Available online at www.sciencedirect.com

ScienceDirect Alternative FeS cluster ligands: tuning redox potentials and chemistry Daniel W Bak1,3 and Sean J Elliott1,2 A subset of biological Fe–S clusters contain protein-based ligands other than cysteine (Cys). The most common alternative ligand is histidine, while aspartate, arginine, and threonine ligation have also been identified. With the exception of the 2-Cys, 2-His ligated Rieske clusters, the functions of these uniquely ligated clusters are, in general, poorly understood. Recent functional studies of a set of 3-Cys, 1-His ligated [2Fe– 2S] clusters have begun to highlight the importance of non-Cys ligation in controlling both the redox and chemical properties of these clusters as well as their physiological stability. Here, a survey of non-Cys ligation motifs is examined along with the possible biological roles of these clusters. Addresses 1 Program in Molecular and Cellular Biology and Biochemistry, Boston University, Boston, MA 02215, United States 2 Department of Chemistry, Boston University, Boston, MA 02215, United States 3 Current address: Department of Chemistry, Merkert Chemistry Center, Boston College, 2609 Beacon Street, Chestnut Hill, MA 02467, United States. Corresponding author: Elliott, Sean J ([email protected])

Current Opinion in Chemical Biology 2014, 19:50–58 This review comes from a themed issue on Bioinorganic chemistry Edited by Elizabeth M Nolan and Mitsuhiko Shionoya

1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.12.015

Introduction Iron–sulfur (Fe–S) clusters and iron–sulfur containing proteins represent an evolutionarily ancient motif for redox chemistry. Though first identified in the 1960s and found to contain both iron and ‘acid-labile’ sulfide, Fe–S clusters are found in all domains of life and have evolved to play roles in many biological processes, both as electron transfer units and as more complex redox catalysts. Canonically, these clusters bind to the protein through the sulfur atoms of cysteinate (Cys) residues, such as in the all-Cys ligated Fe–S clusters occurring in small electron transfer proteins known as ferredoxins [1], or as domains of larger protein complexes where they may form part of a chain of electron-shuttling redox cofactors (e.g., the many clusters found in NADH:ubiquinone oxidoreductase [2], hydrogenases [3,4], and respiratory nitrate reductase [5,6]). Additionally, clusters of this type Current Opinion in Chemical Biology 2014, 19:50–58

are sometimes used as redox/iron sensors (e.g., Escherichia coli Fnr [7], SoxR [8], etc.), where oxidation or oxidative damage results in a change in cellular signaling. It has been increasingly observed that one or more of the cluster ligands can be non-Cys in origin: either (1) another thiolate donating ligand like glutathione (GSH), (2) a nonsulfur-based ligand, such as S-adenosyl methionine (SAM), or (3) another protein-based ligand, such as histidine (His). While the first two of these subcategories have been examined in some detail to date, much less information is available for the last of these emerging families of Fe–S clusters with alternative, non-Cys protein-based ligands (Table 1), the subject of this review. As will be shown, the presence of these non-Cys ligands can strongly influence the physical properties of these clusters, including reduction potential, stability, and reactivity.

His-ligand: redox tuning and PCET While many aspects of protein structure can tune cluster reduction potential [9,10], the nature of the actual cluster ligands has perhaps the largest influence. By far, His is the most common evolutionary choice for an alternative Fe–S cluster ligand. Mostly, these His-ligands are conserved within their respective protein families. Figure 1 shows the range of reduction potentials that have been reported for a variety of iron–sulfur cluster types. At neutral pH, clusters with His-ligands have a higher reduction potential than those with only Cys-ligands, due to the neutral charge of the ligating imidazole compared to the negative thiolate ligand. Unlike Cys-ligands, which are incapable of protonation (due to their depressed pKa values), the distal nitrogen of a ligating His can exist in either a neutral protonated form or a de-protonated imidazolate form, where the negatively charged state of the His-ligand results in a significant depression in the reduction potential at higher pH values. Replacement of these [2Fe–2S] cluster His-ligands with a Cys residue results in an approximately 300 mV decrease in reduction potential [11], suggesting that the charge on the cluster ligand strongly affects reduction potential in these systems. Proton-coupled electron transfer (PCET) has been extensively demonstrated for both Rieske clusters that contain two His-ligands at a single Fe [12,13] and CDGSH clusters, containing a single unique His ligand [14]. In both systems, the potential for protonation of the Hisligand is coupled to cluster reduction, resulting in a significant decrease in reduction potential with increasing solution pH. This relationship is in contrast to most all www.sciencedirect.com

Tuning FeS clusters with alternative ligands Bak and Elliott 51

Table 1 Functions of alternately ligated biological iron-sulfur proteins and clusters Cluster

Protein 2-Cys, 2-His [2Fe–2S] bc1/b6f Rieske dioxygenase 3-Cys, 1-His [2Fe–2S] MitoNEET (CDGSH) IscR Grx3/4-Fra2 a 3-Cys, 1-His [4Fe–4S] Hydrogenase Respiratory nitrate reductase NADH:ubiquinone oxidoreductase 3-Cys, 1-Asp [4Fe–4S] Fnr Dark-operative protochlorophyllide reductase Ferredoxins 3-Cys, 1-Arg [2Fe–2S] Biotin synthase 3-Cys, 1-Thr [4Fe–4S] Dissimilatory sulfite reductase 3-Cys, 1-X NsrR Quinohemoprotein alcohol dehydrogenase IscU a

References

Electron transfer/proton translocation Electron transfer

Electron/proton transfer Electron transfer

[17–21] [21–24]

Unclear (regulates mitochondrial iron?) Transcription factor/iron–sulfur cluster sensor Iron homeostasis

Unknown Presence of cluster modulates DNA binding Presence of cluster modulates Atf1 binding

[25,26,27] [28,29,30]

Hydrogen oxidation/proton reduction Reduction of nitrate Oxidation of NADH, reduction of quinone/ubiquinone

Electron transfer Electron transfer Electron transfer

[3,4] [34,35,36] [2,37,38]

Transcription factor/redox sensor Reductive step in chlorophyll a synthesis Electron transfer

Cluster state controls DNA binding Electron transfer

[39,40,41] [42–45]

Electron transfer

[46,47]

Insertion of sulfur into dethiobiotin

Sulfur donor

[48–51]

Reduction of sulfite to sulfide

Electron donor (coupled to siroheme)

[52]

Transcription factor/NO sensor Ethanol oxidation Scaffold for iron-sulfur biosynthesis

Cluster state controls DNA binding Electron transfer Transient, delivered to apo-target

[53–55] [56,57] [58,59]

[31,32,33]

Actual ligation is 3-sulfur, 1-His, where one sulfur is provided by cysteine, one by GSH, and one by an unknown source.

Figure 1

Rubredoxin [Fe]

4-Cys 2-Cys, 2-His 3-Cys, 1-His 2-Cys, 2-Glutathione

[2Fe-2S] 3-Cys, 1-Arg 4-Cys [3Fe-4S]

3-Cys 3-Cys, 1-His

[4Fe-4S]

3-Cys, 1-Asp 4-Cys -800

-600

4-Cys (HiPiP) -400

-200

0

200

400

600

800

Em (S.H.E.) Current Opinion in Chemical Biology

Fe–S cluster midpoint potential ranges for all known native cluster nuclearities and ligand arrangements. Common coloring denotes a similarity in the ligand environments for those cluster types. The more lightly shaded regions of 2-Cys, 2-His, and 3-Cys, 1-His clusters, represent potential ranges accessed by pH dependence. Data used to compose this figure are taken from Refs. [1,13–15,35,46,50,62,70,76,77,80]. www.sciencedirect.com

Current Opinion in Chemical Biology 2014, 19:50–58

52 Bioinorganic chemistry

Cys-ligated [2Fe-2S] clusters, where reduction potentials are relatively pH invariant [15,16]. Thus, above a pH of 8, Rieske clusters display a steep 120 mV/pH change due to the deprotonation of both His-ligands [12,13]. For these clusters the pKox and pKred values are 7.5–9.5 and >12, respectively (these values were recorded for the soluble versions of these Rieske proteins, and not in their native protein complexes, where they may be shifted slightly) [12,13]. A functional role for PCET has been established for Rieske proteins, which fall into two general types, high potential bc1 and b6f Rieske proteins, and the low-potential Rieske-type clusters, found in Rieske dioxygenases, or as soluble ferredoxin components of dioxygenase systems. In both cases, the main role of the cluster is electron transfer, either shuttling an electron between the quinol pool and the c1 heme of the bc1 complex [18] (analogously for b6f complex) or delivering electrons to the non-heme iron of arene dioxygenases [23] (Figure 2). In both systems, ionization of at least one His-ligand appears essential to protein function. Structural studies suggest that in the bc1 complex, the Rieske domain swings between two unique positions [18,20] (Figure 2a); proton translocation is thought to proceed from the quinol to the matrix, using the His-ligand as a proton carrier through a PCET process. In 2-oxoquinoline-8-monooxygenase, reduction of the Rieske center and subsequent His-ligand ionization, results in the formation of a hydrogen-bonding network between a bridging Asp-residue and both the cluster and non-heme iron His-ligands [22–24] (Figure 2b). Structural rearrangement allows oxygen to bind to the previously sterically crowded non-heme iron center and initiate catalysis. This mechanism appears to be common to many Rieske dioxygenase enzymes [60], and presents an intriguing model for His-ligated clusters in mediating small local structural changes upon cluster reduction. In general, the functional role of PCET in other Hisligated clusters has not been established, though it is clear in many of these systems that His-ligation is essential for protein function, even if little change in midpoint potential is seen upon Cys-substitution (e.g., NiFe hydrogenase distal [4Fe–4S] cluster) [61]. Additionally, protonation and oxidation of the mitoNEET cluster have been shown to decrease cluster stability [62], suggesting a functional role for PCET in CDGSH proteins.

His ligated [2Fe–2S] clusters: dynamics and cellular iron Three distinct types of 1-His, 3-Cys ligated [2Fe–2S] cluster protein can be identified currently, represented by the bacterial transcription factor IscR [30], glutaredoxins (Grx) such as Grx3/4-Fra2 [33] and CDGSH-domain proteins like mitoNEET [27]. All three proteins bind a single Fe–S cluster per polypeptide, and appear to play a role in redox and iron sensing (Figure 3). For all three of Current Opinion in Chemical Biology 2014, 19:50–58

Figure 2

(a)

2

2 Heme c1

1

Stigmatellin Cytochrome bc1 Complex

1

Aspox-218

(b)

Aspred-218 Hisox-221

His-86′ His-108′

Hisred-221

Cys-84′ Cys-105′

His-225 Current Opinion in Chemical Biology

Structural visualization of the role of Rieske Fe–S clusters. (a) The complete crystal structure of the bovine bc1 complex, showing the two orientations of the Rieske domains (1 and 2, PDB entries 1bcc and 3h1j), and inset with putative hydrogen bonds to the electron/proton donor (stigmatellin) and acceptor (cytochrome c). Arrows show the movement of the domain from position 1 to position 2. (b) Oxidized (white, PDB entry 1z01) and reduced (grey, PDB entry 1z02) states of the 2oxoquinoline 8-monooxygenase oxygenase Rieske dioxygenase [2Fe– 2S] cluster and non-heme iron site. Red spheres represent iron atoms, while yellow spheres represent sulfide ions. Dashed lines represent putative hydrogen bonds.

these protein families, it has been suggested that these clusters are unstable or that cluster loss is tied to function and that perhaps His-ligation contributes to cluster instability. IscR (iron–sulfur cluster regulator) is a bacterial transcription factor that regulates the expression of not only the iscoperon but a host of other iron-responsive genes [28,63]. IscR is known to bind to DNA in both its apo-form and holo-form [29,64], though the genes that each state activates are different, suggesting that cluster occupancy/availability directly controls gene expression and cell function (Figure 3a). Some Grx proteins are dimeric and bind a [2Fe–2S] cluster that bridge the dimer interface. This cluster has www.sciencedirect.com

Tuning FeS clusters with alternative ligands Bak and Elliott 53

Figure 3

(a)

(b)

X

Fra1

X

Isc operon Type I DNA Binding Site

Holo-Fra2Grx3/4

Fur Fra2 a2 2

Grx3/4 Grx G

Aft1/2

Aft1/2

Suf operon Type II DNA Binding Site

Fra1

+O2

Holo-IscR

Apo-Fra2Grx3/4 Grx3/4

Fra2

Apo-IscR

-O2

Aft1/2 Aft1/2

Aft1/2

Cytosol Nucleus Fur

Isc operon Type I DNA Binding Site

Aft1/2

Suf operon Type II DNA Binding Site

(c)

-O2

Fe Regulon

+O2

Fe pool

Fe pool

Cytosol

Holo-mitoNEET

Apo-mitoNEET

Mitochondrial Intermembrane Space

Mitochondrial Matrix Current Opinion in Chemical Biology

Roles of 1-His ligated [2Fe–2S] cluster proteins. (a) Function of Apo-IscR and Holo-IscR showing the different binding affinities for the two states to type I and type II DNA sites. (b) Function of the Grx3/4-Fra2 dimer, sequestering Atf1/2 into the cytosol in the presence of cluster. (c) Function of mitoNEET in controlling iron entry into the mitochondria. Red spheres represent iron atoms and yellow spheres represent sulfide ions. PDB entry 4hf0 for IscR and 2qh7 for mitoNEET.

a 4-sulfur coordination provided by a single Cys residue from each monomer and 2 molecules of GSH. It has recently been shown that yeast Grx3/4 (Glrx3 in humans) can preferentially interact with Fra2 (BolA2 in humans) to form a different hetero-dimer with a bridging [2Fe–2S] cluster; one His-ligand and a sulfur-based ligand of unknown origin are provided by Fra2 [32,33,65]. Under iron-replete conditions, this hetero-dimer will sequester www.sciencedirect.com

the transcription factor Atf1/2 in the cytosol [66]. Atf1/2 is considered to be important in controlling expression of iron uptake and storage proteins (Figure 3b). While it is clear that in both the case of IscR and the Grx3/ 4-Fra2 dimer that cluster occupancy is key to protein function and that these clusters have a single His-ligand, no biophysical or biochemical data demonstrate how Current Opinion in Chemical Biology 2014, 19:50–58

54 Bioinorganic chemistry

His-ligation contributes to the redox properties or stability of these clusters. On the other hand a good deal of information exists revealing the importance of His-ligation on PCET and cluster stability for mitoNEET, a human CDGSH protein, which is thought to regulate iron entry into the mitochondria through an unidentified mechanism (Figure 3c) [26]. MitoNEET is the founding member of a diverse class of [2Fe–2S] cluster binding proteins, which appear to be widespread throughout the three kingdoms of life [67]. It has been demonstrated that these proteins all contain a 1-His, 3-Cys ligated [2Fe–2S] cluster [68,69,70,71], which is unstable under oxidative and low pH conditions. Mutation to the 4-Cys ligated cluster leads to significantly increased cluster stability [62,72]. This His-ligand-induced cluster lability seen for mitoNEET might be a common mechanism that could also be at work in the IscR and Grx3/4-Fra2 systems. Additionally, the 1-Arg, 3-Cys ligated auxiliary cluster of biotin synthase has been demonstrated to be functionally unstable as part of the reaction cycle [49,50,73]. Initially, the function of this second auxiliary cluster was debated, but is now known to be the origin of the sulfur atom that is inserted into dethiobiotin during the reaction mechanism. One hypothesis for the conservation of Arg-ligation is to allow for a compromise between cluster reactivity and cluster stability, which is needed if the [2Fe–2S] cluster is thought of as more of a co-substrate for sulfur insertion rather than a traditional redox cofactor [50].

His-ligated [4Fe–4S] clusters: modulating electron transport

in complex I, suggest an important role for this cluster in controlling electron flow through the complex. Dissimilatory nitrate reductases also contain a unique, single His-bearing Fe–S cluster in the electron transport ‘wire’ that enables communication to the molybdopterinbased active site [34]. The FS0 cluster (with a single Hisligand) of membrane-associated dissimilatory nitrate reductases is the most proximal cluster to the molybdenum cofactor (Figure 4b) [5]. The role of this Hisligand is unknown, and while the native cluster appears to have a midpoint potential of 55 mV, the His-to-Cys variant, did not display a typical EPR spectrum. The lack of EPR signal for this mutant suggests that the reduction potential was greatly reduced, and that standard reductants are no longer capable of generating the reduced, EPR-active cluster [36,76]. This variant also displayed significantly reduced activity, suggesting that His-ligation is necessary for proper enzyme function, whether by tuning the cluster reduction potential or some further function. Finally, some hydrogenases (both NiFe and FeFe) also contain a 1-His-ligated [4Fe–4S] cluster. Unlike the FS0 cluster, which is proximal to the active site, the hydrogenase 1-His-ligated cluster is often the most distal cluster in these ET chains [3,4]. The His-ligand is located at the protein surface, and in a recent crystal structure of the complete membrane-bound E. coli Hyd1 protein (Figure 4c), the distal nitrogen of the His-ligand is oriented towards one of the carboxylate groups of the heme b cofactor (not within hydrogen bonding distance), suggesting an efficient route for electron transfer. It has been shown for the Desulfovibrio fructosovorans hydrogenase that His-to-Cys mutation results in no significant change in reduction potential, but a dramatic decrease in the rates of both inter-molecular and intra-molecular electron transfer [61]. This observation once again highlights the importance the His-ligand plays in the function of the protein, specifically in helping control the flow of electrons in these systems. It is interesting that Hisligated [4Fe–4S] clusters are only found in these large electron transfer chains, and not in more isolated forms.

1-His ligated [4Fe–4S] clusters have also been identified in a number of protein complexes: NADH:ubiquinone oxidoreductase (Complex I), NiFe and FeFe hydrogenases, and the membrane-associated dissimilatory nitrate reductases (NarGHI). Interestingly, each of these complexes contains a series of Fe-S clusters, of which the 1His ligated cluster is but one (Figure 4). Biophysical analyses for these clusters are complicated by the sheer number of spectroscopically observable and redox active cofactors present, providing an obfuscating background. However, their appearance in key positions for modulating ET chemistry suggests required functions as summarized below.

Other patterns of alternative cluster ligation

In the crystal structure of the respiratory Complex I from Thermus thermophilus, cluster N5 is located in subunit Nqo3 (E. coli NuoG, H. sapien NDUFS1) (Figure 4a), about halfway along the electron transfer pathway (NADH-FMN-N3-N1b-N4-N5-N6a-N6b-N2-quinone) [2,38]. Recent EPR (DEER) and Mo¨ssbauer data suggest that, along with clusters N1a and N6b, cluster N5 is not reduced by NADH, and that this cluster is found in the oxidized state [74,75]. The N5 cluster’s unique ligation (and oxidization state) and the fact that it is 14 A˚ away from the N6a cluster (Figure 4a), the longest ET distance

Unlike Fe–S cluster His-ligands, Asp-ligation is not nearly as well conserved across protein families. Aspligation has been confirmed only for [4Fe–4S] clusters. The [4Fe–4S] cluster of P. furiousus ferredoxin was the first identified 1-Asp-ligated cluster [47]. Additionally, the [4Fe–4S] cluster of the [7Fe–8S] D. africanus ferredoxin III was also found to possess Asp ligation [77]. In other ferredoxins the same Cys-to-Asp substitution is seen, but these form [3Fe–4S] clusters [1]. Additionally, the dark-operative photochlorophyllide reductase (DPOR) from Rhodobacter capsulatus [45] and the FNR bacterial oxygen sensor from Bacillus subtilis [41] were

Current Opinion in Chemical Biology 2014, 19:50–58

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Tuning FeS clusters with alternative ligands Bak and Elliott 55

Figure 4

N4 [4Fe-4S]

(a) FMN

Nqo3

N1a

N1b

N3 N4

N7

N5

14Å

N6a N6b N2

NADH:ubiquinone Oxidoreductase

N5 [4Fe-4S]

N6a [4Fe-4S]

(b)

Moco

bis-MGD Moco

FS0 FS1 FS2 FS4

Nitrate Reductase

FS3

FS0 [4Fe-4S]

heme b heme b

protein surface

(c)

medial cluster [4Fe-4S] b-type heme

NiFe medial

proximal distal

Hydrogenase

heme b

distal cluster [4Fe-4S]

subunit interface Current Opinion in Chemical Biology

Full (left) and localized (right) structures of (a) T. thermophilus NADH:ubiquinone oxidoreductase (PDB entry 4hea), (b) E. coli respiratory nitrate reductase (PDB entry 1q16), and (c) E. coli NiFe hydrogenase (Hyd1, PDB entry 4gd3). On both the left and right panels, the 1-His ligated Fe–S cluster is circled. Red spheres represent iron atoms, yellow spheres represent sulfide ions, and teal spheres represent molybdenum atoms. Dashed lines between amino acid side chains indicate possible hydrogen bonds or proton transfer paths.

each identified as binding a [4Fe–4S] cluster with an Aspligand. Interestingly, in the E. coli FNR protein, the cluster is ligated by all Cys residues [7], which may explain the difference in mode of action for the two proteins [40,78]. It is thought that Asp-ligation in these proteins may decrease reduction potential [79] or control cluster stability [78], though more data are needed to confirm either of these functions.

Conclusions Iron–sulfur clusters display a large range of functionalities, from electron transfer to catalysis to redox sensors, www.sciencedirect.com

all of which can be performed by all-Cys-ligated clusters. As a greater number of iron sulfur clusters with unique non-Cys-ligation are identified, it will be necessary to understand how alternative ligation influences cluster function and reactivity. As described in this review, His-ligation is disproportionately represented in these non-Cys-ligated clusters. Studies of Reiske clusters have demonstrated the functional importance of these His-ligands in controlling reduction potential and facilitating PCET, and that ionization of the His-ligand can help reposition elements of protein structure to control reactivity. Additionally, studies of Current Opinion in Chemical Biology 2014, 19:50–58

56 Bioinorganic chemistry

3-Cys, 1-His ligated [2Fe–2S] clusters (IscR, mitoNEET, and GRx3/4-Fra2) suggest that His-ligation helps tune cluster stability. Further work in these systems is needed to connect His-ligation and cluster instability in these proteins to biological function. Despite the evolutionary distance between these three proteins, the conservation of the 3-Cys, 1-His-ligand set suggests a conserved mechanism for cluster lability. Finally, the least well characterized class of His-ligated clusters are the [4Fe–4S] clusters of large respiratory complexes. These clusters are extremely difficult to study, but their positioning within their respective electron transfer chains suggests an important role in controlling the flow of electrons within these systems. It will be necessary in future studies to be able to parse out the role of these individual clusters from the rest of the electron transfer system. As highlighted here, it is clear that novel Fe–S cluster ligation patterns are emerging in bioinorganic chemistry, suggesting as-of-yet unexplored functions and properties of Fe–Sclusters.

Acknowledgements The authors are grateful for the support of the National Science Foundation (MCB 1122977).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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2.

Sazanov LA, Hinchliffe P: Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 2006, 311:1430-1436.

3.

Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, FontecillaCamps JC: Crystal structure of the nickel–iron hydrogenase from Desulfovibrio gigas. Nature 1995, 373:580-587.

4.

Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC: X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 A˚ resolution. Science 1998, 282:18531858.

5.

Dias JM, Than ME, Humm A, Huber R, Bourenkov GP, Bartunik HD, Bursakov S, Calvete J, Caldeira J, Carneiro C et al.: Crystal structure of the first dissimilatory nitrate reductase at 1.9 A˚ solved by MAD methods. Structure 1999, 7:65-79.

6.

Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, Strynadka NC: Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat Struct Biol 2003, 10:681-687.

7.

Khoroshilova N, Beinert H, Kiley PJ: Association of a polynuclear iron–sulfur center with a mutant FNR protein enhances DNA binding. Proc Natl Acad Sci U S A 1995, 92:2499-2503.

8.

Watanabe S, Kita A, Kobayashi K, Miki K: Crystal structure of the [2Fe–2S] oxidative-stress sensor SoxR bound to DNA. Proc Natl Acad Sci U S A 2008, 105:4121-4126.

9.

Holm RH, Kennepohl P, Solomon EI: Structural and functional aspects of metal sites in biology. Chem Rev 1996, 96:22392314.

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Tuning FeS clusters with alternative ligands Bak and Elliott 57

27. Paddock ML, Wiley SE, Axelrod HL, Cohen AE, Roy M, Abresch EC, Capraro D, Murphy AN, Nechushtai R, Dixon JE, Jennings PA: MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone. Proc Natl Acad Sci U S A 2007, 104:14342-14347. 28. Schwartz CJ, Giel JL, Patschkowski T, Luther C, Ruzicka FJ, Beinert H, Kiley PJ: IscR, an Fe–S cluster-containing transcription factor, represses expression of Escherichia coli genes encoding Fe–S cluster assembly proteins. Proc Natl Acad Sci U S A 2001, 98:14895-14900. 29. Rajagopalan S, Teter SJ, Zwart PH, Brennan RG, Phillips KJ,  Kiley PJ: Studies of IscR reveal a unique mechanism for metaldependent regulation of DNA binding specificity. Nat Struct Mol Biol 2013, 20:740-747. First crystal structure of apo-IscR showing how the IscR protein can bind to two unique DNA binding motifs and providing insight into how cluster occupancy can influence IscR-DNA interactions. 30. Fleischhacker AS, Stubna A, Hsueh KL, Guo Y, Teter SJ, Rose JC,  Brunold TC, Markley JL, Munck E, Kiley PJ: Characterization of the [2Fe–2S] cluster of Escherichia coli transcription factor IscR. Biochemistry 2012, 51:4453-4462. Biophysical characterization of the IscR [2Fe–2S] cluster demonstrating for the first time that His-107 is the fourth cluster ligand.

41. Gruner I, Fradrich C, Bottger LH, Trautwein AX, Jahn D, Hartig E: Aspartate 141 is the fourth ligand of the oxygen-sensing  [4Fe–4S]2+ cluster of Bacillus subtilis transcriptional regulator Fnr. J Biol Chem 2011, 286:2017-2021. Identification of Asp-141 as the fourth clusters ligand of the B. subtilis FNR transcription factor [4Fe–4S] cluster. 42. Nomata J, Swem LR, Bauer CE, Fujita Y: Overexpression and characterization of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus. Biochim Biophys Acta 2005, 1708:229-237. 43. Nomata J, Ogawa T, Kitashima M, Inoue K, Fujita Y: NB-protein (BchN–BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe–S clusters. FEBS Lett 2008, 582:1346-1350. 44. Brocker MJ, Schomburg S, Heinz DW, Jahn D, Schubert WD, Moser J: Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase catalytic complex (ChlN/ChlB). J Biol Chem 2010, 285:27336-27345. 45. Muraki N, Nomata J, Ebata K, Mizoguchi T, Shiba T, Tamiaki H, Kurisu G, Fujita Y: X-ray crystal structure of the lightindependent protochlorophyllide reductase. Nature 2010, 465:110-114.

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46. George SJ, Armstrong FA, Hatchikian EC, Thomson AJ: Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus. Identification of a [4Fe–4S] cluster with one non-cysteine ligand. Biochem J 1989, 264:275-284.

32. Li H, Mapolelo DT, Randeniya S, Johnson MK, Outten CE: Human  glutaredoxin 3 forms [2Fe–2S]-bridged complexes with human BolA2. Biochemistry 2012, 51:1687-1696. Characterization of the Glrx3–BolA2 complex in humans, the homologous complex to the yeast Grx3/4-Fra2. Results confirm that a [2Fe–2S] cluster forms between Glrx3 and BolA2, and spectroscopic properties suggest partial histidine ligation.

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33. Li H, Mapolelo DT, Dingra NN, Keller G, Riggs-Gelasco PJ,  Winge DR, Johnson MK, Outten CE: Histidine 103 in Fra2 is an iron–sulfur cluster ligand in the [2Fe–2S] Fra2–Grx3 complex and is required for in vivo iron signaling in yeast. J Biol Chem 2011, 286:867-876. Biophysical characterization of yeast Fra2, demonstrating that His-103 is one of the 4 iron-sulfur ligands in the Grx3/4-Fra2 complex. 34. Richardson DJ, Berks BC, Russell DA, Spiro S, Taylor CJ: Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 2001, 58:165-178. 35. Rothery RA, Bertero MG, Cammack R, Palak M, Blasco F, Strynadka NC, Weiner JH: The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe–4S] cluster with a high-spin ground state. Biochemistry 2004, 43:5324-5333. 36. Rothery RA, Bertero MG, Spreter T, Bouromand N,  Strynadka NC, Weiner JH: Protein crystallography reveals a role for the FS0 cluster of Escherichia coli nitrate reductase A (NarGHI) in enzyme maturation. J Biol Chem 2010, 285:88018807. Examination of the His-ligated [4Fe–4S] cluster, FS0 in E. coli NarA. Specifically the S = 3/2 signal was used to monitor the FS0 cluster, and in His to Cys variants this signal was lost, but allowed for the determination of a very high 55 mV reduction potential of the cluster in the WT protein. 37. Baradaran R, Berrisford JM, Minhas GS, Sazanov LA: Crystal structure of the entire respiratory complex I. Nature 2013, 494:443-448. 38. Sazanov L (Ed): A Structural Perspective on Respiratory Complex I: Structure and Function of NADH:Ubiquinone Oxidoreductase. Springer; 2012. 39. Reents H, Munch R, Dammeyer T, Jahn D, Hartig E: The Fnr regulon of Bacillus subtilis. J Bacteriol 2006, 188:1103-1112. 40. Reents H, Gruner I, Harmening U, Bottger LH, Layer G, Heathcote P, Trautwein AX, Jahn D, Hartig E: Bacillus subtilis Fnr senses oxygen via a [4Fe–4S] cluster coordinated by three cysteine residues without change in the oligomeric state. Mol Microbiol 2006, 60:1432-1445. www.sciencedirect.com

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62. Bak DW, Elliott SJ: Conserved hydrogen bonding networks of  mitoNEET tune Fe–S cluster binding and structural stability. Biochemistry 2013, 52:4687-4696. Thorough examination of how protein structural elements, the oxidation state, and the protonation state of mitoNEET can control cluster stability and how this impacts overall protein structure. 63. Giel JL, Rodionov D, Liu M, Blattner FR, Kiley PJ: IscR-dependent gene expression links iron–sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol Microbiol 2006, 60:1058-1075. 64. Nesbit AD, Giel JL, Rose JC, Kiley PJ: Sequence-specific binding to a subset of IscR-regulated promoters does not require IscR Fe–S cluster ligation. J Mol Biol 2009, 387:28-41. 65. Li H, Mapolelo DT, Dingra NN, Naik SG, Lees NS, Hoffman BM, Riggs-Gelasco PJ, Huynh BH, Johnson MK, Outten CE: The yeast iron regulatory proteins Grx3/4 and Fra2 form heterodimeric complexes containing a [2Fe–2S] cluster with cysteinyl and histidyl ligation. Biochemistry 2009, 48:9569-9581. 66. Li H, Outten CE: Monothiol CGFS glutaredoxins and BolA-like  proteins: [2Fe–2S] binding partners in iron homeostasis. Biochemistry 2012, 51:4377-4389. Review article about the Grx3/4-Fra2 interactions across multiple species; E. coli, yeast, and human. Specifically the role these complexes play in the overall homeostasis of iron in the cell is examined. 67. Lin J, Zhang L, Lai S, Ye K: Structure and molecular evolution of CDGSH iron–sulfur domains. PLoS ONE 2011, 6:e24790. 68. Tamir S, Zuris JA, Agranat L, Lipper CH, Conlan AR, Michaeli D, Harir Y, Paddock ML, Mittler R, Cabantchik ZI et al.: Nutrientdeprivation autophagy factor-1 (NAF-1): biochemical properties of a novel cellular target for anti-diabetic drugs. PLoS ONE 2013, 8:e61202. 69. Zuris JA, Harir Y, Conlan AR, Shvartsman M, Michaeli D, Tamir S,  Paddock ML, Onuchic JN, Mittler R, Cabantchik ZI et al.: Facile transfer of [2Fe–2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein. Proc Natl Acad Sci U S A 2011, 108:13047-13052. Here cluster transfer from mitoNEET to an apo-acceptor protein was shown in vitro, and that cluster transfer was prevented in the reduced protein and in the H87C mutant.

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