Comprehensive Coordination Chemistry II 2003 BRODERICK.pdf

7/29/2019 Comprehensive Coordination Chemistry II 2003 BRODERICK.pdf

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8.27

IronSulfur Clusters in Enzyme

Catalysis

J. B. BRODERICKMichigan State University, East Lansing, Michigan, USA

8.27.1 INTRODUCTION 7398.27.1.1 Roles for IronSulfur Clusters in Biology 7398.27.1.2 Types of Catalytic Biological IronSulfur Clusters 740

8.27.2 ACONITASE 7408.27.2.1 Background and Early Studies 7408.27.2.2 The IronSulfur Cluster of Aconitase 7418.27.2.3 The Unique Iron Site 7418.27.2.4 The Role of the Unique Iron Site in Catalysis 743

8.27.2.4.1 Mode of substrate binding to the unique site 7438.27.2.4.2 X-ray structural studies of aconitase 743

8.27.2.4.3 Catalytic mechanism of aconitase 7448.27.2.5 Aconitase and Iron Homeostasis 7468.27.2.6 Other Hydrolytic Enzymes Containing [4Fe4S] Clusters 747

8.27.3 S-ADENOSYLMETHIONINE-DEPENDENT RADICAL ENZYMES 7488.27.3.1 Introduction to the Radical-SAM Superfamily 748

8.27.3.1.1 Lysine 2,3-aminomutase 7488.27.3.1.2 Pyruvate formate-lyase activating enzyme 7488.27.3.1.3 Anaerobic ribonucleotide reductase activating enzyme 7498.27.3.1.4 Biotin synthase and lipoate synthase 7498.27.3.1.5 Spore photoproduct lyase 750

8.27.3.2 Properties of the IronSulfur Clusters 7508.27.3.3 Involvement of the Clusters in Radical Catalysis 7518.27.3.4 Interaction of S-Adenosylmethionine with the Clusters 7528.27.3.5 A Second IronSulfur Cluster in Biotin Synthase 755

8.27.4 REFERENCES 755

8.27.1 INTRODUCTION

8.27.1.1 Roles for IronSulfur Clusters in Biology

Ironsulfur clusters are among the most ubiquitous and diverse metal-containing structures inbiology. Since the early 1960s they have been known for their role in electron transfer, includingmost notably in the ferredoxins, the mitochondrial electron transport chain, and photosynthe-sis.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 ofFeS 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 theseclusters, reflecting the fascinating diversity of chemistry accessible to ironsulfur clusters.2,3 For

739


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example, ironsulfur clusters function in regulatory roles,4 turning gene expression on or off inresponse to levels of iron (the iron-responsive element-binding protein or IRE-BP),5,6 oxygen (theFNR protein),79 or superoxide (SoxR).1012 Evidence also points to an essential structuralrole forironsulfur clusters in several enzymes, including the DNA repair enzymes endonuclease III13 andMutY.14 Ironsulfur clusters can also be used directly in catalysis of redox chemistry on smallmolecules, as they are, for example, in carbon monoxide dehydrogenase,15 hydrogenase (seeChapter 8.21), and nitrogenase (see Chapter 8.22). The focus of this chapter will be on recentdevelopments towards understanding the role of the coordination chemistry of ironsulfur clustersin enzymatic catalysis, with an emphasis on systems in which coordination of substrate to theironsulfur 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 (seeChapter 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 involvedin enzymatic catalysis. A common feature among catalytic [4Fe4S] clusters is the presence of asite-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 bediscussed in further detail in the following sections.

8.27.2 ACONITASE

8.27.2.1 Background and Early Studies

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

Fe

S Fe

S Fe

SFe

S

L

LL

L Fe

S Fe

S

SFe

S

L

LL

L L

LLFe

S

Fe

S

3+: 3FeIII, 1FeII

2+: 2FeIII, 2FeII

1+: 1FeIII, 3FeII

[3Fe4S]

1+: 3FeIII

0 : 2FeIII, 1FeII2+: 2FeIII

1+: 1FeIII, 1FeII

[2Fe2S][4Fe4S]

Figure 1 The three most common types of biological ironsulfur clusters, with the common oxidationstates indicated for each. For biological ironsulfur clusters, cluster oxidation states are typically stated asthe 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.

OH

H

OOC

H

COOCOO

H2OOOCH COO

COO +H2O

+H2O H2O

H

HO

OOC

H

COOCOO

*

*

Citrate cis-aconitate IsocitrateFigure 2 The reactions catalyzed by aconitase.

740 IronSulfur Clusters in Enzyme Catalysis


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removed during dehydration does not readily exchange with solvent, and therefore can reappearon the adjacent carbon during the rehydration reaction.19 Citrate and isocitrate are intermediatesin what is known as the citric acid cycle (or alternatively the TCA (tricarboxylic acid) or Krebscycle), a central process of energy metabolism in aerobic organisms. Aconitase was first suggestedto be an iron enzyme in 1951, when it was shown that it could be re-activated by addition ofiron(II) and a reducing agent.20 The form of the iron and its role in catalysis was not known a t 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-labilesulfide,21 as well as a g= 2.01 EPR signal,22,23 in the purified protein. The ironsulfur clustergiving rise to this g= 2.01 signal was later shown to be a [3Fe4S] cluster, and was characteristicof inactive enzyme.2427 Activation by addition of iron and reductant, or by addition of reductantalone, provided the enzymatically active aconitase, although in the latter case only approximately75% of full activity was obtained.28 As the active form of aconitase was subsequently shown to bethe [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 theclusters, 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), theenzyme becomes purple and develops a distinctive UVvisible spectrum29 which had previouslybeen observed for [Fe3S4(SEt)4]

3, a linear three-iron cluster.30 EPR and Mo ssbauer studiesconfirmed the presence of a linear three-iron cluster in purple aconitase.29 Biochemical studiessuggested that, unlike in the case of the cuboidal cluster, the linear 3Fe cluster was coordinated byfour cysteinyl ligands.31 Two of the ligands to the cuboidal cluster (C421 and C424) are retainedin the linear cluster, while the third original cysteine ligand (C358) is lost. Two cysteines from anadjacent helix (C250 and C257) provide the other two ligands to the linear cluster. X-raycrystallographic 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 significantstructural 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 theothers. Stoichiometric oxidation of [4Fe4S]2/aconitase with ferricyanide also results in quanti-tative formation of the [3Fe4S] cluster of aconitase, while addition of Fe2 and a thiol reagentregenerates the [4Fe4S]2 cluster.28,34 Studies with a series of iron isotopes demonstrated thatiron 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 of57FeII to natural-abundance [3Fe4S]-aconitase allowed Mo ssbauer spectroscopic characteriza-tion 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 labilesite (Fea) and the three nonlabile sites (Feb) could be probed independently, and it was demon-strated that each had unique Mo ssbauer parameters, as shown in Table 1.35 Therefore, the labilesite 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 freesulfhydryl upon converting the [4Fe4S] to the [3Fe4S] form,36 as well as the evidence that onlythree cysteine residues were protected from labeling in both the [3Fe4S] and [4Fe4S] forms ofaconitase,37 suggested that the labile iron was coordinated by a noncysteinyl ligand. EPR linebroadening in H2

17O had shown the presence of an exchangeable solvent in the vicinity of the

cluster, and subsequent ENDOR studies in H217

O demonstrated coordination of solvent to thecluster.38 2H ENDOR spectra of aconitase in 2H2O reveal only a single type of exchangeableproton coupled to the cluster, indicating that the solvent is bound as hydroxide rather thanwater.38 Thus the coordination environment of the cluster in the absence of substrate can bedepicted as shown in Figure 3, with the unique site coordinated by a solvent-derived hydroxyl.

IronSulfur Clusters in Enzyme Catalysis 741


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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 newdoublets with parameters given in Table 1. It was suggested, and then later supported on the basisof rapid-freeze-quench Mo ssbauer experiments,39 that the two doublets arose from binding ofisocitrate and citrate due to substrate turnover during sample preparation.39 This conclusion wasconfirmed by Mo ssbauer spectroscopy of structurally characterized single crystals of aconitasewith isocitrate bound, which showed a single doublet.40 Regardless of the differences in theMo ssbauer parameters upon binding citrate vs. isocitrate, in both cases the addition of substrateresults in a dramatic perturbation of the Mo ssbauer parameters of the Fea (Table 1), to para-

meters that are no longer consistent with an iron in a tetrahedral sulfur environment, or even atetrahedral iron with one thiolate replaced by a nonsulfur ligand.41 The Mo ssbauer results insteadpointed to the Fea going to five- or six-coordinate upon binding of substrate, suggesting thatsubstrate was coordinating to the unique Fea.

35

Further evidence for direct interaction of the substrate with the cluster was provided by EPRspectroscopy.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 EPRsignal with g= 2.06, 1.93, 1.86, which is perturbed upon addition of substrates or analogs to amore rhombic signal with g= 2.04, 1.85, and 1.78.34 The increased rhombicity upon addition ofsubstrate supported the conclusions from Mo ssbauer spectroscopy,35 that addition of substratecauses 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 evenmore dramatic effect on the unique site than in the case of [4Fe4S]2 aconitase, with the Feaparameters indicating largely ferrous character and a localized valence at this site (Table 1).39 TheFeb 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 sitesshow increased ferrous character, while the Feb2 and Feb3 sites are essentially unchanged uponsubstrate binding, requires that electron density be withdrawn either from the bound substrate or

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

Sample EQ(mm s1)

(mm s1) References

[4Fe4S]2 aconitaseFea 0.83 0.46 35

Feb 1.30 0.44 24Fea citrate 1.261.83

0.840.89

35

Feb citrate 1.15 0.44 35

[4Fe4S] aconitaseFea 2.6 1.00 39Feb1 2.6 0.64 39Feb2=Feb3 1.15 0.49 39

Reference valuesFeIIIS4

b$0.25

FeIIS4b

$0.70Typical [4Fe-4S]2 $0.45

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 indicatedsite is labeled with 57Fe. Indicated parameters are for T= 4 K. b For the localized valence states in an ironsulfur cluster.

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

OH

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

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

42

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

A series of elegant 17O-ENDOR experiments using substrates site-specifically labeled with 17Oprovided 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 specifically17O-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, with17O. ENDOR spectroscopic studies of these labeled substrates bound to aconitase provided clearevidence 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, showedcoupling from 17O labels at both the hydroxyl (9 MHz) and the carboxyl (13 MHz) at the Cposition. These results provided evidence for both the citrate mode (i.e., coordination through thehydroxyl and carboxyl at C) and the isocitrate mode (coordination through the hydroxyl andcarboxyl 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, demonstra-

ting solvent coordination to the unique site.38 This 17O ENDOR signal is also observed afteraddition 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 toaccommodate the two additional oxygen ligands. This suggestion is supported by the Mo ssbauerresults 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, derivedfrom 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 tothe [4Fe4S]2 form by addition of ferrous ammonium sulfate.32 Alternatively, anaerobic crystal-lization methods were used to crystallize the [4Fe4S]2 form directly.40 The ironsulfur cluster ofaconitase 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 openiron 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 vacantsite in the cluster. The fourth iron is tetrahedrally coordinated, but with a solvent hydroxiderather 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 withonly a single proton.)

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

OH HO COO

OOC

COO

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

O

H

OH2 O

OOC

COO

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



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The X-ray crystallographic studies also confirmed the previous spectroscopic results indicatingthat substrate binds to this unique iron site.40,4446 Although the equilibrium mixture of substratesshould contain 0.88:0.04:0.08 citrate:cis-aconitate:isocitrate, when crystals were grown in thepresence of cis-aconitate, only isocitrate was observed to be bound to the cluster.40 The isocitratebound form was also obtained when crystals were grown in the presence of citrate. The structuresshow 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 enzymesubstratecomplex, as was previously shown by ENDOR.38 Although six-coordinate, the unique ironenvironment is distorted from octahedral, with SFeS angles averaging 101 and OFeO anglesaveraging 74.40 The unique iron is also displaced by approximately 0.2 A from the site it wouldoccupy in a typical [4Fe4S] cluster.

As crystals with citrate bound in the active site could not be obtained, crystals with the analoguenitrocitrate were characterized.40 The structure shows that nitrocitrate binds to the unique ironvia the carboxylate and hydroxyl groups of C. This structure confirmed the binding modesdeduced from spectroscopic methods, in which isocitrate binds through carboxyl and hydroxylmoieties on C, while citrate binds through the same groups on C.43 In other words, the twobinding modes (denoted the citrate mode and the isocitrate mode) are related by a twofold axisof rotation about the CC bond (Figure 5).

The observation that isocitrate and nitrocitrate bind with the carboxyl and hydroxyl of C andC, respectively, bound to the unique site, and that the binding modes are related by a twofoldaxis of rotation about the CC bond, requires that the C carboxyl be situated in oppositeorientations in these two binding modes (Figure 5). As cis-aconitate is the intermediate productfrom which both citrate and isocitrate are formed, this requires that cis-aconitate be able to bindin 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 evidencefor these dual binding modes.45,46 The binding of this substrate analogue is nearly identical to thatof 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 aconitasein which the unique iron site of the [4Fe4S] cluster serves as a Lewis acid in catalysis, binding andpolarizing substrate to facilitate the dehydration/rehydration reactions. The use of an ironsulfurcluster 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 beyondelectron transfer.

The overall catalytic mechanism of aconitase is shown in Figure 6. Starting with citrate orisocitrate as substrate, substrate binding occurs via coordination of the carboxylate and hydroxylof Cor 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 thehydroxyl 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, tosix-coordinate, with three 3-bridging sulfides, a water, and the carboxylate and hydroxyl ofsubstrate, coordinated to the iron. This change in coordination number was shown by ENDOR

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH

OH2 OO

O

O

O

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH

OH2 O

O

O

O

O

Citrate Isocitrate

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



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and by X-ray crystallographic studies,38,40,44 and is consistent with the dramatically alteredMo 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 putativeaci-acid intermediates and are also good inhibitors of aconitase provides additional evidence forthe intermediacy of the aci-acid intermediates. The collapse of the aci-acid intermediate ultimately

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH

OH2 OO

O

O

O

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH

OH2 O

O

O

O

O

Citrate Isocitrate

HH

B

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH

OH2 OO

O

O

O HBHH

+

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH2

OH2 OO

O

O

OBH

B

H

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH

OH2 O

O

O

O

O

BHH+

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

OH2

OH2 O

O

O

O

O

BHCitrate mode Isocitrate mode

flip

flip

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

OHO

OH2 OO

O

O

OB

H

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

O

HO

OH2 O

O

O

O

O

B

H

**

**

**

**

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



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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-aconitateremain bound to the unique iron of the [4Fe4S] cluster.

As discussed previously, enzymatic action of aconitase on cis-aconitate can produce eithercitrate or isocitrate, and it is the mode of binding of cis-aconitate in the enzyme active site thatdetermines 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 detailedspectroscopic and structural studies that gave so much insight into the aconitase mechanism (videsupra). 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 CCbond, as shown in Figure 6. Once cis-aconitate is formed in the active site, it can dissociate and re-bind in either conformation, oralternatively 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 uniquesite, as shown in Figure 6. If cis-aconitate is bound in the citrate mode, C is closest to the iron-bound water and thus water adds to this position, producing citrate. Alternatively, if cis-aconitateis bound in the isocitrate mode, C is closest to the iron-bound water and hydroxyl adds to thisposition. In either case, the pKa of the bound water is lowered by coordination to the unique ironof the cluster, making water a better nucleophile in the reaction. Coordination of the cis-aconitatealso makes C or C (depending on binding mode) a better electrophile. Attack of the boundwater/hydroxyl at C or C is accompanied by protonation at Cor C, respectively, to produceisocitrate 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, tritium-labeling experiments have shown that the proton abstracted from substrate to initiate the reactionis 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 Lewisacid, coordinating the organic substrates and water, stabilizing reaction intermediates, andmaking 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 redoxstate during catalysis. In fact, other than the unique iron site, none of the rest of the cluster isinvolved 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 hydrolyticchemistry, the type of chemistry that in other biological systems is adequately performed bymononuclear metal centers, is an intriguing question. It has been suggested that the clusterprovides a unique scaffold that allows the iron to undergo the dramatic changes in coordinationnumber required by the proposed mechanism.40 However, this may not be an adequate explan-ation, since mononuclear iron enzymes are known which can accommodate up to three exo-genous 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 cellulariron homeostasis. Two types of aconitase are present in cells: mitochondrial aconitase, responsiblefor the key role in the Krebs cycle as discussed previously, and cytosolic aconitase, which wasshown in the 1990s to be the same protein as the iron-responsive protein (IRP, also known as theiron-responsive element-binding protein, IRE-BP).5,49 IRP is a protein that senses iron levels inthe cell and translates this information into increased or decreased production of ferritin, the iron-storage protein, and transferrin, the iron-transport protein, as well as other iron-regulatedgenes.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 mRNAand prevents further synthesis of ferritin, which is not needed under low iron conditions. In thecase of transferrin, IRP binding is at the 30 end of the mRNA and prevents degradation of themRNA, therefore allowing more transferrin synthesis, which results in more iron being trans-

ported into the cell. Under high iron levels, IRP does not bind to either ferritin or transferrinmRNA, and as such it allows synthesis of ferritin, needed under high iron conditions, andprevents further synthesis of transferrin, since the transferrin mRNA is rapidly degraded in theabsence of IRP binding. It had been known for some time that the binding or lack of binding ofIRP to mRNA was related not to the presence or absence of IRP, but rather to its redox state



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and/or its state of metallation. Subsequent detailed studies into IRP and its identification as thecytosolic aconitase revealed the difference between active and inactive IRP: IRP incapable ofmRNA binding contained a [4Fe4S] or [3Fe4S] cluster, with the former showing significantaconitase activity, while IRP capable of mRNA binding contained no ironsulfur cluster and noaconitase activity.5,49 Thus IRP/c-aconitase presented one of the first examples of an ironsulfurcluster 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 functionmetalloregulatory 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 reactionwith 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 thatthey use an ironsulfur cluster to catalyze dehydratase reactions (see Figure 7).51 Among these areisopropylmalate 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 beconverted 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 aunique 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 forturnover. In addition to the enzymes just mentioned, several others have been proposed to belongto the [4Fe4S]-dehydratase group of enzymes.51

H

H

OOC

COO

OH

H2O

H

OOC COO

+H2OH2O+H2O

H

OOC H

COO

H2O

+H2O

H

HO

H

OOC

COO

H

COO

HO

R

OHH

H2O

+H2O R

COO

OH

H

OOC COO

H H2O

+H2O H

H

HO

OOC

COO

H

O

OOC H

COO

H

H2O

+H2O

OH

HO

H

OOC

COO

H

A

B

D

COO

H

R

O

H

HO

OOCCOO

H

E

C

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.



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8.27.3 S-ADENOSYLMETHIONINE-DEPENDENT RADICAL ENZYMES

8.27.3.1 Introduction to the Radical-SAM Superfamily

The recently identified radical-SAM61 superfamily comprises a diverse group of enzymes whichat 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 super-family 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 cysteinesalone are responsible for cluster binding.62 The presence of only three cysteines in the cluster-binding motif is reminiscent of aconitase and related enzymes in that it suggests the presence of aunique, noncysteinyl-coordinated iron in a [4Fe4S] cluster, although the putative noncysteinylligand 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 andutilize S-adenosylmethionine (SAM or AdoMet) as a required cofactor or co-substrate.61 Theseenzymes also contain catalytically essential ironsulfur clusters.62 The best-studied members of thisfamily, including lysine aminomutase, pyruvate formate-lyase activating enzyme, anaerobic ribo-nucleotide reductase, lipoate synthase, and biotin synthase, were being investigated long beforethe superfamily was named, and have provided important clues to the catalytic mechanism andthe role of the ironsulfur cluster and AdoMet.62 Additional members of this family are beingidentified and isolated at a fairly rapid pace. In fact, the bioinformatics approach that was used toidentify the superfamily has revealed hundreds of other putative members throughout the phylo-genetic 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 rearrangementreaction 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 bycoenzyme B12-dependent enzymes,6366 and in fact in both the B12 and the AdoMet-dependentenzymes the key initial step in catalysis is the abstraction of a hydrogen atom to yield a substrateradical intermediate.67,68 In the case of the B12-dependent enzymes, the hydrogen atom isabstracted by a B12-derived 50-deoxyadenosyl radical intermediate generated via homolyticCoC bond cleavage. In the case of the AdoMet-dependent lysine aminomutase, the hydrogenatom is abstracted by an AdoMet-derived 50-deoxyadenosyl radical. Such a radical mechanismwas originally proposed by Moss and Frey,69 and later supported experimentally by the observa-tion of a kinetically competent lysine radical,7072 as well as by the spectroscopic observation ofan allylic analogue of the putative 50-deoxyadenosyl radical intermediate.73,74 This intriguingparallel between the B12 and the AdoMet-dependent radical enzymes, the intermediacy of a50-deoxyadenosyl radical, extends to other members of the radical-SAM superfamily as well. Inaddition to the requirement for an ironsulfur cluster and AdoMet, LAM also requires pyridoxal50-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-dependentprotein required to activate pyruvate formate-lyase (PFL) for its essential function in anaerobicglucose metabolism in bacteria, the conversion of pyruvate and coenzyme-A (CoA) to formateand acetyl-CoA.7577 The activation of PFL for this central function involves the generation of astable and catalytically essential glycyl radical,76 which is generated under anaerobic conditionsby 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 byPFL-AE, unlike the LAM reaction, involves the stoichiometric consumption of AdoMet, which isconverted to methionine and 50-deoxyadenosine by reductive cleavage of the sulfurcarbon bondof AdoMet.76 Isotopic labeling studies showed early on that the hydrogen atom abstracted fromthe glycyl residue of PFL ended up at the 50-carbon of 50-deoxyadenosine,79 thereby implicating



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an intermediate adenosyl radical in catalysis. The nature of the iron requirement for PFL-AEremained 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 bacterialenzyme 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 22 holoen-zyme,83 is now known to function catalytically with respect to the aRNR (i.e., the 2 compon-ent).84 However, the activase (2) is tightly associated with the reductase (2), unlike theactivating enzyme of PFL-AE, which is not tightly associated with PFL. The aRNR activatingenzyme 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

C

H bonds to generate essential cofactors (Figures 8C and 8D). The substrate in the case ofbiotin synthase is dethiobiotin, with a single sulfur inserted into two CH bonds to generate thetetrahydrothiophene ring of biotin. In the case of lipoate synthase, two atoms of sulfur areinserted, one each into the CH bonds at positions 6 and 8 of octanoic acid to producedihydrolipoate, which is typically isolated in the oxidized form shown in Figure 8D. (The actual

NH3+

COO

H

H

H+H3N

NH3+

COO

NH3+

H

HH

HN

NHO

HH

A B

DC

COOH

HN

NH

O

COOH

HN

NH

OS

COOH

NNH

O

O

NNH

O

O

NNH

O

O

NNH

O

ODNA

backbone

DNA

backbone

HN

NHO

H

COOH

S

S

E

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



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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 containironsulfur 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 impli-cating an intermediate 50-deoxyadenosyl in the catalytic mechanism.92 The deoxyadenosyl radicalpresumably abstracts a hydrogen atom from substrate to generate a substrate radical intermedi-

ate, to which sulfur is added. Evidence points to an ironsulfur cluster being the source of theadded 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 containan ironsulfur cluster and to utilize AdoMet in catalysis.96,97 Like lysine aminomutase but unlikethe 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 mechanismof DNA repair has been obtained, as repair of damaged DNA labeled at C-6 of thymine resultsin specific label transfer into AdoMet, suggesting that C-6 H atom abstraction by a 5 0-deoxyadeno-syl 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 adistinctive three-cysteine cluster-binding motif,62 as was also seen in aconitase. This conservedcluster-binding motif leads to similarities in the cluster properties among the members of thisfamily. As will be discussed in more detail below, the [4Fe4S] cluster is now known to be thecatalytically relevant cluster. However, a distinctive feature of the clusters in these enzymes is theirlability; 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 clusterlability, though somewhat confusing in the early literature on these enzymes, is reminiscent ofaconitase, in which all these cluster forms were also observed. However even among the radical-SAM enzymes the degree of cluster lability is quite variable.62

Pyruvate formate-lyase activating enzyme is the member of the radical-SAM family whosecluster properties are most similar to those of aconitase. The cluster in pyruvate formate-lyaseactivating enzyme is quite labile, and in fact until 1997 it was not known that the enzymecontained 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 amixture of [2Fe2S] and [4Fe4S] clusters,80 and subsequent reconstitution studies of the apoenzyme provided evidence for a [4Fe4S] cluster.99 Further studies showed that anaerobic isol-ation 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 ofaconitase (see Section 8.27.2.2), and suggested a labile cluster site. Adding to the similarity toaconitase, Mo ssbauer spectroscopy provided evidence for a linear [3Fe4S] cluster in PFL-AEisolated under appropriate conditions.101 Therefore all of the cluster forms previously identified inaconitase were also found in PFL-AE, and like aconitase it appeared to be relatively simple tointerconvert between these cluster forms.101

The other members of the radical-SAM family have shown some, but not all, of the clusterproperties 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 purifiedaerobically 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 biotinsynthase, although lipoate synthase appears to contain some [3Fe4S] depending on isolationconditions.87

It had been previously proposed that the reductive cluster conversions (in the absence of addediron) in the radical-SAM enzymes occurred by cluster cannibalization, as had also been

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proposed for aconitase. That is, reduction results in labilization of the metalligand bonds,essentially releasing iron and sulfide which can then reassemble into the thermodynamicallyfavored cluster form under reducing conditions. Direct evidence for such release and reabsorptionof 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 fo rm

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 memberof this family in which a [4Fe4S]3 cluster has been observed.107 The ironsulfur cluster in sporephotoproduct 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 enzymesconfers 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 toenzyme.

8.27.3.3 Involvement of the Clusters in Radical CatalysisThe variety of clusters observed in these enzymes, and the cluster lability, led to difficulties indetermining unequivocally the active cluster form and oxidation state. Fontecave and co-workersshowed that, in the absence of the aRNR (also known as the 2 domain), the aRNR-AE (or 2domain) in the [4Fe4S] state reacts with AdoMet to generate methionine concomitant withcluster oxidation, with a stoichiometry of 23 methionines per cluster oxidized.85 Although this isnot the physiologically relevant reaction of radical generation on aRNR, it does demonstrate theability of the [4Fe4S]-aRNR-AE to reductively cleave AdoMet. Later studies on aRNR con-firmed 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 0.50.9glycyl 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 assaysthat the [4Fe4S]-LAM generated in the presence of AdoMet was catalytically active.108 UnlikeaRNR-AE, however, LAM catalyzes a reversible reductive cleavage of AdoMet, and thusmethionine production and cluster oxidation could not be monitored as evidence of turnover. Itis 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 dithionitein the absence of AdoMet, while the presence of AdoMet or its analogue S-adenosylhomocysteinedramatically increases the quantity of [4Fe4S] produced.108 It is not clear whether the presenceof AdoMet affects the redox potential of the cluster or whether some other effect, such asaccessibility of the cluster by the reductant, is at work.

The most direct demonstration of the involvement of the [4Fe4S] cluster in radical catalysisby 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 reductivelycleave AdoMet at an appreciable rate in the absence of the other substrate, PFL.) Parallelsamples to which PFL had been added (addition was performed in the dark to eliminate theexogenous reductant) showed increasing quantities of a glycyl radical EPR signal with increasingillumination time. Spin quantitation of the EPR signals from the parallel PFL-AE/AdoMet andPFL-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, therebysuggesting 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 theelectron necessary for reductive cleavage of AdoMet, either reversibly (as in the case of LAM) orirreversibly (as in the cases of the two activating enzymes), as illustrated in Figure 9.62 Therefore,



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

8.27.3.4 Interaction of S-Adenosylmethionine with the ClustersThe involvement of a [4Fe4S] cluster and its role in reductive cleavage of AdoMet led to thequestion of precisely how the [4Fe4S] cluster was involved in catalysis. Based on the evidencedescribed in the previous section, it was conceivable that the cluster served as a remote electron-transfer center, reducing AdoMet via long-range electron transfer to generate an AdoMet radicalwhich subsequently underwent reductive CS bond cleavage. Alternatively, the cluster mightinteract directly with AdoMet to mediate this unusual radical generation reaction. A number ofpieces 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] uponaddition of AdoMet110,111 and increased ability to reduce the cluster in the presence of Ado-Met.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 amino-mutase 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 ironof the cluster. However, in the presence of DTT and the substrate analog trans-4,5-dehydrolysine,the cofactor was cleaved to deoxyadenosine and selenomethionine, and a close (2.7 A ) contact toan iron of the [4Fe4S] cluster was observed in the selenium EXAFS.112 This led the authors topropose a close association between AdoMet and the [4Fe4S] cluster of LAM, with the sulfoniumsulfur situated near to the presumed unique iron site of the cluster (Figure 10). Substrate bindingwould then bring AdoMet closer to the ironsulfur cluster, allowing electron transfer from thecluster and ultimately sulfurcarbon bond cleavage, which would leave methionine in close contactwith 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 andthe cluster in this enzyme.111113 Deuterium ENDOR spectra of PFL-AE in the [4Fe4S] statecomplexed with methyl-D2-AdoMet showed a pair of peaks centered at the deuteron Larmorfrequency and split by the hyperfine coupling to the spin of the cluster.111 Examination of the field-dependence of the coupling showed that it was dipolar in nature, and gave an estimation of the

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

X

[4Fe4S]+

OOC

NH3+

S

OAH

HH3C

HO OH

OOC

NH3+

S

CH3

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

X

[4Fe

4S]

2+

OAH

H

HO OH

OAH

H

HO OH

H

+ SH + S

Figure 9 A generalized reaction scheme for the radical-SAM enzymes. The [4Fe4S] providesthe 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.



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distance from the nearest deuteron to the closest iron of the cluster of approximately 3.03.8 A .111

Fully consistent results were obtained from 13C-ENDOR studies of [4Fe-4S]/PFL-AE inthe presence of AdoMet labeled at the methyl carbon with 13C.111 In this case the estimateddistance to the nearest iron of the cluster was 45 A , and the coupling could best be modeledthrough a combination of through-space and through-bond contributions. The presence ofisotropic through-bond contributions to the 13C coupling requires that there be some orbitaloverlap, in order to provide a pathway for delocalization of unpaired spin density from thecluster. Due primarily to electrostatic considerations, it was proposed that the orbital overlapoccurs via a close association of the AdoMet sulfonium with one of the 3-bridging sulfides of thecluster (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 beprobed directly using ENDOR spectrsocopy, since the [4Fe4S]2 cluster is diamagnetic. In orderto probe the interaction with the 2 cluster, therefore, PFL-AE in the [4Fe4S]2 state was mixed

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

X

OOC NH3+

Se

O

A

CH3 OH

OH

[4Fe4S]+ [4Fe4S]+

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

X

OOC NH3+

SeCH3

[4Fe4S]2+ [4Fe4S]2+

O

A

OH

OH

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

X

OOC NH3+

Se

O

A

CH3 OH

OH

S Fe

SFe

S

Fe

Fe

S

RS

RS

SR

X

OOC NH3+

Se

O

A

CH3 OH

OH

substrate

2.7

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

S Fe

SFe

S

Fe

Fe

S

RS

RS

O

NH2

S

C Ado

O

HH H

4-5 33.8

Figure 11 The interaction of AdoMet with the [4Fe4S] cluster of PFL-AE as deduced from 2H, 13C, 17O,and 15N ENDOR measurements and 54Fe Mo ssbauer studies.



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with isotopically labeled AdoMet, and then frozen. At 77 K, with the samples frozen in theconformation of the 2 state, the samples were -irradiated to cryoreduce the clusters to the1 state. Both 2H and 13C-ENDOR with methyl-labeled AdoMet give essentially identical resultsto those for the [4Fe4S]/PFL-AE/AdoMet complex, suggesting that AdoMet interacts withboth 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 factdemonstrated in PFL-AE via specific isotopic labeling of the unique iron site with 57Fe, inthe same way described previously for aconitase (see Section 8.27.2.3), which allowed probingof the unique site by Mo ssbauer spectroscopy.114 The Mo ssbauer parameters for this unique sitein the absence of AdoMet were typical for iron in [4Fe4S]2 clusters (Table 2). Addition ofAdoMet, however, dramatically altered the Mo ssbauer parameters (Table 2). The new parametersare inconsistent with coordination of a sulfur to the unique site, but are consistent with anincrease in coordination number of the unique iron to 5 or 6, and/or coordination of an ionicligand.114 Thus it was proposed that the carboxylate of AdoMet coordinated to the unique ironsite of the cluster.114

Further ENDOR studies have confirmed such an interaction with the unique site, and point toa role for the unique iron in anchoring AdoMet for catalysis.113 ENDOR studies with thecarboxylate 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 17Ocoupling in the PFL-AE/AdoMet complex is consistent with a direct coordination of the carboxy-late to the unique iron site of the [4Fe4S] cluster. 13C-ENDOR with the carboxylate carbonlabeled with 13C also showed coupling (0.71 MHz) to the cluster, confirming the coordination ofthe carboxylate group. By comparison, a coupling of 1 MHz was observed for aconitase in thepresence of substrate labeled with 13C at the carboxyl group.43 A broad ENDOR signal observedat$8 MHz in [4Fe4S]/PFL-AE samples made with natural abundance AdoMet disappears insamples in which AdoMet was labeled with 15N at the amino position, while a new signal appearscorresponding to an 15N coupling of 5.8 MHz.113 This coupling of the amino nitrogen to thecluster clearly demonstrates that the amino group of AdoMet is also coordinated to the uniqueiron 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 inaconitase.43 The sulfonium of AdoMet is thus anchored in place, closely associated with one ofthe 3-bridging sulfides of the cluster, for an inner-sphere electron transfer from the cluster toAdoMet 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 andPFL-AE, the role for the unique site appears to be distinctly different in these two enzymes. Inaconitase, the unique site is a catalytic site, binding and activating substrate for the dehydratasereaction. In PFL-AE, the unique site is an anchor, holding substrate in place so that the reactiveend 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 thequestion 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 differenttype of interaction, in which the sulfonium is closely associated with the unique iron site, ratherthan a 3-bridging sulfide.

112 The potentially different modes of interaction between AdoMet andthe [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 Mo ssbauer parameters for the unique site of PFL-AE.a

Sample EQ(mm s1)

(mm s1) References

[4Fe4S]2 PFL-AE[4Fe4S]2 b 1.15 0.45 101

1.00 0.45 101Fea

c 1.12 0.42 114Fea

cAdoMet 1.15 0.72 114

a Data recorded at 4.2 K. b All sites are labeled with 57Fe. c Only the unique site is labeled with 57Fe.

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catalytic cofactor. Perhaps these different modes of interaction will be found to be characteristicof the distinct ways AdoMet is used by the radical-SAM enzymes, with the enzymes usingAdoMet stoichiometrically (aRNR-AE, biotin synthase, lipoate synthase) showing a PFL-AE-type 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 berequired 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 incatalytically active biotin synthase. Using a combination of spectroelectrochemical studies andenzymatic assays, Jarrett and co-workers have shown that the form of biotin synthase present intypical anaerobic assays of the enzyme contains one [4Fe4S]2 and one [2Fe2S]2 cluster.105 Theseresults have been further substantiated by Mo ssbauer spectroscopic experiments that provideevidence for two distinct cluster binding sites in biotin synthase.115 Jarrett and co-workers havealso shown that the [2Fe2S]2 cluster is degraded during turnover.116 Together, these results areinterpreted 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 thecatalytic 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 thesulfur 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.

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