8.

27 IronSulfur Clusters in Enzyme Catalysis

J. B. BRODERICK Michigan State University, East Lansing, Michigan, USA
8.27.1 INTRODUCTION 8.27.1.1 Roles for IronSulfur Clusters in Biology 8.27.1.2 Types of Catalytic Biological IronSulfur Clusters 8.27.2 ACONITASE 8.27.2.1 Background and Early Studies 8.27.2.2 The IronSulfur Cluster of Aconitase 8.27.2.3 The Unique Iron Site 8.27.2.4 The Role of the Unique Iron Site in Catalysis 8.27.2.4.1 Mode of substrate binding to the unique site 8.27.2.4.2 X-ray structural studies of aconitase 8.27.2.4.3 Catalytic mechanism of aconitase 8.27.2.5 Aconitase and Iron Homeostasis 8.27.2.6 Other Hydrolytic Enzymes Containing [4Fe4S] Clusters 8.27.3 S-ADENOSYLMETHIONINE-DEPENDENT RADICAL ENZYMES 8.27.3.1 Introduction to the Radical-SAM Superfamily 8.27.3.1.1 Lysine 2,3-aminomutase 8.27.3.1.2 Pyruvate formate-lyase activating enzyme 8.27.3.1.3 Anaerobic ribonucleotide reductase activating enzyme 8.27.3.1.4 Biotin synthase and lipoate synthase 8.27.3.1.5 Spore photoproduct lyase 8.27.3.2 Properties of the IronSulfur Clusters 8.27.3.3 Involvement of the Clusters in Radical Catalysis 8.27.3.4 Interaction of S-Adenosylmethionine with the Clusters 8.27.3.5 A Second IronSulfur Cluster in Biotin Synthase 8.27.4 REFERENCES 739 739 740 740 740 741 741 743 743 743 744 746 747 748 748 748 748 749 749 750 750 751 752 755 755

8.27.1 8.27.1.1

INTRODUCTION Roles for IronSulfur Clusters in Biology

Ironsulfur clusters are among the most ubiquitous and diverse metal-containing structures in biology. Since the early 1960s they have been known for their role in electron transfer, including most notably in the ferredoxins, the mitochondrial electron transport chain, and photosynthesis.13 Electron transfer is perhaps the most obvious role for these clusters, due to the presence of at least two redox states readily accessible under normal biological conditions for most types of FeS clusters. The role of ironsulfur clusters in electron transport is covered in detail elsewhere (see Chapter 8.3). In addition to electron transport, however, a number of other roles have emerged for these clusters, reflecting the fascinating diversity of chemistry accessible to ironsulfur clusters.2,3 For 739

740

IronSulfur Clusters in Enzyme Catalysis

L S Fe S Fe S S L Fe L L

L S Fe S S S L Fe L L Fe S S Fe L L

Fe L

Fe

[4Fe4S] 3+: 3FeIII, 1FeII 2+: 2Fe , 2Fe 1+: 1FeIII,

III II

[3Fe4S] 1+: 3FeIII 0 : 2FeIII, 1FeII

[2Fe2S] 2+: 2FeIII 1+: 1FeIII, 1FeII

3FeII

Figure 1 The three most common types of biological ironsulfur clusters, with the common oxidation states indicated for each. For biological ironsulfur clusters, cluster oxidation states are typically stated as the sum of the charges on the iron ions and the sulfide ions, ignoring the charges on the exogenous ligands (L in this figure). The nominal oxidation states of the iron ions in each cluster are indicated, although it should be emphasized that delocalized mixed-valent states are common in these clusters.

example, ironsulfur clusters function in regulatory roles,4 turning gene expression on or off in response to levels of iron (the iron-responsive element-binding protein or IRE-BP),5,6 oxygen (the FNR protein),79 or superoxide (SoxR).1012 Evidence also points to an essential structural role for ironsulfur clusters in several enzymes, including the DNA repair enzymes endonuclease III13 and MutY.14 Ironsulfur clusters can also be used directly in catalysis of redox chemistry on small molecules, as they are, for example, in carbon monoxide dehydrogenase,15 hydrogenase (see Chapter 8.21), and nitrogenase (see Chapter 8.22). The focus of this chapter will be on recent developments towards understanding the role of the coordination chemistry of ironsulfur clusters in enzymatic catalysis, with an emphasis on systems in which coordination of substrate to the ironsulfur cluster has been demonstrated.

8.27.1.2

Types of Catalytic Biological IronSulfur Clusters

The common types of ironsulfur clusters in biology have been discussed elsewhere (see Chapter 8.3), as have the more complex clusters involved in chemistry such as nitrogen fixation (see Chapter 8.22). Although the [2Fe2S], [3Fe4S], and [4Fe4S] structural types (Figure 1) are common in biology, only the [4Fe4S] cluster is currently known to be directly involved in enzymatic catalysis. A common feature among catalytic [4Fe4S] clusters is the presence of a site-differentiated cluster, with one iron in a unique position coordinated by a noncysteinyl ligand. This unique iron is the site of coordination of substrate during enzymatic catalysis, as will be discussed in further detail in the following sections.

8.27.2 8.27.2.1

ACONITASE Background and Early Studies

Aconitase (citrate(isocitrate)hydro-lyase) catalyzes the isomerization of citrate and isocitrate via cis-aconitate, as shown in Figure 2. The reaction is stereospecific, as shown, with the hydration reactions occurring in a trans orientation across the double bond.1618 Also as shown, the H
OOC H

OH

COO

H2O +H2O

*H COO Citrate

H OOC

COO +H2O COO H2O

OOC H

H*

COO

cis-aconitate
Figure 2 The reactions catalyzed by aconitase.

HO COO Isocitrate

IronSulfur Clusters in Enzyme Catalysis

741

removed during dehydration does not readily exchange with solvent, and therefore can reappear on the adjacent carbon during the rehydration reaction.19 Citrate and isocitrate are intermediates in what is known as the citric acid cycle (or alternatively the TCA (tricarboxylic acid) or Krebs cycle), a central process of energy metabolism in aerobic organisms. Aconitase was first suggested to be an iron enzyme in 1951, when it was shown that it could be re-activated by addition of iron(II) and a reducing agent.20 The form of the iron and its role in catalysis was not known at the time, although it was suggested that binding of substrate to the iron might facilitate catalysis.20

8.27.2.2

The IronSulfur Cluster of Aconitase

Aconitase was first suggested to have an ironsulfur cluster by virtue of the presence of acid-labile sulfide,21 as well as a g = 2.01 EPR signal,22,23 in the purified protein. The ironsulfur cluster giving rise to this g = 2.01 signal was later shown to be a [3Fe4S] cluster, and was characteristic of inactive enzyme.2427 Activation by addition of iron and reductant, or by addition of reductant alone, provided the enzymatically active aconitase, although in the latter case only approximately 75% of full activity was obtained.28 As the active form of aconitase was subsequently shown to be the [4Fe4S]2 form,24 addition of iron and reductant provided the iron needed to occupy the fourth metal site in the cluster, while addition of reductant alone resulted in rearrangement of the clusters, labilizing iron to allow reassembly of [4Fe4S] clusters in 3/4 of the cluster sites. It is of interest to note that yet another known cluster type has been observed in aconitase. When the as-isolated, [3Fe4S] form of aconitase is placed in alkaline conditions (pH > 9), the enzyme becomes purple and develops a distinctive UVvisible spectrum29 which had previously been observed for [Fe3S4(SEt)4]3, a linear three-iron cluster.30 EPR and Mo ssbauer studies confirmed the presence of a linear three-iron cluster in purple aconitase.29 Biochemical studies suggested that, unlike in the case of the cuboidal cluster, the linear 3Fe cluster was coordinated by four cysteinyl ligands.31 Two of the ligands to the cuboidal cluster (C421 and C424) are retained in the linear cluster, while the third original cysteine ligand (C358) is lost. Two cysteines from an adjacent helix (C250 and C257) provide the other two ligands to the linear cluster. X-ray crystallographic studies of aconitase with the cuboidal cluster show that formation of the linear 3Fe cluster and the ligand replacement involved in this transformation require a fairly significant structural reorganization of the protein.32

8.27.2.3

The Unique Iron Site

That aconitase is isolated with a cuboidal [3Fe4S] cluster,33 rather than the catalytically active [4Fe4S]2 cluster, suggested that one of the irons in the [4Fe4S]2 cluster is more labile than the others. Stoichiometric oxidation of [4Fe4S]2/aconitase with ferricyanide also results in quantitative formation of the [3Fe4S] cluster of aconitase, while addition of Fe2 and a thiol reagent regenerates the [4Fe4S]2 cluster.28,34 Studies with a series of iron isotopes demonstrated that iron used to reconstitute the [3Fe4S] enzyme was found only in the unique labile site,28 and thus this site-specific labeling could be used to investigate the unique site.35 For example, addition of 57 FeII to natural-abundance [3Fe4S]-aconitase allowed Mo ssbauer spectroscopic characterization of the unique site.35 Alternatively, 57Fe-enriched aconitase could be oxidized to generate the [3Fe4S] form, and then 56Fe could be substituted into the unique site.24 In this way the labile site (Fea) and the three nonlabile sites (Feb) could be probed independently, and it was demonstrated that each had unique Mo ssbauer parameters, as shown in Table 1.35 Therefore, the labile site was spectroscopically distinct from the three iron sites that constitute the [3Fe4S] cluster.35 The presence of a labile iron site, together with the lack of evidence for the liberation of a free sulfhydryl upon converting the [4Fe4S] to the [3Fe4S] form,36 as well as the evidence that only three cysteine residues were protected from labeling in both the [3Fe4S] and [4Fe4S] forms of aconitase,37 suggested that the labile iron was coordinated by a noncysteinyl ligand. EPR line broadening in H217O had shown the presence of an exchangeable solvent in the vicinity of the cluster, and subsequent ENDOR studies in H217O demonstrated coordination of solvent to the cluster.38 2H ENDOR spectra of aconitase in 2H2O reveal only a single type of exchangeable proton coupled to the cluster, indicating that the solvent is bound as hydroxide rather than water.38 Thus the coordination environment of the cluster in the absence of substrate can be depicted as shown in Figure 3, with the unique site coordinated by a solvent-derived hydroxyl.

742

IronSulfur Clusters in Enzyme Catalysis

Table 1 Mo ssbauer parameters for the iron sites in aconitase.a

Sample [4Fe4S]2 aconitase Fea Feb Fea citrate Feb citrate [4Fe4S] aconitase Fea Feb1 Feb2 = Feb3 Reference values FeIIIS4b FeIIS4b Typical [4Fe-4S]2

EQ (mm s1) 0.83 1.30 1.26 1.83 1.15 2.6 2.6 1.15

 (mm s1) 0.46 0.44 0.84 0.89 0.44 1.00 0.64 0.49 $0.25 $0.70 $0.45

References 35 24 35 35 39 39 39

a Fea refers to the unique iron site. Feb refers to the three other iron sites in the [4Fe4S] cluster. For each set of data, only the indicated site is labeled with 57Fe. Indicated parameters are for T = 4 K. b For the localized valence states in an ironsulfur cluster.

Addition of substrate had a dramatic effect on the Mo ssbauer parameters of the unique (Fea) site, but not the Feb sites (Table 1).35,39 The unique site in the presence of citrate shows two new doublets with parameters given in Table 1. It was suggested, and then later supported on the basis of rapid-freeze-quench Mo ssbauer experiments,39 that the two doublets arose from binding of isocitrate and citrate due to substrate turnover during sample preparation.39 This conclusion was confirmed by Mo ssbauer spectroscopy of structurally characterized single crystals of aconitase with isocitrate bound, which showed a single doublet.40 Regardless of the differences in the Mo ssbauer parameters upon binding citrate vs. isocitrate, in both cases the addition of substrate results in a dramatic perturbation of the Mo ssbauer parameters of the Fea (Table 1), to parameters that are no longer consistent with an iron in a tetrahedral sulfur environment, or even a tetrahedral iron with one thiolate replaced by a nonsulfur ligand.41 The Mo ssbauer results instead pointed to the Fea going to five- or six-coordinate upon binding of substrate, suggesting that substrate was coordinating to the unique Fea.35 Further evidence for direct interaction of the substrate with the cluster was provided by EPR spectroscopy.35 Although the catalytically most active state of aconitase is the EPR-silent [4Fe4S]2, this state could be reduced to the [4Fe4S] state for examination by EPR. The [4Fe4S] state of aconitase appears to retain approximately 30% activity, and exhibits a rhombic EPR signal with g = 2.06, 1.93, 1.86, which is perturbed upon addition of substrates or analogs to a more rhombic signal with g = 2.04, 1.85, and 1.78.34 The increased rhombicity upon addition of substrate supported the conclusions from Mo ssbauer spectroscopy,35 that addition of substrate causes one iron site to become distinct from the other three sites in the cluster. Mo ssbauer spectroscopy of the [4Fe4S] aconitase in the presence of substrate shows an even more dramatic effect on the unique site than in the case of [4Fe4S]2 aconitase, with the Fea parameters indicating largely ferrous character and a localized valence at this site (Table 1).39 The Feb sites also differentiate, with two (Feb2 and Feb3) essentially unaffected by substrate addition, and the third (Feb1), which shows increased ferrous character. That both the Fea and Feb1 sites show increased ferrous character, while the Feb2 and Feb3 sites are essentially unchanged upon substrate binding, requires that electron density be withdrawn either from the bound substrate or

RS Fe OH Fe S Fe Fe S SR S S

RS
Figure 3 The coordination environment of the [4Fe4S] cluster of aconitase as predicted based on biochemical and spectroscopic measurements. The coordinating cysteine residues are indicated as RS.

IronSulfur Clusters in Enzyme Catalysis

743

from the cluster sulfides upon substrate binding.39 The former possibility would be consistent with the role of the cluster in Lewis acid catalysis (vide infra). 57Fe-ENDOR spectroscopy of the site-specifically labeled cluster confirmed these Mo ssbauer results: four inequivalent iron sites were observed, with the Fea undergoing dramatic perturbations upon substrate binding, while the three Feb sites show only minor changes.42

8.27.2.4 8.27.2.4.1

The Role of the Unique Iron Site in Catalysis Mode of substrate binding to the unique site

A series of elegant 17O-ENDOR experiments using substrates site-specifically labeled with 17O provided a clear picture of the interaction of substrate with the unique iron site of the [4Fe4S] cluster of aconitase.43 By a combination of enzymatic and chemical means, citrate specifically 17 O-labeled at the - and -carboxyls, and isocitrate labeled at the -carboxyl, were synthesized. Nitroisocitrate was similarly labeled at the -carboxyl and hydroxyl, or at the hydroxyl alone, with 17 O. ENDOR spectroscopic studies of these labeled substrates bound to aconitase provided clear evidence for the two proposed modes of substrate binding. In the case of 17O-labeled citrate, only samples with label at the C carboxylate showed coupling (15 MHz) to the [4Fe4S] cluster. Nitroisocitrate, an analogue of isocitrate that is incapable of binding in the citrate mode, showed coupling from 17O labels at both the hydroxyl (9 MHz) and the carboxyl (13 MHz) at the C position. These results provided evidence for both the citrate mode (i.e., coordination through the hydroxyl and carboxyl at C) and the isocitrate mode (coordination through the hydroxyl and carboxyl at C) of substrate binding to the unique iron site of aconitase.43 As stated previously, [4Fe4S]-aconitase in H217O exhibits an 17O ENDOR signal, demonstrating solvent coordination to the unique site.38 This 17O ENDOR signal is also observed after addition of substrate, suggesting that substrate binding does not displace the bound solvent.38 Instead, the coordination number of the unique iron is expanded upon substrate binding to accommodate the two additional oxygen ligands. This suggestion is supported by the Mo ssbauer results described previously, which provided evidence for an increase in coordination number of the Fea site.35,39 A schematic representation of substrate binding to the active site of aconitase, derived from the ENDOR studies just described, is shown in Figure 4.

8.27.2.4.2

X-ray structural studies of aconitase

Aconitase was initially crystallized in the [3Fe4S] form, and these crystals could be converted to the [4Fe4S]2 form by addition of ferrous ammonium sulfate.32 Alternatively, anaerobic crystallization methods were used to crystallize the [4Fe4S]2 form directly.40 The ironsulfur cluster of aconitase sits within a solvent-filled cleft in the protein structure and is coordinated by Cys 358, Cys 421, and Cys 424. The [3Fe4S] cluster shows the typically cuboidal geometry, with the open iron site directed toward the active site cleft.32 Conversion to the active [4Fe4S]2 form results in almost no structural perturbation, with the extra iron atom simply being inserted into the vacant site in the cluster. The fourth iron is tetrahedrally coordinated, but with a solvent hydroxide rather than a cysteine as the fourth ligand. (The electron density clearly reveals a bound solvent, and previous ENDOR studies38 had demonstrated that the bound species was associated with only a single proton.)

COO RS Fe Fe S Fe Fe S SR S S OH
OOC

RS COO

HO

Fe S RS

S Fe

S Fe

OH2 O Fe S O SR H
OOC

O COO

RS

Figure 4 Citrate binding to the active site [4Fe4S] cluster of aconitase, as deduced from ENDOR measurements.

744

IronSulfur Clusters in Enzyme Catalysis

RS Fe S RS S Fe S Fe

OH2 Fe S

O O

RS O O RS Fe S S Fe S Fe

OH2 Fe S SR

O O H

O O O O

O SRH O

O Isocitrate

Citrate

Figure 5 Binding of citrate and isocitrate to the catalytic [4Fe4S] cluster.

The X-ray crystallographic studies also confirmed the previous spectroscopic results indicating that substrate binds to this unique iron site.40,4446 Although the equilibrium mixture of substrates should contain 0.88:0.04:0.08 citrate:cis-aconitate:isocitrate, when crystals were grown in the presence of cis-aconitate, only isocitrate was observed to be bound to the cluster.40 The isocitrate bound form was also obtained when crystals were grown in the presence of citrate. The structures show that isocitrate coordinates to the unique iron via one oxygen of the C-carboxylate and the hydroxyl group on C.40 A water is also coordinated to the unique iron in the enzymesubstrate complex, as was previously shown by ENDOR.38 Although six-coordinate, the unique iron environment is distorted from octahedral, with SFeS angles averaging 101 and OFeO angles from the site it would averaging 74 .40 The unique iron is also displaced by approximately 0.2 A occupy in a typical [4Fe4S] cluster. As crystals with citrate bound in the active site could not be obtained, crystals with the analogue nitrocitrate were characterized.40 The structure shows that nitrocitrate binds to the unique iron via the carboxylate and hydroxyl groups of C. This structure confirmed the binding modes deduced from spectroscopic methods, in which isocitrate binds through carboxyl and hydroxyl moieties on C, while citrate binds through the same groups on C.43 In other words, the two binding modes (denoted the citrate mode and the isocitrate mode) are related by a twofold axis of rotation about the CC bond (Figure 5). The observation that isocitrate and nitrocitrate bind with the carboxyl and hydroxyl of C and C, respectively, bound to the unique site, and that the binding modes are related by a twofold axis of rotation about the CC bond, requires that the C carboxyl be situated in opposite orientations in these two binding modes (Figure 5). As cis-aconitate is the intermediate product from which both citrate and isocitrate are formed, this requires that cis-aconitate be able to bind in two different orientations, which have been called the citrate mode and the isocitrate mode.44,47 Dual binding modes for cis-aconitate would also be required to explain the trans addition/ elimination of H2O across the double bond, as shown in Figure 2.19 Use of the substrate analogue -methyl-cis-aconitate, which is converted only to -methyl-isocitrate, provided further evidence for these dual binding modes.45,46 The binding of this substrate analogue is nearly identical to that of isocitrate, with the -carboxylate coordinated to the unique iron of the cluster.

8.27.2.4.3

Catalytic mechanism of aconitase

Together, the biochemical, spectroscopic, and structural data suggest a mechanism for aconitase in which the unique iron site of the [4Fe4S] cluster serves as a Lewis acid in catalysis, binding and polarizing substrate to facilitate the dehydration/rehydration reactions. The use of an ironsulfur cluster in a nonredox role, as well as its function in binding substrates, was novel and unexpected, and was the first indication of the remarkable diversity of these clusters in functions beyond electron transfer. The overall catalytic mechanism of aconitase is shown in Figure 6. Starting with citrate or isocitrate as substrate, substrate binding occurs via coordination of the carboxylate and hydroxyl of C or C, respectively, to the unique iron of the [4Fe4S]2 cluster. In doing so, substrate does not displace the coordinated hydroxyl, although the ENDOR studies demonstrate that the hydroxyl becomes protonated upon substrate binding.38 Therefore, the unique iron of the [4Fe4S] cluster goes from four-coordinate, with three 3-bridging sulfides and a hydroxyl ligand, to six-coordinate, with three 3-bridging sulfides, a water, and the carboxylate and hydroxyl of substrate, coordinated to the iron. This change in coordination number was shown by ENDOR

IronSulfur Clusters in Enzyme Catalysis

745

RS Fe S RS S Fe S Fe

OH2 Fe S

RS O O Fe S B RS S Fe S Fe

OH2 Fe S

O O O B O

O SRH O O

H H*

O SR H

H* Isocitrate O

Citrate

RS Fe S RS S Fe S Fe

OH2 Fe S H

RS O O Fe S RS S Fe S Fe

OH2 Fe S

O O O *H B O

O SR H O O

O SRH O

H *H B

H+

RS Fe S RS S Fe S Fe

OH2 Fe

RS O OH2 O O O O O *H B flip flip RS Fe S S Fe S Fe

OH2 Fe

O OH2 O

O O

S SR

S SR O

Citrate mode

Isocitrate mode

*H B

RS Fe S S

RS OH2 Fe O O *H O B O O RS Fe S S Fe S Fe

OH2 Fe

O HO

O *H O O

O O

HO S Fe SR Fe S O RS

S SR

Figure 6 Catalytic mechanism of aconitase starting from citrate (left) or isocitrate (right).

and by X-ray crystallographic studies,38,40,44 and is consistent with the dramatically altered Mo ssbauer parameters upon substrate binding.35,39 Once substrate is bound, a base in the active site, thought to be the alkoxide form of serine 642,40 abstracts a proton from either C (in the case of citrate) or C (in the case of isocitrate). This proton abstraction produces initially a carbanion intermediate, depicted here as the aci-acid. The fact that the nitro analogs of the substrates are both good structural mimics of the putative aci-acid intermediates and are also good inhibitors of aconitase provides additional evidence for the intermediacy of the aci-acid intermediates. The collapse of the aci-acid intermediate ultimately

746

IronSulfur Clusters in Enzyme Catalysis

results in the protonation (by His 101) and elimination of the C (citrate) or C (isocitrate) hydroxyl as water, leaving cis-aconitate as the other product. Both the water and the cis-aconitate remain bound to the unique iron of the [4Fe4S] cluster. As discussed previously, enzymatic action of aconitase on cis-aconitate can produce either citrate or isocitrate, and it is the mode of binding of cis-aconitate in the enzyme active site that determines the product of the reaction.40,44,47 The involvement of two modes of cis-aconitate binding in aconitase was first suggested by kinetic studies,47 and later supported by the detailed spectroscopic and structural studies that gave so much insight into the aconitase mechanism (vide supra). The two modes of cis-aconitate binding, termed the citrate mode and the isocitrate mode, are related by a twofold axis of rotation about the CC bond, as shown in Figure 6. Once cisaconitate is formed in the active site, it can dissociate and re-bind in either conformation, or alternatively it can be displaced by another cis-aconitate molecule binding in either orientation. The cis-aconitate bound in the active site can then be hydrated by the water bound to the unique site, as shown in Figure 6. If cis-aconitate is bound in the citrate mode, C is closest to the ironbound water and thus water adds to this position, producing citrate. Alternatively, if cis-aconitate is bound in the isocitrate mode, C is closest to the iron-bound water and hydroxyl adds to this position. In either case, the pKa of the bound water is lowered by coordination to the unique iron of the cluster, making water a better nucleophile in the reaction. Coordination of the cis-aconitate also makes C or C (depending on binding mode) a better electrophile. Attack of the bound water/hydroxyl at C or C is accompanied by protonation at C or C, respectively, to produce isocitrate or citrate, respectively, as shown in Figure 6. The protonation accompanying water/ hydroxyl attack appears to occur via Ser 642, the same residue that, in its alkoxyl form, abstracted a proton from citrate or isocitrate to initiate the reaction. In fact, remarkably, tritiumlabeling experiments have shown that the proton abstracted from substrate to initiate the reaction is not readily exchangeable, and is returned to the product upon completion of the reaction.19 Thus, the overall mechanism for aconitase involves an ironsulfur cluster acting as a Lewis acid, coordinating the organic substrates and water, stabilizing reaction intermediates, and making water a better nucleophile and the double bond in cis-aconitate a better electrophile. This is a remarkable role for an ironsulfur cluster, since the cluster itself does not change redox state during catalysis. In fact, other than the unique iron site, none of the rest of the cluster is involved in catalysis in any obvious way. Why nature chose to utilize a relatively complex, labile, and biosynthetically expensive species like an ironsulfur cluster to perform hydrolytic chemistry, the type of chemistry that in other biological systems is adequately performed by mononuclear metal centers, is an intriguing question. It has been suggested that the cluster provides a unique scaffold that allows the iron to undergo the dramatic changes in coordination number required by the proposed mechanism.40 However, this may not be an adequate explanation, since mononuclear iron enzymes are known which can accommodate up to three exogenous ligands for catalysis.48

8.27.2.5

Aconitase and Iron Homeostasis

Perhaps one of the most surprising developments regarding aconitase is its relationship to cellular iron homeostasis. Two types of aconitase are present in cells: mitochondrial aconitase, responsible for the key role in the Krebs cycle as discussed previously, and cytosolic aconitase, which was shown in the 1990s to be the same protein as the iron-responsive protein (IRP, also known as the iron-responsive element-binding protein, IRE-BP).5,49 IRP is a protein that senses iron levels in the cell and translates this information into increased or decreased production of ferritin, the ironstorage protein, and transferrin, the iron-transport protein, as well as other iron-regulated genes.6,50 IRP binds to the messenger RNA (mRNA) of ferritin and transferrin (or other proteins) under low iron conditions; in the case of ferritin mRNA the binding is at the 50 end of the mRNA and prevents further synthesis of ferritin, which is not needed under low iron conditions. In the case of transferrin, IRP binding is at the 30 end of the mRNA and prevents degradation of the mRNA, therefore allowing more transferrin synthesis, which results in more iron being transported into the cell. Under high iron levels, IRP does not bind to either ferritin or transferrin mRNA, and as such it allows synthesis of ferritin, needed under high iron conditions, and prevents further synthesis of transferrin, since the transferrin mRNA is rapidly degraded in the absence of IRP binding. It had been known for some time that the binding or lack of binding of IRP to mRNA was related not to the presence or absence of IRP, but rather to its redox state

IronSulfur Clusters in Enzyme Catalysis

747

and/or its state of metallation. Subsequent detailed studies into IRP and its identification as the cytosolic aconitase revealed the difference between active and inactive IRP: IRP incapable of mRNA binding contained a [4Fe4S] or [3Fe4S] cluster, with the former showing significant aconitase activity, while IRP capable of mRNA binding contained no ironsulfur cluster and no aconitase activity.5,49 Thus IRP/c-aconitase presented one of the first examples of an ironsulfur cluster as a sensor, in which building up or destruction of the cluster was used as a signal for required metabolic changes. The IRP/c-aconitase is also an unusual example of a dual function metalloregulatory protein/metalloenzyme. The mechanism by which the [4Fe4S] cluster of IRP/ c-aconitase is degraded and re-synthesized in response to changing iron levels in the cell, however, is still not well understood, although cluster degradation has been suggested to involve reaction with NO.6

8.27.2.6

Other Hydrolytic Enzymes Containing [4Fe4S] Clusters

Several additional enzymes have been identified that appear to be related to aconitase in the sense that they use an ironsulfur cluster to catalyze dehydratase reactions (see Figure 7).51 Among these are isopropylmalate isomerase, which catalyzes a key reaction in leucine biosynthesis, has sequence homology with aconitase,52 and has spectroscopic features of a [4Fe4S] cluster that can be converted to a [3Fe4S] cluster.53 Fumarase A also contains a catalytically essential [4Fe4S] cluster, which can be converted to a [3Fe4S] cluster upon oxidation, suggesting the presence of a unique iron site.54 Dihydroxyacid dehydratase,55 maleic acid hydratase,56 tartrate dehydratase,57 serine dehydratase,58,59 and phosphogluconate dehydratase60 also appear to belong to this group of [4Fe4S] cluster-containing dehydratase enzymes. In all these cases it is thought that the [4Fe4S] cluster contains a unique iron site whose function is to coordinate and activate substrate for turnover. In addition to the enzymes just mentioned, several others have been proposed to belong to the [4Fe4S]-dehydratase group of enzymes.51

OOC H H

H2O COO OH +H2O

OOC

COO

+H2O H2O

OOC

HO H

COO H

OOC

HO

H COO H COO OH H COO H

H2O +H2O

OOC

H COO COO H O

R HO

H2O +H2O R
OOC HO H

COO OH

OOC

+H2O H2O

D H
OOC

H COO H

H HO

OH COO H

H2O +H2O

OOC

H COO H

Figure 7 Other dehydratases that may use a [4Fe4S] cluster in an analogous manner to aconitase. A. Isopropylmalate isomerase. B. Fumarase. C. Dihydroxyacid dehydratase. D. Maleic acid hydratase. E. Tartrate dehydratase.

748 8.27.3 8.27.3.1

IronSulfur Clusters in Enzyme Catalysis S-ADENOSYLMETHIONINE-DEPENDENT RADICAL ENZYMES Introduction to the Radical-SAM Superfamily

The recently identified radical-SAM61 superfamily comprises a diverse group of enzymes which at first glance bear little resemblance to aconitase and the related dehydratases. However, as we shall see, the radical-SAM enzymes, like aconitase, appear to use a unique iron site in a [4Fe4S] cluster to coordinate substrate. A common feature of the members of the radical-SAM superfamily is the presence in the amino acid sequence of a characteristic cluster-binding motif (CX3CX2C) containing only three cysteines, and evidence suggests that these three cysteines alone are responsible for cluster binding.62 The presence of only three cysteines in the clusterbinding motif is reminiscent of aconitase and related enzymes in that it suggests the presence of a unique, noncysteinyl-coordinated iron in a [4Fe4S] cluster, although the putative noncysteinyl ligand has not been identified for any of the enzymes in this family. The radical-SAM superfamily is so-called because its members catalyze radical reactions and utilize S-adenosylmethionine (SAM or AdoMet) as a required cofactor or co-substrate.61 These enzymes also contain catalytically essential ironsulfur clusters.62 The best-studied members of this family, including lysine aminomutase, pyruvate formate-lyase activating enzyme, anaerobic ribonucleotide reductase, lipoate synthase, and biotin synthase, were being investigated long before the superfamily was named, and have provided important clues to the catalytic mechanism and the role of the ironsulfur cluster and AdoMet.62 Additional members of this family are being identified and isolated at a fairly rapid pace. In fact, the bioinformatics approach that was used to identify the superfamily has revealed hundreds of other putative members throughout the phylogenetic kindgom, from simple unicellular organisms to humans.61

8.27.3.1.1

Lysine 2,3-aminomutase

Lysine 2,3-aminomutase, known alternatively as LAM or KAM, catalyzes a rearrangement reaction of lysine, the interconversion of L-lysine and L--lysine (Figure 8A). The reaction, which is catalytic in AdoMet, is directly analogous to rearrangement reactions catalyzed by coenzyme B12-dependent enzymes,6366 and in fact in both the B12 and the AdoMet-dependent enzymes the key initial step in catalysis is the abstraction of a hydrogen atom to yield a substrate radical intermediate.67,68 In the case of the B12-dependent enzymes, the hydrogen atom is abstracted by a B12-derived 50 -deoxyadenosyl radical intermediate generated via homolytic CoC bond cleavage. In the case of the AdoMet-dependent lysine aminomutase, the hydrogen atom is abstracted by an AdoMet-derived 50 -deoxyadenosyl radical. Such a radical mechanism was originally proposed by Moss and Frey,69 and later supported experimentally by the observation of a kinetically competent lysine radical,7072 as well as by the spectroscopic observation of an allylic analogue of the putative 50 -deoxyadenosyl radical intermediate.73,74 This intriguing parallel between the B12 and the AdoMet-dependent radical enzymes, the intermediacy of a 50 -deoxyadenosyl radical, extends to other members of the radical-SAM superfamily as well. In addition to the requirement for an ironsulfur cluster and AdoMet, LAM also requires pyridoxal 50 -phosphate, which forms an adduct with the substrate during turnover.

8.27.3.1.2

Pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) was identified early on as an iron-dependent protein required to activate pyruvate formate-lyase (PFL) for its essential function in anaerobic glucose metabolism in bacteria, the conversion of pyruvate and coenzyme-A (CoA) to formate and acetyl-CoA.7577 The activation of PFL for this central function involves the generation of a stable and catalytically essential glycyl radical,76 which is generated under anaerobic conditions by PFL-AE (Figure 8B) and is quenched once conditions become increasingly oxic (PFL is not needed under aerobic conditions) by the PFL deactivating enzyme.78 The activation of PFL by PFL-AE, unlike the LAM reaction, involves the stoichiometric consumption of AdoMet, which is converted to methionine and 50 -deoxyadenosine by reductive cleavage of the sulfurcarbon bond of AdoMet.76 Isotopic labeling studies showed early on that the hydrogen atom abstracted from the glycyl residue of PFL ended up at the 50 -carbon of 50 -deoxyadenosine,79 thereby implicating

IronSulfur Clusters in Enzyme Catalysis

749

NH3+

NH3+ HN NH3+ H H H COO H H NH HN B H O NH

H H + H3N H COO

H N O N H C S

H N O N H D COOH

S S

COOH

COOH

COOH

DNA backbone

DNA backbone

N O N NH O N H

O E O

N NH O N NH O

Figure 8 Reactions catalyzed by the radical-SAM superfamily. A. Lysine 2,3-aminomutase. B. Activating enzymes. C. Biotin synthase. D. Lipoic acid synthase. E. Spore photoproduct lyase.

an intermediate adenosyl radical in catalysis. The nature of the iron requirement for PFL-AE remained a mystery until PFL-AE was identified as an ironsulfur protein.80

8.27.3.1.3

Anaerobic ribonucleotide reductase activating enzyme

The anaerobic ribonucleotide reductase (aRNR), like pyruvate formate-lyase, is a bacterial enzyme that operates only under anaerobic conditions. Like PFL, aRNR must be activated for catalysis, which involves the generation of a stable glycyl radical on the enzyme (Figure 8B).81,82 The aRNR activating enzyme, once considered to be merely the 2 subunits of an 22 holoenzyme,83 is now known to function catalytically with respect to the aRNR (i.e., the 2 component).84 However, the activase ( 2) is tightly associated with the reductase (2), unlike the activating enzyme of PFL-AE, which is not tightly associated with PFL. The aRNR activating enzyme has been shown to contain an ironsulfur cluster and to utilize AdoMet in catalysis, converting it stoichiometrically to methionine and 50 -deoxyadenosine.85,86

8.27.3.1.4

Biotin synthase and lipoate synthase

The biotin and lipoate synthases catalyze similar reactions, the insertion of sulfur into unactivated CH bonds to generate essential cofactors (Figures 8C and 8D). The substrate in the case of biotin synthase is dethiobiotin, with a single sulfur inserted into two CH bonds to generate the tetrahydrothiophene ring of biotin. In the case of lipoate synthase, two atoms of sulfur are inserted, one each into the CH bonds at positions 6 and 8 of octanoic acid to produce dihydrolipoate, which is typically isolated in the oxidized form shown in Figure 8D. (The actual

750

IronSulfur Clusters in Enzyme Catalysis

substrate of lipoate synthase is not free octanoic acid, but octanoate bound to acyl-carrier protein (octanoyl-ACP).87) Both biotin synthase88 and lipoate synthase89,90 have been shown to contain ironsulfur clusters, and both require AdoMet for catalysis.87,91 In the case of biotin synthase, there is evidence for hydrogen atom transfer from substrate into deoxyadenosine, thereby implicating an intermediate 50 -deoxyadenosyl in the catalytic mechanism.92 The deoxyadenosyl radical presumably abstracts a hydrogen atom from substrate to generate a substrate radical intermediate, to which sulfur is added. Evidence points to an ironsulfur cluster being the source of the added sulfur, as is discussed further in Section 8.27.3.4.

8.27.3.1.5

Spore photoproduct lyase

Spore photoproduct lyase (SPL) catalyzes the repair of UV-induced DNA damage in Bacillus, and possibly other spore-forming microorganisms (Figure 8E).9395 It has been shown to contain an ironsulfur cluster and to utilize AdoMet in catalysis.96,97 Like lysine aminomutase but unlike the other radical-SAM enzymes discussed above, SPL appears to use AdoMet catalytically; i.e., AdoMet is not consumed during substrate turnover.98 Evidence for a radical mechanism of DNA repair has been obtained, as repair of damaged DNA labeled at C-6 of thymine results in specific label transfer into AdoMet, suggesting that C-6 H atom abstraction by a 50 -deoxyadenosyl radical intermediate is the initial step in DNA repair.98

8.27.3.2

Properties of the IronSulfur Clusters

As stated previously, a common feature among the radical-SAM enzymes is the presence of a distinctive three-cysteine cluster-binding motif,62 as was also seen in aconitase. This conserved cluster-binding motif leads to similarities in the cluster properties among the members of this family. As will be discussed in more detail below, the [4Fe4S] cluster is now known to be the catalytically relevant cluster. However, a distinctive feature of the clusters in these enzymes is their lability; thus, the literature on these enzymes provides evidence for [2Fe2S] clusters, cuboidal and linear [3Fe4S] clusters, and [4Fe4S] clusters in a variety of oxidation states.62 This cluster lability, though somewhat confusing in the early literature on these enzymes, is reminiscent of aconitase, in which all these cluster forms were also observed. However even among the radicalSAM enzymes the degree of cluster lability is quite variable.62 Pyruvate formate-lyase activating enzyme is the member of the radical-SAM family whose cluster properties are most similar to those of aconitase. The cluster in pyruvate formate-lyase activating enzyme is quite labile, and in fact until 1997 it was not known that the enzyme contained an ironsulfur cluster, as all preparations to that time had been done aerobically, under which conditions the cluster falls apart.75 It was initially reported that PFL-AE contained a mixture of [2Fe2S] and [4Fe4S] clusters,80 and subsequent reconstitution studies of the apo enzyme provided evidence for a [4Fe4S] cluster.99 Further studies showed that anaerobic isolation resulted in purification of a form of PFL-AE that contained primarily [3Fe4S] clusters, which upon reduction converted to [4Fe4S] clusters.100,101 This reductive cluster conversion from [3Fe4S] to [4Fe4S]2 clusters even in the absence of added iron was remarkably reminiscent of aconitase (see Section 8.27.2.2), and suggested a labile cluster site. Adding to the similarity to aconitase, Mo ssbauer spectroscopy provided evidence for a linear [3Fe4S] cluster in PFL-AE isolated under appropriate conditions.101 Therefore all of the cluster forms previously identified in aconitase were also found in PFL-AE, and like aconitase it appeared to be relatively simple to interconvert between these cluster forms.101 The other members of the radical-SAM family have shown some, but not all, of the cluster properties observed for PFL-AE. For example, evidence for both [2Fe2S]2 and [4Fe4S]2 clusters have been reported for both biotin synthase102104 and lipoate synthase.89,90,104 In fact, the [2Fe2S] cluster in biotin synthase is quite stable, and thus biotin synthase can be purified aerobically with the [2Fe2S] cluster intact,88 and then reconstituted anaerobically to generate the [4Fe4S] cluster.102105 No significant amount of [3Fe4S] cluster has been observed in biotin synthase, although lipoate synthase appears to contain some [3Fe4S] depending on isolation conditions.87 It had been previously proposed that the reductive cluster conversions (in the absence of added iron) in the radical-SAM enzymes occurred by cluster cannibalization, as had also been

IronSulfur Clusters in Enzyme Catalysis

751

proposed for aconitase. That is, reduction results in labilization of the metalligand bonds, essentially releasing iron and sulfide which can then reassemble into the thermodynamically favored cluster form under reducing conditions. Direct evidence for such release and reabsorption of iron and sulfide during reductive cluster conversions was provided for biotin synthase,106 supporting this general mechanism of cluster conversion in the radical-SAM enzymes. In contrast to these examples is lysine aminomutase, in which the only significant cluster form observed is the [4Fe4S] form, although a [3Fe4S] cluster can be generated upon oxidation.107 However, no [2Fe2S] cluster has been reported for this enzyme. LAM is also the only member of this family in which a [4Fe4S]3 cluster has been observed.107 The ironsulfur cluster in spore photoproduct lyase has not been characterized, although some evidence for [4Fe4S] and [3Fe4S] forms has been obtained.97 In summary, the common CX2CX3C cluster-binding motif found in the radical-SAM enzymes confers some common properties to the clusters in these enzymes, including cluster lability. However, the details of the lability and the precise cluster forms observed vary from enzyme to enzyme.

8.27.3.3

Involvement of the Clusters in Radical Catalysis

The variety of clusters observed in these enzymes, and the cluster lability, led to difficulties in determining unequivocally the active cluster form and oxidation state. Fontecave and co-workers showed that, in the absence of the aRNR (also known as the 2 domain), the aRNR-AE (or 2 domain) in the [4Fe4S] state reacts with AdoMet to generate methionine concomitant with cluster oxidation, with a stoichiometry of 23 methionines per cluster oxidized.85 Although this is not the physiologically relevant reaction of radical generation on aRNR, it does demonstrate the ability of the [4Fe4S]-aRNR-AE to reductively cleave AdoMet. Later studies on aRNR confirmed the stoichiometry of approximately two methionines produced per [4Fe-4S] oxidized, although for aRNR-AE in the presence of the aRNR only one methionine, along with 0.50.9 glycyl radicals, was produced per [4Fe4S] oxidized.86 Evidence for the [4Fe4S] cluster as the active form of lysine aminomutase was obtained by Frey and co-workers, who showed by a combination of EPR spectroscopy and enzyme assays that the [4Fe4S]-LAM generated in the presence of AdoMet was catalytically active.108 Unlike aRNR-AE, however, LAM catalyzes a reversible reductive cleavage of AdoMet, and thus methionine production and cluster oxidation could not be monitored as evidence of turnover. It is of interest to note that in the case of LAM, the presence of AdoMet facilitates reduction to the [4Fe4S] state; very little [4Fe4S] cluster is produced by the reduction of LAM with dithionite in the absence of AdoMet, while the presence of AdoMet or its analogue S-adenosylhomocysteine dramatically increases the quantity of [4Fe4S] produced.108 It is not clear whether the presence of AdoMet affects the redox potential of the cluster or whether some other effect, such as accessibility of the cluster by the reductant, is at work. The most direct demonstration of the involvement of the [4Fe4S] cluster in radical catalysis by the radical-SAM enzymes was obtained in the case of PFL-AE.109 PFL-AE was reduced from the [4Fe4S]2 to the [4Fe4S] state using photoreduction in the presence of 5-deazariboflavin. EPR samples of the enzyme in the presence of AdoMet alone show increasing amounts of [4Fe4S] with increasing times of illumination. (Unlike the aRNR-AE, PFL-AE does not reductively cleave AdoMet at an appreciable rate in the absence of the other substrate, PFL.) Parallel samples to which PFL had been added (addition was performed in the dark to eliminate the exogenous reductant) showed increasing quantities of a glycyl radical EPR signal with increasing illumination time. Spin quantitation of the EPR signals from the parallel PFL-AE/AdoMet and PFL-AE/AdoMet/PFL samples demonstrated a 1:1 correspondence between the amount of [4Fe4S] in the former to the amount of glycyl radical in the latter.109 Furthermore, the [4Fe4S] EPR signal disappears upon addition of PFL and generation of the glycyl radical, thereby suggesting that the [4Fe4S] cluster is oxidized to [4Fe4S]2. It was proposed that the [4Fe4S] cluster provided the electron necessary for reductive cleavage of AdoMet to generate methionine and the putative 50 -deoxyadenosyl radical intermediate.109 Together, the results described for aRNR-AE, LAM, and PFL-AE point to the [4Fe4S] cluster as the catalytically active cluster, and they point to a role for this cluster in providing the electron necessary for reductive cleavage of AdoMet, either reversibly (as in the case of LAM) or irreversibly (as in the cases of the two activating enzymes), as illustrated in Figure 9.62 Therefore,

752
RS Fe X Fe S Fe Fe S SR S [4Fe4S] + S

IronSulfur Clusters in Enzyme Catalysis

RS Fe X Fe S Fe Fe S SR S [4Fe4S] 2+ S

RS

RS

H 3C OOC NH3+ S

H H A O OH

OOC

CH3 S NH3+

H H A O HO OH

H + SH

H H A O

+ S

HO OH Figure 9 A generalized reaction scheme for the radical-SAM enzymes. The [4Fe4S] provides the electron necessary for the reductive cleavage of AdoMet to generate the intermediate adenosyl radical. The adenosyl radical abstracts a hydrogen atom from substrate (SH) to initiate the radical reaction.

HO

although many of the cluster properties of the radical-SAM enzymes are similar to those of aconitase, the precise role of the cluster in catalysis is not. In aconitase, the [4Fe4S]2 cluster serves as a Lewis acid, binding and activating substrate for the dehydratase reaction, but not acting in a redox role. In contrast, the cluster in the radical-SAM enzymes is a redox-active cluster, with the reduced [4Fe4S] state being catalytically active. The oxidized [4Fe4S]2 state is produced either as an intermediate (e.g., LAM) or a product (e.g., aRNR-AE and PFL-AE) as AdoMet is used catalytically or stoichiometry, respectively.

8.27.3.4

Interaction of S-Adenosylmethionine with the Clusters

The involvement of a [4Fe4S] cluster and its role in reductive cleavage of AdoMet led to the question of precisely how the [4Fe4S] cluster was involved in catalysis. Based on the evidence described in the previous section, it was conceivable that the cluster served as a remote electrontransfer center, reducing AdoMet via long-range electron transfer to generate an AdoMet radical which subsequently underwent reductive CS bond cleavage. Alternatively, the cluster might interact directly with AdoMet to mediate this unusual radical generation reaction. A number of pieces of evidence had hinted at the possibility that AdoMet interacted directly with the cluster, including the observation of dramatic changes in the EPR signal line shape of the [4Fe4S] upon addition of AdoMet110,111 and increased ability to reduce the cluster in the presence of AdoMet.108 Additional evidence for a close association between AdoMet and the [4Fe4S] cluster in the radical-SAM enzymes came from selenium K-edge X-ray absorption studies of lysine aminomutase in the presence of the cleaved cofactor S-adenosyl-L-selenomethionine (Se-AdoMet).112 Selenium EXAFS of the LAM/Se-AdoMet complex itself did not show a close contact to an iron of the cluster. However, in the presence of DTT and the substrate analog trans-4,5-dehydrolysine, ) contact to the cofactor was cleaved to deoxyadenosine and selenomethionine, and a close (2.7 A 112 an iron of the [4Fe4S] cluster was observed in the selenium EXAFS. This led the authors to propose a close association between AdoMet and the [4Fe4S] cluster of LAM, with the sulfonium sulfur situated near to the presumed unique iron site of the cluster (Figure 10). Substrate binding would then bring AdoMet closer to the ironsulfur cluster, allowing electron transfer from the cluster and ultimately sulfurcarbon bond cleavage, which would leave methionine in close contact with the unique iron site (Figure 10). Electron-nuclear double resonance (ENDOR) studies of PFL-AE complexed to specifically isotopically labeled AdoMets has revealed the details of the interaction between AdoMet and the cluster in this enzyme.111113 Deuterium ENDOR spectra of PFL-AE in the [4Fe4S] state complexed with methyl-D2-AdoMet showed a pair of peaks centered at the deuteron Larmor frequency and split by the hyperfine coupling to the spin of the cluster.111 Examination of the fielddependence of the coupling showed that it was dipolar in nature, and gave an estimation of the

IronSulfur Clusters in Enzyme Catalysis

OOC

753
OOC

NH3+ CH3 O OH OH substrate S RS RS Fe S Fe S

NH3+ CH3 O A OH OH

RS Fe S RS S Fe S Fe X

Se

Se Fe X

Fe

S SR

Fe

S SR

[4Fe4S]+

[4Fe4S]+

OOC

NH3+ CH3 OH O A RS OH

OOC

NH3+ CH3 O A OH OH

2.7 RS Fe S S Se X Fe S Fe Fe S SR [4Fe4S]2+

RS Fe S S

Se X Fe S Fe Fe S SR [4Fe4S]2+

RS

Figure 10 Interaction of AdoMet with the iron sulfur cluster in LAM to generate the intermediate deoxyadenosyl radical. Adapted from ref. 112.

.111 distance from the nearest deuteron to the closest iron of the cluster of approximately 3.03.8 A Fully consistent results were obtained from 13C-ENDOR studies of [4Fe-4S]/PFL-AE in the presence of AdoMet labeled at the methyl carbon with 13C.111 In this case the estimated , and the coupling could best be modeled distance to the nearest iron of the cluster was 45 A through a combination of through-space and through-bond contributions. The presence of isotropic through-bond contributions to the 13C coupling requires that there be some orbital overlap, in order to provide a pathway for delocalization of unpaired spin density from the cluster. Due primarily to electrostatic considerations, it was proposed that the orbital overlap occurs via a close association of the AdoMet sulfonium with one of the 3-bridging sulfides of the cluster (Figure 11).111 The results just described probed the interaction of AdoMet with the catalytically active [4Fe4S] cluster of PFL-AE. Interaction of AdoMet with the oxidized [4Fe4S]2 cluster cannot be probed directly using ENDOR spectrsocopy, since the [4Fe4S]2 cluster is diamagnetic. In order to probe the interaction with the 2 cluster, therefore, PFL-AE in the [4Fe4S]2 state was mixed

RS Fe S S S Fe O Fe N H2 O

Fe S 4-5 S RS 33.8 C H Ado H H

Figure 11 The interaction of AdoMet with the [4Fe4S] cluster of PFL-AE as deduced from 2H, and 15N ENDOR measurements and 54Fe Mo ssbauer studies.
13

C,

17

O,

754

IronSulfur Clusters in Enzyme Catalysis

with isotopically labeled AdoMet, and then frozen. At 77 K, with the samples frozen in the conformation of the 2 state, the samples were -irradiated to cryoreduce the clusters to the 1 state. Both 2H and 13C-ENDOR with methyl-labeled AdoMet give essentially identical results to those for the [4Fe4S]/PFL-AE/AdoMet complex, suggesting that AdoMet interacts with both oxidation states of the cluster in essentially the same manner.111 The proposal that the sulfonium of AdoMet interacts with a 3-bridging sulfide of the cluster left open the question of the role for the putative unique iron site. A unique iron site was in fact demonstrated in PFL-AE via specific isotopic labeling of the unique iron site with 57Fe, in the same way described previously for aconitase (see Section 8.27.2.3), which allowed probing of the unique site by Mo ssbauer parameters for this unique site ssbauer spectroscopy.114 The Mo in the absence of AdoMet were typical for iron in [4Fe4S]2 clusters (Table 2). Addition of AdoMet, however, dramatically altered the Mo ssbauer parameters (Table 2). The new parameters are inconsistent with coordination of a sulfur to the unique site, but are consistent with an increase in coordination number of the unique iron to 5 or 6, and/or coordination of an ionic ligand.114 Thus it was proposed that the carboxylate of AdoMet coordinated to the unique iron site of the cluster.114 Further ENDOR studies have confirmed such an interaction with the unique site, and point to a role for the unique iron in anchoring AdoMet for catalysis.113 ENDOR studies with the carboxylate labeled with 17O show a strong coupling (12.2 MHz) to the [4Fe4S] cluster, much like what was observed with aconitase with 17O-labeled substrate (1315 MHz).43 This 17O coupling in the PFL-AE/AdoMet complex is consistent with a direct coordination of the carboxylate to the unique iron site of the [4Fe4S] cluster. 13C-ENDOR with the carboxylate carbon labeled with 13C also showed coupling (0.71 MHz) to the cluster, confirming the coordination of the carboxylate group. By comparison, a coupling of 1 MHz was observed for aconitase in the presence of substrate labeled with 13C at the carboxyl group.43 A broad ENDOR signal observed at $8 MHz in [4Fe4S]/PFL-AE samples made with natural abundance AdoMet disappears in samples in which AdoMet was labeled with 15N at the amino position, while a new signal appears corresponding to an 15N coupling of 5.8 MHz.113 This coupling of the amino nitrogen to the cluster clearly demonstrates that the amino group of AdoMet is also coordinated to the unique iron of the cluster. The picture that emerges from the ENDOR results is one in which AdoMet forms a classical five-member ring N/O chelate with the unique iron of the [4Fe4S] cluster, as shown in Figure 11.113 This is analogous to the chelation of substrate hydroxyl and carboxyl to the unique iron in aconitase.43 The sulfonium of AdoMet is thus anchored in place, closely associated with one of the 3-bridging sulfides of the cluster, for an inner-sphere electron transfer from the cluster to AdoMet to initiate catalysis.113 Therefore, although the presence of a unique site in the [4Fe4S] cluster and the chelation of substrate to that unique site are common features of aconitase and PFL-AE, the role for the unique site appears to be distinctly different in these two enzymes. In aconitase, the unique site is a catalytic site, binding and activating substrate for the dehydratase reaction. In PFL-AE, the unique site is an anchor, holding substrate in place so that the reactive end of the substrate, the sulfonium, is positioned properly for the subsequent chemistry to occur. The evidence for coordination of AdoMet to the unique iron site in PFL-AE leads to the question of how these results relate to the interaction of AdoMet in the other radical-SAM enzymes. As described previously, the EXAFS evidence for LAM points to an entirely different type of interaction, in which the sulfonium is closely associated with the unique iron site, rather than a 3-bridging sulfide.112 The potentially different modes of interaction between AdoMet and the [4Fe4S] cluster in these two enzymes may reflect a key difference between these two enzymes: in PFL-AE, AdoMet is consumed as a substrate during turnover, while in LAM AdoMet is a
Table 2 Sample [4Fe4S]2 PFL-AE [4Fe4S]2 b Feac Feac AdoMet
a

Mo ssbauer parameters for the unique site of PFL-AE.a EQ (mm s1) 1.15 1.00 1.12 1.15  (mm s1) 0.45 0.45 0.42 0.72
Fe.
c

References 101 101 114 114

57

Data recorded at 4.2 K.

All sites are labeled with

57

Only the unique site is labeled with

Fe.

IronSulfur Clusters in Enzyme Catalysis

755

catalytic cofactor. Perhaps these different modes of interaction will be found to be characteristic of the distinct ways AdoMet is used by the radical-SAM enzymes, with the enzymes using AdoMet stoichiometrically (aRNR-AE, biotin synthase, lipoate synthase) showing a PFL-AEtype interaction and the enzymes using AdoMet catalytically (e.g., spore photoproduct lyase) showing LAM-type interaction. Further detailed studies on the radical-SAM enzymes will be required to address this intriguing question.

8.27.3.5

A Second IronSulfur Cluster in Biotin Synthase

It should be noted that recent evidence points to a second ironsulfur cluster being present in catalytically active biotin synthase. Using a combination of spectroelectrochemical studies and enzymatic assays, Jarrett and co-workers have shown that the form of biotin synthase present in typical anaerobic assays of the enzyme contains one [4Fe4S]2 and one [2Fe2S]2 cluster.105 These results have been further substantiated by Mo ssbauer spectroscopic experiments that provide evidence for two distinct cluster binding sites in biotin synthase.115 Jarrett and co-workers have also shown that the [2Fe2S]2 cluster is degraded during turnover.116 Together, these results are interpreted as the requirement for two distinct clusters for catalysis by biotin synthase.116 The [4Fe4S] cluster, like the related clusters in the other radical-SAM enzymes, is thought to be the catalytic cluster, generating an adenosyl radical intermediate via reductive cleavage of AdoMet. The [2Fe2S] cluster has been proposed to be the sulfur donor in the biotin synthase reaction, which is consistent with previous suggestions that an ironsulfur cluster was the source of the sulfur for biotin synthesis.117119 Whether similar results will be obtained with lipoic acid synthase, which also catalyzes a sulfur insertion reaction, remains to be determined.

8.27.4

REFERENCES

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Comprehensive Coordination Chemistry II ISBN (set): 0-08-0437486 Volume 8, (ISBN 0-08-0443303); pp 739757