 1993,
Schalinske KL, Anderson SA, Tuazon PT, Chen OS, Kennedy MC, Eisenstein RS: The iron-sulfur cluster of iron regulatory protein modulates the accessibility of RNA binding and phosphorylation sites. Biochemistry 1997, 36:3950-3958. Pantopoulos by oxidative @5:10559-l
K, Hentze MW: stress in vitro. 0563.
is
1
Activation of iron regulatory protein-i Proc Nat/ Acad Sci USA 1998,
Eisentstein RS, Kennedy MC, Beinert HB: The iron responsive element (IRE), the iron regulatory protein (IRP), and the cytosolic aconitase; postranscriptional regulation of mammalian iron metabolism. In Metal ions in Gene Regulation. Edited by Walden W, Silver S. New York: Chapman and Hall; 1998:157-216. This is an extensive survey and discussion of the subject.
37. ..
Fe-S
38.
Wilson TJG, Bertrand N, Tang J-L, Feng J-X, Pan M-Q, Barber GE, Dow JM, Daniels MJ: The rpfA gene of Xantbomonas campestfis pathovar campesfris, which is involved in the regulation of pathogenic@ factor production, encodes an aconitase. MO/ M;crobio/ 1998, 28:961-970.
39.
Michaels ML. Pham L. Nahiem Y. Cruz C. Miller JH: MutY, glycosylaseactive on &A misPairs, has homology to endonuclease III. Nucleic Acids Res 1990, 18:3841-3845.
40.
Porello SL, Cannon MJ, David SS: A substrate recognition the 14Fe-4Sl*+ cluster of the DNA repair glycosylase Biochemistry 1998, 37:6465-6475.
41.
proteins
in sensing
and
regulatory
functions
Beinert
and
Kiley
157
potentials
of
50.
Lanzilotta WN, Seefeldt LC: Changes in the midpoint the nitrogenase metal centers as a result of iron protein-molybdenum-iron protein complex formation. Biochemistry 1997, 36:12976-l 2983.
51.
Lanzilotta WN, Fisher K, Seefeldt LC: Evidence for electron from the nitrogenase iron protein to the molybdenum-iron without MgATP hydrolysis: characterization of a tight protein-protein complex. Biochemistry 1996, 35:7188-7196.
for
52.
lkeda S, Biswas T, Roy R, lzumi T, Boldogh I, Kurosky A, Sarker AH, Seki S, Mitra S: Purification and characterization of human NTHI, a homolog of Escherichia cc/i endonuclease Ill. J Biol Chem 1998, 273:21585-21593.
Spee JH, Arendsen AF, Wassink Redox properties and electron spectroscopy of the transition vinelandii nitrogenase. FEBS
53
Dailey HA, Finnegan iron-sulfur protein.
54
Lloyd SG, Franc0 R, Moura JJG, Moura I, Ferreira GC, Huynh BH: Functional necessity and physicochemical characteristics of the [2Fe-2Sl cluster in mammalian ferrochelatase. J Am Chem Sot 1996, 118:9892-9900.
an adenine
role MutY.
transfer protein
H, Marritt SJ. Hagen WR, Haaker paramagnetic resonance state complex of Azotobacfer Let! 1998,432:55-58.
MG, Johnson Biochemistry
MK: Human ferrochelatase 1994, 33:403-407.
H:
is an
42.
Wu J, Dunham WR, Weiss B: Overproduction and physical characterization of SoxR, a [2Fe-2Sl protein that governs an oxidative response reguion in fscherichia co/i. J B/o/ Chem 1995, 270:10323-i 0327.
43.
Hidalgo E, Ding HG, Demple B: Redox signal transduction mutations shifting C2FE-2Sl centers of the SoxR sensor-regulator to the oxidized form. Cell 1997, 88:121-l 29.
55
Sellers cluster oxide
44.
Liochev SI, Hausladen A, Beyer WFJ, Fridovich oxidoreductase acts as a paraquat diaphorase the soxRS regulon. Proc /Vat/ Acad SC; USA
56
Furukawa inactivation possible Eiochem
45.
Howard fixation.
57.
Ollagnier S, Mulliez E, Schmidt PP, Eliasson R, Gaillard J, Deronzier C, Bergman T, Graslund A, Reichard P, Fontecave M: Activation of the anaerobic ribonucleotide reductase from Escberkhia co/i. J Biol Cheri, 1997,272:24216-24223.
58
Kulzer R, Pils T, Kappl R, Hiittermann characterization of the polynuclear formate-lyase-activating enzyme. 273:4897-4903.
59
Duin EC, Lafferty ME, Crouse BR, Allen RM, Sanyal I, Flint DH, Johnson MK: [2Fe-2S1 to [4Fe-4Sl cluster conversion in Escherichia co/i biotin synthase. Biochemistry 1997, 36:i 181 l-l 1820.
18, Rees DC: Structural basis Chem Rev 1996, 96:2965-2982.
I: NADPH: ferredoxin and is a member of 1994, 91:1328-l 331.
of biological
Burgess BK, Lowe DJ: Mechanism Chem Rev 1996,96:2983-3011.
of molybdenum
47.
Ferguson SJ: Nitrogen 1998, 2:182-193.
48.
Angove HC, Yoo SJ, Mijnck E: An all-ferrous state of nitrogenase. J Biol Chem 1998, 273:26330-26337.
49.
Musgrave KB, Angove HC, Burgess BK, Hedman B, Hodgson KO: All-ferrous titanium (Ill) citrate reduced Fe protein of nitrogenase: an XAS study of electronic and metrical structure. J Am Chem Sot 1998, 120:5325-5326.
enzymology.
Curr
T, Kohno H, Tokunaga R, Taketani of mammalian ferrochelatase involvement of the iron-sulphur J 1995, 310:533-538.
S: Nitric oxide-mediated in viva and in vitro: cluster of the enzyme.
nitrogen
46.
cycle
VM, Johnson MK, Dailey HA: Function of the [2Fe-2Sl in mammalian ferrochelatase: a possible role as a nitric sensor. Biochemistry 1996, 35:2699-2704.
nitrogenase. Opin
Chem of the
Biol Fe protein
J, Knappe J: Reconstitution and iron-sulfur cluster in pyruvate J Biol Chem 1998,
Fe-S proteins
in sensing and regulatory
functions
Helmut Beinert* and Patricia J Kileyt In the past five to ten years, it has become apparent that the function of Fe-S clusters
increasingly is not limited
electron
discovery.
transfer,
a function
implicit
in their
know that the vulnerability of these structures destruction is used by nature in sensing O,, also
nitric
clusters
oxide.
Changes
can also
serve
in the oxidation as a reversible
We
to now
to oxidative iron, and possibly
state
of Fe-S
switch.
Addresses *Institute for Enzyme Research, Graduate School, and the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, 1710 University Avenue, Madison, WI 53705, USA; e-mail: [email protected] Department of Biomolecular Chemistry, Medical School, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA; e-mail: [email protected]
Current
Opinion
in Chemical
Biology
1999,
3:152-l
57
http://biomednet.com/elecref/1367593100300152 0
Elsevier
Science
Ltd
ISSN
1367-5931
Abbreviations C
EPR FNR
IRE IRPl m NO
PRPP
cytoplasmic electron paramagnetic resonance fumarate nitrate reduction iron-responsive element iron regulatory protein 1 mitochondrial nitric oxide phosphoribosylpyrophosphate
Introduction It should barely come as a surprise that FeS clusters modular structures made up from two of the most versatile and abundant elements -should have found numerous and divergent uses during the billenia of our planet’s existence. It is only in the past few years, however, that their implication in sensing, signaling and regulation of gene expression has attracted attention and is now being explored. Because of their structure and chemical properties [ 1**,2**J5 Fe-S clusters are ideal agents for electron uptake and donation, intramolecular electron shifts and polarization. These are also the properties that can make them sensors of molecules in their immediate environment. Because the redox potentials of the couples formed by the majority of Fe-S clusters are in the range of -500-150 mV, one would expect that in biological systems Fe-S clusters are most responsive to available agents that could serve as oxidants under these low-potential conditions. Thus, one might expect that Fe-S clusters could have been recruited by nature to act as sensors of cellular oxidants; in vitro, they may also respond to strong reducing agents or ligands for iron that are stronger than their sulfur ligands. Oxidation of Fe-S clusters may have the following consequences: first, cluster rearrangement or total disassembly, which is generally irreversible and requires auxiliary agents or enzymes for reversal (category 1); second,
one-electron oxidation, which is readily reversible, depending on the prevalent redox potential or the specific oxidant present (category 2). The effects of this sensing of environmental influences by oxidation (and its eventual reversal) according to either mechanism would then have to be communicated to the surrounding protein structure for the regulatory effect to occur. The regulatory response may involve facilitating changes in the oligomeric state or may destabilize structures required for binding to other molecules such as nucleotides or proteins, which may in turn alter the resistance to proteolysis. In this review, we will describe examples for all these possibilities. We do not consider here the many simple electron transfer functions of Fe-S clusters in proteins that have been explored and described since their discovery (for recent reviews, see [ l”J”.3,4]). Rather, we will deal largely with those cases where specific oxidation (or reductive reversal) reactions are involved and a regulatory circuit is directly influenced. We also consider some examples where oxidation/reduction or destruction of an Fe-S cluster influences the regulation of a metabolic pathway. While we know for most examples described below that Fe-S clusters are involved in regulatory e\-ents, critical details of the kind of involvement are still missing at this time. Such cases will be discussed briefly, after we have covered in detail several more thoroughly studied examples (e.g. FNR [fumarate nitrate reduction] of Escbidia roji [SO], aconitaseliron regulatory protein 1 [IRPl] [6-S], glutamine phosphoribosylpyrophosphate [PRPP] amidotransferase of Bad/us suhfil’ic [9,10], endonuclease III of E. CO/~[ll] and SoxR of fi:. co/i [12’.13]). It may be useful here to remind the reader that the terms ‘oxidized’ or ‘reduced’, generally used to describe the state of a particular cluster, can be ambiguous because in many cases more than two possible oxidation states are involved. Ambiguity can be avoided by stating the charge of the cluster core (ignoring the ligands). Then, we recognize that even the most oxidized naturally occurring 4Fe cluster (e.g. in the high-potential Fe-S protein [HiPIP]), [4Fe--4S]“+, formally still contains one Fez+ iron, and the most reduced 4Fe cluster ([4FellS]+ ; see ‘the Fe-protein of nitrogenase’ section below, however), as seen in reduced ferredoxin, contains one Fe”+ ion. While the electrons (or the respective ‘holes’ in the more oxidized forms) are largely delocalized over the whole or parts of the structure, on reaction with any suitable outside molecule, these electrons (or holes) are available for reaction at any position in the cluster structure.
Sensing
by Fe-S
cluster
destruction
The FNR protein of E. co/i, an oxygen sensor This is the simplest case that falls into category 1 because, at least with respect to the sensing function, there does not
Fe-S
seem to be any compound or protein involved other than the sensor and oxygen. This is not likely to be so for the recovery phase (i.e. when FNR returns to its active [4Fe-4S] state) of the sensor in YEW, however. FNR is a global regulator controlling the synthesis of proteins required for anaerobic respiration with nitrate, fumarate, trimethylamine oxide and similar electron acceptors replacing oxygen (reviewed in 114,151). This -30 kDa protein is present in E. co/i at a fairly constant level, irrespective of the oxygen concentration. Only the anaerobically purified protein contains a [4Fe--4S]Z+ cluster (4Fe-FNR) and this cluster is required for FNR function. Also, FNR is functional only in its dimeric form, binding DNA with high affinity and thus controlling gene expression [16,17]. It seems, therefore, that the [4FeAS]z+ cluster stabilizes the dimeric form. Initially, it was considered that the oxidation state of iron in a mononuclear chelate (FeCysJ might control FNR activity (reviewed in [14]); however, when it was shown that the iron was actually part of an Fe-S cluster, namely [4Fe-4S], it was then found that the Fe-S cluster in the active form of FNR is actually present in E. coli in its 2+ form [l&19]), and not in what is generally considered the ‘reduced’, +l form (see discussion above on oxidation states and core charges). The redox potential of the 2+/l+ couple in FNR has been estimated to be 1-6.50 mV, which would rule out any function of the l+ form in V&XI [S’]. The [4Fe--4S]2+ cluster is very sensitive to oxygen, which rapidly converts it to the more 02-stable [ZFe-ZS]z+ form (the 2Fe form) [18]; this occurs with extrusion of two Fez+ ions and two sulfides, which may then become further oxidized. In the 2Fe form, FNR is inactive and appears to be monomeric. Upon addition of the reductant dithionite to the [‘ZFe-2S]2+ form, one does not obtain the [ZFe-ZS]+ form; rather, the [4Fe-4S]Z+ form is reconstituted in good yield [18]. It is not known so far, however, whether this reconstitution proceeds directly from the 2Fe form or via smaller fragments derived from it. Under these reaction conditions, there is no evidence that any other agents are involved in these processes; they are spontaneous under the conditions mentioned [18], but it seems likely that in viva the reconstitution will require additional proteins such as enzyme(s) and carriers of iron and sulfide [ZO]. In this regard, reconstitution of 4Fe-FNR can also be achieved from apoprotein [21&Z] and from the 2Fe form in the presence of iron and sulfide [18]. It is of particular interest that the sensitivity of FNR to oxygen depends dramatically on the environment of the cluster. The LeuZ8-+His FNR mupant (CysZ9 is one of the iron ligands) is stable toward oxygen for many hours. Experimental results similar to those with purified FNR, demonstrating conversion of the 4Fe to the 2Fe form and its reversal, have also been obtained with whole cells of 8. co/i, in which overexpressed FNR in either form was readily detected by s7Fe Miissbauer spectroscopy [19]. There are a number of FNR homologs found throughout the prokaryotic world [14,23”], and it is almost certain that those containing a cysteine cluster similar to that of FNR function by ligating
proteins
in sensing
and regulatory
functions
Beinert
and Kiley
153
an Fe-S cluster, perhaps with an oxygen sensitivity somewhat modulated by the detailed protein structure. It has yet to be demonstrated for the majority of the FNR homologs that are involved in regulating anaerobic processes which molecules are being sensed, however. Aconitaseliron
regulatory
protein,
an iron sensor
An intact [4Fe--4S] cluster is required for the enzymatic function of mitochondrial (m-) aconitase in the tricarboxylic acid cycle [6]. Thus, destruction or (re)synthesis of this cluster will determine the activity of aconitase and hence control the citric acid cycle if aconitase should become the limiting enzyme in the cycle. Cytoplasmic (c-) aconitase, a close relative of the mitochondrial enzyme (-30% amino acid identity), is converted to IRPl through loss of its Fe-S cluster [6,24,25]. Despite its similarity to IRPl, the closely related IRP?. has not been found to incorporate an Fe-S cluster [26,27]. IRPs regulate translation of ferritin mRNA (ferritin is an iron storage protein) and transferrin receptor mRNA (transferrin receptor transfers iron into the cell) in an opposing sensevia binding to iron-responsive elements (IREs), which are specific stemloop RNA structures present in untranslated regions of selected mRNAs. Recently, alternative splicing of the IRE has been observed in ferritin mRNA from Drosop&/n such that some of the spliced mRNAs lack the IRE and escape iron regulation [28]. IRPl has also been found to bind to IREs present in the mRNAs of m-aconitase. erythroid &aminolevulinic acid synthase, succinate dehydrogenase subunit b (known as ‘iron protein’) of DrosopUz [7] and a mammalian proton-coupled metal-ion transporter [29]. IRPl is therefore able to regulate the translation of the corresponding mRNAs. The Fe-S clustersof the aconitasesare alsoreadily attacked by oxidants, particularly by O,- and H,O,, and converted to [3Fe-4S]+ clusters [6,7,30,31]. One could therefore. in a broader sense,consider the aconitasesassensorsfor oxygen and its derivatives, which destroy the 4Fe cluster, and also assensorsfor iron, asiron is a prerequisite for resynthesisof the cluster. In its form asIRPl, the cluster-free cytoplasmic aconitaseclearly is an iron sensor;however, it owesthis sensing ability to the very fact that it is no longer an Fe-S protein. Thus, we have the opposing examples: FNR sensing oxygen via its Fe-S cluster and IRPl regulating translation and sensingiron becauseof its absence. The Fe-S cluster of m-aconitase - and by analogy presumably alsothat of c-aconitase- is located (in the crystal structure) in a deep cleft and is surrounded by cluster ligandsor substrate-binding residuesfrom all four domainsof the molecule [6]. Some of these residues (e.g. arginines that bind the tricarboxylic acid substrates)have also been identified as ligands to IREs [6,32]. Moreover, it has been shown that human IRPl cannot bind to IREs when Cys437 is encumbered through disulfide bond formation or reaction with AT-ethylmaleimide. As Cys437 is an obligatory Fe-S cluster ligand in c-aconitase, it is obvious that
154
Bio-inorganic
chemistry
the functions of the protein as aconitase and as IRPl are mutually exclusive. The conversion of c-aconitase to IRP can proceed spontaneously in the presence of oxidants, and the resynthesis of aconitase from IRPl is readily accomplished in the presence of reducing agents and appropriate reagents such as sulfide and iron. However, the rate of these processes is probably too slow to be useful in nature; additional reactants, such as nitric oxide (NO) [7,33] in the disassembly of the cluster, and enzymes and carriers of Fe or sulfide in the resynthesis [20,34] are likely to play a role. It has also been shown that phosphorylation of serine residues within the cleft of the aconitase influences the ease with which the conversion between the two forms, aconitase and IRP, can proceed [35]. Thus, for instance. IRPl is four to five times more efficiently phosphorylated by protein kinase C in vitro than is c-aconitase. Moreover, the phosphorylation state of IRPl may have an influence on cluster stability. Furthermore, there is evidence for additional pathways of iron-independent modulation of IRP function [36,37”]. Of particular interest has been a cross-relation between oxidative stressand iron-response systems involving the IRP/c-aconitase interconversion [36]. H,O, has been found to stimulate formation of active IRPl; however, this does not occur via direct oxidative attack and conversion of c-aconitase to IRP, but proceeds via a separate signaling pathway involving at least one membrane-bound component [36]. A consequence of this is that extracellular H,O, acts differently compared with intracellularly generated H,O, and, once the stimulus has occurred, the presence of H,O, is no longer required for the effect to persist. This is in contrast to the effects of NO or iron chelators in generating IRP from c-aconitase; these agents are only effective for the duration of their presence [7,36]. In addition, it must be remembered that (iso)citrate is fairly effective in chelating iron and at the same time in binding and protecting aconitase from loss of its Fe-S cluster and may thus play a role. These observations and the fact that IREs are also able to control the translation of components of the energy-producing system and of heme synthesis (see above), provide a fascinating -while still blurred - vista of the delicate cross-connections that seem to exist between various control systems in living organisms. Recent results have shown that regulation of pathogenicity factor production was altered in mutants of the cruciferous plant pathogen Xanthomonas campestris lacking rpfA, which encodes for a homologue of aconitase, indicating that an aconitase may also be involved in regulation in prokaryotes [38]. Phosphoribosylpyrophosphate-amidotransferase B. subtilis and endonuclease III of E. co/i
of
PRPP-amidotransferase and endonuclease III can also be considered as belonging to category 1 (described above). In both proteins, a [4Fe-4S]Z+ cluster stabilizes structures
required for maintaining activity. This is strongly suggested by the X-ray structures of these proteins: in PRPP-amidotransferase, the Fe-S cluster ligands are located in different domains of the structure, which is held together through the Fe-S center [lo]. Similarly, in endonuclease III the cluster stabilizes a fold that positions several positively charged residues for DNA binding [ 111.In PRPP-amidotransferase, the Fe-S cluster is oxygen-sensitive and it was observed that cluster oxidation occurs in parallel to enzyme inactivation [9]. Thus, through destruction of the Fe-S cluster, the protein becomes vulnerable towards proteolytic degradation. In the context of regulatory functions, it is of interest that in glucose-starved cells, the Fe-S cluster decays at a much higher rate than in growing cells. The Fe-S cluster of endonuclease III, however, has not been found to be particularly unstable toward oxygen. Endonuclease III has many similarities to another protein involved in DNA repair in E. coli, the glycosylase MutY protein [39]. MutY also contains an Fe-S cluster with the same pattern of cysteine ligation, but the two enzymes recognize different substrates. As with endonuclease III, the Fe-S cluster of MutY is required for activity [40]. Human endonuclease III also contains an Fe-S cluster [41].
Sensing by reversible oxidation/reduction of an Fe-S cluster: the SoxR protein of E. co/i, a sensor of oxidative stress This is a prime example for category 2 (described above), that is, sensing via a reversible one-electron redox system [ 12’,13]. SoxR is activated on exposure of cells specifically to 02-and NO, but not to H,O, or OH’. Active SoxR activates transcription of only a single gene, SOXS,leading to the formation of the SoxS protein. SoxS regulates expression of a number of genes whose products function in the defense against 02--generating agents such as paraquat, as well as against NO. In addition, activating SoxR/SoxS provides generalized antibiotic resistance. The SoxR protein is a homodimer of relative molecular mass(M,) 34,000 that contains one [2Fe-ZS] cluster per monomer. Under physiological conditions, the cluster is in the l+ state (redox potential at PH 7.6, E”7.6 - -28.5 mV). Both the l+ and 2+ form, and, even the apoprotein, binds to the soxS promoter DNA. Thus, the Fe-S cluster is not required for this function; however, only SoxR containing the cluster in the 2+ form can lead to transcription initiation by RNA polymerase [12’]. The response of the Fe-S cluster to oxidation has been demonstrated using electron paramagnetic resonance (EPR) on the purified SoxR protein [ 12’,42], as well as on whole cells, by the disappearance of the EPR signal that is typical of the [ZFe-ZS]+ form [12’]. It was also ascertained that the EPR signal of the l+ form could be restored on addition of dithionite to the sample that had been exposed to O,-, showing that the SoxR protein had not been destroyed on oxidation. The notion that the redox state of the Fe-S cluster is critical to SoxR function is further supported by experiments using
Fe-S
constitutive mutations that activate SoxR/SoxS-dependent genes in the absence of O,- generating agents. While -40-95s of the Fe-S clusters of wild type SoxR are in the l+ form, in the mutants less than 4% were in this state [43]. In one such mutant protein, the midpoint redox potential was shifted to --350 mV, which would make the Fe-S cluster more readily oxidizable, thus increasing the sensitivity of the sensor. Thus far, there is no clear indication that any more specific mechanism is at work in the reduction of the Fe-S cluster of SoxR after the oxidative stress has been removed; however, such a possibility must certainly be kept in mind. It has also been suggested that a ferredoxin or a flavodoxin might be required to keep SoxR in its reduced state [44]. If that is indeed the case, then SoxR activity might respond in GUO both to the level of specific oxidants that can directly oxidize the SoxR [ZFe-ZS]+ form, as well as to agents that could affect the oxidation state of a ferredoxin.
Other
proteins
The ‘Fe-protein’
to be considered of nitrogenase
In the context of what is often called a ‘structural role’ for Fe-S clusters, the ‘Fe-protein’ of the nitrogen-fixation enzyme nitrogenase should be mentioned. (The principle catalysts in nitrogen fixation are the so-called ‘Fe-protein’ and the ‘MO-Fe’ protein.) Fe-protein is a homodimer of M, 60,000, which contains a single [4Fe-4S] cluster located at the interface between two subunits, with two pairs of identical cysteine residues from each monomer furnishing the ligands [45]. The cluster can be removed from the dimeric protein, however, without dissociation of the subunits. One cannot, therefore, unambiguously attribute a primary role to the cluster in the formation of the dimer. The Fe-S cluster is, however, required for electron transfer to the ‘MoFeprotein’ of nitrogenase, which ultimately reduces dinitrogen [46,47]. In its reduced state, the Fe-protein binds two molecules of MgATP in the same intersubunit channel, in which the Fe-S cluster is located some 20 A away. Significant structural changes occur in the Fe-protein during binding. The ensuing conformation is not, however, able to hydrolyze ATP This can only occur after complex formation between the Fe- and the MoFeprotein. Then follows formation of MgADP and electron transfer to the P-clusters of the MoFe-protein, the protein complex dissociates and the Fe-protein can then be reduced for the next cycle. Recent results indicate that the Fe-S cluster of the Fe-protein can be reduced to the fully reduced, all ferrous [4Fe-4S]O state [48,49], which is not observed with other Fe-S proteins, but may be significant in the context of the multielectron process of dinitrogen reduction. The Fe-S cluster is essential for the reaction steps involved to occur; however, details of the involvement of the various components in these reaction sequences are at best only partially known and are the subject of intensive current investigations [SO-521. The Fe-protein is also required for the biosynthesis of FeMoco, the complex Fe-S cluster of the
proteins
in sensing
and regulatory
functions
Beinert
and
Kiley
155
MoFe-protein that also contains MO, and for the incorporation of preformed FeMoco into the apo-MoFeprotein [45]. There are a number of Fe-S proteins involved in the complicated synthesis of the MoFe-protein that have unusual functions which could be considered as regulatory [45]. Mammalian
ferrochelatase
This protein is the last enzyme involved in heme biosynthesis. It catalyzes the insertion of ferrous iron into protoporphyrin IX. Mammalian ferrochelatase (M, -50,000) has been found to contain a [2Fe-2S] cluster [53,54]. This cluster has not been found in ferrochelatases from bacteria, yeast or plants, whereas the human and mouse enzymes are inactive unlessan intact Fe-S cluster is present. Apparently, the Fe-S cluster has no function in the reduction of ferric iron in the Fe insertion reaction. It has been observed, however, that ferrochelatase is strongly inhibited by NO and that it is destruction of the Fe-S cluster by NO that causesenzyme inactivation [55,56]. It therefore seemspossible that the cluster is used as a sensor for NO, which would establish a link to other intracellular signaling pathways that depend on NO as messenger(see the aconitase/IRP section). It is suggested that inactivation of ferrochelatase, which would bring heme synthesis to a halt, may be a defense mechanism against bacterial infections [55]. Other
unexpected
functions
of Fe-S
proteins
In recent years, a number of enzymes have been encountered that require Fe-S clusters, either within the same molecule or in an associated protein (i.e. the ‘activating enzyme’), to carry out reactions in which free radicals are obligatory intermediates [2”]. Fe-S clusters are well suited to supply single electrons, as required in homolytic reactions. Examples of these enzymes include anaerobic ribonucieotide reductase [57], pyruvate-formate lyase [58] and biotin synthase [59] in E. cdi. As these enzymes have been discussedat some length in [2’*], we refer the reader to this article for detail. This area of Fe-S protein involvement will probably receive much attention in the near future.
Conclusions Fe-S proteins, ubiquitous in living matter, do not only have a versatile, modular structure that can be adapted to various conditions and requirements, they are also unexpectedly flexible in the reactivities that they display: from agents of electron storage,transfer and polarization, to signaling and regulating agents, via their tunable sensitivity to various oxidants or reductants.
Acknowledgements We acknowledge with gratitude the comments on our manuscript by BK Bnrsxss. B Demnle. RE Eisenstein, Y Hat&, MK Johnson, MC Ken&& TA Rouault~a~d RL Switzer. This work was supported by the United States Public Health Service and by a National Institutes for Health grant GM45844 (to PJ Kiley), who was also a recipient of a Young Investigator Award from the National Science Foundation and the Shaw Scientist Award from the Milwaukee Foundation.
156
Bio-inorganic
References Papers of particular have been highlighted l
**of
chemistry
and recommended interest, as:
published
within
reading the annual
IQ.
period
of review,
of special interest outstanding interest
20.
1.
Beinert H, Holm RH, Mijnck E: Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 1997, 277:653-659. &s paper and [2*] represent the most recent concise surreys of the Fe-S field, with emphasis on new insights concerning chemical, physical and biological aspects. 2. .. See
Johnson MK: iron-sulfur Opin Chem Bio/1998, annotation [l “I.
3.
Johnson MK: Iron-sulfur proteins. In Encyclopedia Chemistry. Edited by King RB. Chichester: John 1994:1896-1913.
4.
proteins: 2:173-181.
Cammack R, Sykes AG (Eds): Advances 38. San Diego: Academic Press; 1992.
5. .
Kiley PJ, Beinert H: Oxygen sensing the role of the iron-sulfur cluster. 22:341-352. This work summarizes the present status tion of Fe-S clusters in FNR of E. co/i. 6.
new
Beinert H, Kennedy MC, Stout DC: protein, enzyme, and iron-regulatory 96:2335-2373.
roles
for old
Wiley
in inorganic
Aconitase protein.
Curr
of inorganic & Sons; vol
regulator, FNR: Rev 1999, concerning
the func-
as iron-sulfur Chem Rev
1996,
7.
Hentze MW, Kuhn LC: Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Nat/ Acad Sci USA 1996, 93:8175-8182.
8.
Klausner RD, Rouault the control of cellular
9.
Grandoni JA, Switzer RL, Makaroff CA, Zalkin H: Evidence that the iron-sulfur cluster of Bacillus subfilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in viva. J Biol Chem 1989, 264:6058-6064.
IO.
11.
Thayer MM, Ahern H, Xing D, Cunntngham binding motiis in the DNA repair enzyme structure. EM60 J 1995,14:4108-4120.
H, Satow
Khoroshilova N, Beinert H, Kiley PJ: Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding. Proc Nat/ Acad Sci USA 1995, 92:2499-2503.
22.
Green J, Bennett B, Jordan P, Ralph E, Thomson A, Guest J: Reconstitution of the [4Fe-4Sl cluster in FNR and demonstration of the aerobic-anaerobic switch in vitro. Biochem 11996, 316:887-892.
Van Spanning Stouthamer denifrificans transcriptional adaptation 23:893-907. an This contams . .. of transcnptronar
RP, Tainer JA: Novel DNA endonuclease III crystal
Hidalgo E, Ding H, Demple B: Reclox signal transduction via iron-sulfur clusters in the SoxR transcription activator. Trends Biochem Sci 1997,22:207-210. _. This contams a concrse presentatron and dtscussron ot knowledge ot me SoxRlSoxS system up to early 1997. 13.
Gaudu P, Weiss B: SoxR, only in its oxidized form. 93:10094-l 0098.
a [2Fe-2Sl transcription Proc Nat/ Acad Sci USA
factor, 1996,
14.
Guest JR, Green J, Irvine AS, Spiro S: The FNR modulon and FNRregulated gene expression. In Regulation of Gene Expression in Escherichia co/i Edited by Lin ECC, Lynch AS. Austin, Texas: RG Landes Co; 1996:317-342.
15.
Unden G, Bongaerts J: Alternative fscherichia co/k energetics and response to electron acceptors. 1320:217-234.
17.
Lazauera BA, Beinert H, Khoroshilova N, Kennedy MC, Kiley PJ: DNA binding and dimerization of the Fe-S containing FNR protein from Escherichia co/i are regulated by oxygen, J B/o/ Chem 1996, 271:2762-2768. Khoroshilova N, Popescu C, Mijnck E, Beinert H, Kiley P: Iron-sulfur cluster disassembly in the FNR protein of Escherichia co/i by 0,: [4Fe-4S1 to [2Fe-2Sl conversion with loss of biological activity. Proc Nat/ Acad Sci USA 1997, 94:6087-6092.
RJ, De Boer APN, Reijnders WNM, Westerhoff HV, AH, Van Der Oost J: FnrP and NNR of Paracoccus are both members of the FNR family of activators but have distinct roles in respiratory in response to oxygen limitation. MO/ Microbial 1997, excellent ., acttvators.
survey
and
discussion
of the FNR
protein
family
25.
Haile DJ, Rouault TA, Harford JB, Kennedy MC, Blondin GA, Beinert H, Klausner RD: Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc Nat/ Acad Sci USA 1992,89:11735-l 1739.
26.
Guo B, Brown FM, Phillips JD, Yu Y, Leibold and expression of iron regulatory protein 1995, 270:16529-l 6535.
27.
lwai K, Klausner RD, Rouault TA: Requirements degradation of the RNA binding protein, iron EM80 J 1995, 14~5350-5357.
for iron-regulated regulatory protein
29.
Gunshin H, MacKenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA: Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 1997, 388:482-488.
30.
Gardner PR, Fridovich co/i aconitase. J Biol
31.
Flint D, Tuminello J, Emptage M: The inactivation containing hydro-lyases by superoxide. J Biol 268:22369-22376.
32.
Philpott CC, Klausner RD, Rouault TA: The bifunctional ironresponsive element binding proteinkytosolic aconitase: the role of active-site residues in ligand binding and regulation. froc Nafl Acad Sci USA 1994,91:7321-7325.
33.
Drapier J-C, Hirling H, Wietzerbin J, Kaldy P, Kuhn LC: Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMEO J 1993,12:3643-3649.
34.
Land T, Rouault TA: Targeting of a human iron-sulfur cluster assembly enzyme, nifS, to different subcellular compartments regulated through alternative AUG utilization. MO/ Cell 1998, 2:807-815.
36.
I: Superoxide Chem 1991,
K: Drosophile the presence 436:476-482.
2.
Lind MI, Ekengren mRNA: alternative iron-responsive
35.
S, Melefors 0, Soderhall RNA splicing regulates element. FEB.9 Leff 1998,
EA: Characterization 2 (IRP2). J Biol Chem
28.
respiratory pathways of transcriptional regulation in BBA - Bioenergetics 1997,
Lazazzera BA, Bates DM, Kiley PJ: The activity of the Escherichia co/i transcription factor FNR is regulated by a change in oligomeric state. Genes Dev 1993, 7:1993-2005.
from
Kennedy MC, Mende-Mueller L, Blondin GA, Beinert H: Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Nat/ Acad Sci USA 1992,89:11730-11734.
is active
16.
froc
24.
Y:
12. .
18.
21.
TA, Harford JB: Regulating the fate of mRNA: iron metabolism. Cell 1993, 72:19-28.
Smith JL, Zaluzec EJ, Wery J-P, Niu L, Switzer RL, Zalkin Structure of the allosteric regulatory enzyme of purine biosynthesis. Science 1994, 264:1427-l 433.
Zheng L, Cash VL, Flint DH, Dean DR: Assembly of iron-sulfur clusters: identification of an iscSUA-hscBA-fdx gene cluster Azotobacter vinelandii. J Biol Chem 1998, 273:13264-l 3272.
23. ..
Chemistry,
by the global FEMS Microbial of knowledge
clusters.
Popescu C, Bates D, Mijnck E, Beinert H: Mijssbauer spectroscopy as a tool for the study of activation/inactivation of the transcription regulator FNR in whole cells of Escherichia co/i. Nat/ Acad Sci USA 1998, 9613431-l 3435.
sensitivity of the 266:i 9328-19333.
ferritin of the
Escherichia
of Fe-S cluster Chem 1993,
Schalinske KL, Anderson SA, Tuazon PT, Chen OS, Kennedy MC, Eisenstein RS: The iron-sulfur cluster of iron regulatory protein modulates the accessibility of RNA binding and phosphorylation sites. Biochemistry 1997, 36:3950-3958. Pantopoulos by oxidative @5:10559-l
K, Hentze MW: stress in vitro. 0563.
is
1
Activation of iron regulatory protein-i Proc Nat/ Acad Sci USA 1998,
Eisentstein RS, Kennedy MC, Beinert HB: The iron responsive element (IRE), the iron regulatory protein (IRP), and the cytosolic aconitase; postranscriptional regulation of mammalian iron metabolism. In Metal ions in Gene Regulation. Edited by Walden W, Silver S. New York: Chapman and Hall; 1998:157-216. This is an extensive survey and discussion of the subject.
37. ..
Fe-S
38.
Wilson TJG, Bertrand N, Tang J-L, Feng J-X, Pan M-Q, Barber GE, Dow JM, Daniels MJ: The rpfA gene of Xantbomonas campestfis pathovar campesfris, which is involved in the regulation of pathogenic@ factor production, encodes an aconitase. MO/ M;crobio/ 1998, 28:961-970.
39.
Michaels ML. Pham L. Nahiem Y. Cruz C. Miller JH: MutY, glycosylaseactive on &A misPairs, has homology to endonuclease III. Nucleic Acids Res 1990, 18:3841-3845.
40.
Porello SL, Cannon MJ, David SS: A substrate recognition the 14Fe-4Sl*+ cluster of the DNA repair glycosylase Biochemistry 1998, 37:6465-6475.
41.
proteins
in sensing
and
regulatory
functions
Beinert
and
Kiley
157
potentials
of
50.
Lanzilotta WN, Seefeldt LC: Changes in the midpoint the nitrogenase metal centers as a result of iron protein-molybdenum-iron protein complex formation. Biochemistry 1997, 36:12976-l 2983.
51.
Lanzilotta WN, Fisher K, Seefeldt LC: Evidence for electron from the nitrogenase iron protein to the molybdenum-iron without MgATP hydrolysis: characterization of a tight protein-protein complex. Biochemistry 1996, 35:7188-7196.
for
52.
lkeda S, Biswas T, Roy R, lzumi T, Boldogh I, Kurosky A, Sarker AH, Seki S, Mitra S: Purification and characterization of human NTHI, a homolog of Escherichia cc/i endonuclease Ill. J Biol Chem 1998, 273:21585-21593.
Spee JH, Arendsen AF, Wassink Redox properties and electron spectroscopy of the transition vinelandii nitrogenase. FEBS
53
Dailey HA, Finnegan iron-sulfur protein.
54
Lloyd SG, Franc0 R, Moura JJG, Moura I, Ferreira GC, Huynh BH: Functional necessity and physicochemical characteristics of the [2Fe-2Sl cluster in mammalian ferrochelatase. J Am Chem Sot 1996, 118:9892-9900.
an adenine
role MutY.
transfer protein
H, Marritt SJ. Hagen WR, Haaker paramagnetic resonance state complex of Azotobacfer Let! 1998,432:55-58.
MG, Johnson Biochemistry
MK: Human ferrochelatase 1994, 33:403-407.
H:
is an
42.
Wu J, Dunham WR, Weiss B: Overproduction and physical characterization of SoxR, a [2Fe-2Sl protein that governs an oxidative response reguion in fscherichia co/i. J B/o/ Chem 1995, 270:10323-i 0327.
43.
Hidalgo E, Ding HG, Demple B: Redox signal transduction mutations shifting C2FE-2Sl centers of the SoxR sensor-regulator to the oxidized form. Cell 1997, 88:121-l 29.
55
Sellers cluster oxide
44.
Liochev SI, Hausladen A, Beyer WFJ, Fridovich oxidoreductase acts as a paraquat diaphorase the soxRS regulon. Proc /Vat/ Acad SC; USA
56
Furukawa inactivation possible Eiochem
45.
Howard fixation.
57.
Ollagnier S, Mulliez E, Schmidt PP, Eliasson R, Gaillard J, Deronzier C, Bergman T, Graslund A, Reichard P, Fontecave M: Activation of the anaerobic ribonucleotide reductase from Escberkhia co/i. J Biol Cheri, 1997,272:24216-24223.
58
Kulzer R, Pils T, Kappl R, Hiittermann characterization of the polynuclear formate-lyase-activating enzyme. 273:4897-4903.
59
Duin EC, Lafferty ME, Crouse BR, Allen RM, Sanyal I, Flint DH, Johnson MK: [2Fe-2S1 to [4Fe-4Sl cluster conversion in Escherichia co/i biotin synthase. Biochemistry 1997, 36:i 181 l-l 1820.
18, Rees DC: Structural basis Chem Rev 1996, 96:2965-2982.
I: NADPH: ferredoxin and is a member of 1994, 91:1328-l 331.
of biological
Burgess BK, Lowe DJ: Mechanism Chem Rev 1996,96:2983-3011.
of molybdenum
47.
Ferguson SJ: Nitrogen 1998, 2:182-193.
48.
Angove HC, Yoo SJ, Mijnck E: An all-ferrous state of nitrogenase. J Biol Chem 1998, 273:26330-26337.
49.
Musgrave KB, Angove HC, Burgess BK, Hedman B, Hodgson KO: All-ferrous titanium (Ill) citrate reduced Fe protein of nitrogenase: an XAS study of electronic and metrical structure. J Am Chem Sot 1998, 120:5325-5326.
enzymology.
Curr
T, Kohno H, Tokunaga R, Taketani of mammalian ferrochelatase involvement of the iron-sulphur J 1995, 310:533-538.
S: Nitric oxide-mediated in viva and in vitro: cluster of the enzyme.
nitrogen
46.
cycle
VM, Johnson MK, Dailey HA: Function of the [2Fe-2Sl in mammalian ferrochelatase: a possible role as a nitric sensor. Biochemistry 1996, 35:2699-2704.
nitrogenase. Opin
Chem of the
Biol Fe protein
J, Knappe J: Reconstitution and iron-sulfur cluster in pyruvate J Biol Chem 1998,