Iron Sulfur Clusters

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IRON-SULFUR CLUSTERS
Affiliation
Joseph T. Jarrett
Department of Chemistry
University of Hawaii at Manoa
Honolulu, Hawaii
United States
jtj@hawaii.edu
Synonyms
FeS Clusters
Definition
Iron-sulfur clusters are polynuclear inorganic cofactors composed of clusters of ferric (Fe3+) and ferrous
(Fe2+) cations and sulfide (S2-) anions.
Basic Characteristics
The most common FeS cluster geometries are the rhomboidal [Fe2S2] cluster* and the cuboidal [Fe3S4]
and [Fe4S4] clusters. These clusters are coordinated within proteins by coordination bonds between the
Fe2+/3+ ions and various heteroatom-containing protein residues, esp. cysteine. FeS clusters are commonly
found as an electron transfer cofactor in small proteins known as ferredoxins, as well as in electron
transport chains or pathways within a variety of redox enzymes. Within a protein environment, the
clusters typically have only two accessible redox states and thus function as one-electron acceptors and
donors. FeS clusters can also have non-redox functions in proteins, including chemical catalysis,
signalling and transcriptional regulation, as well as general electrostatic and structural roles.
Structures and Properties
The structures of FeS clusters are largely dictated by the preference of Fe2+/3+ ions for a tetrahedral
coordination geometry, in which Fe2+/3+ is at the center and the S2- ions and protein ligands are at
alternating corners of a cube (Figure 1). Although technically not an iron-sulfur cluster, the rubredoxin
cofactor is a single Fe2+/3+ ion coordinated by four cysteine thiolate ligands in a tetrahedral geometry, and
is often referenced as a simple model for FeS cluster properties. [Fe2S2]2+ clusters contain two Fe3+ ions
and two S2- ions in a planar rhombus, while the coordinating protein ligands bond to Fe in a plane
perpendicular to the rhombus. Symmetrical clusters with four cysteine thiolate ligands have Fe–S bond
lengths in the range 2.2 – 2.3 Å, while the Fe–Fe distance is in the range 2.70 – 2.75 Å. One common
variant found in nature is known as the Rieske iron-sulfur cluster, in which one Fe is coordinated by
*Various notations commonly used to designate the nuclearity and charge of a cluster include: (Fe2S2)2+, [Fe2S2]2+, or
[2Fe-2S]2+. Explicit inclusion of anionic ligands would alter the stated charge, eg. [Fe2S2(SCys)4]2– would designate a [Fe2S2]2+
cluster coordinated by four cysteine thiolate ligands.

nitrogen atoms from two histidine residues, and the shorter Fe-N bonds (~2.1 Å) result in a distortion of
the otherwise symmetric rhombus.
Larger FeS clusters found in nature generally consist of one or more cuboidal (or cubane-like) structures.
The [Fe4S4]2+ cluster consists of 2 Fe3+ ions and 2 Fe2+ ions in complex with 4 S2- ions, with the atoms at
the corners of a distorted cube, and the cluster is typically ligated by four protein residues coordinated to
each of the Fe sites. Fe–S bond lengths and Fe–Fe distances are essentially indistinguishable from the
range found in [Fe2S2] clusters. The [Fe4S4]2+ cube can be thought of as a fusion of two [Fe2S2]+ rhombs
that have unique nondelocalized electronic properties that can be distinguished using Mössbauer
spectroscopy. The [Fe3S4]+ cluster is generally found in a cuboidal geometry that appears similar to a
[Fe4S4] cluster with one of the Fe atoms removed. Linear [Fe3S4] clusters have also been noted as
products of oxidative degradation but are not commonly found in native proteins. Larger FeS clusters and
mixed clusters that incorporate other metals have been observed in specialized enzymes. For example,
nitrogenase contains an [Fe8S7] cluster (the P cluster) and a [MoFe7S9(O/N)] cluster (the FeMoCo cluster),
carbon monoxide dehydrogenase contains a [NiFe4S4] cluster, and acetyl CoA synthase contains an
[Fe4S4]2+/+ cluster bridged via a cysteine thiolate ligand to a binuclear Ni cofactor. In each of these
examples, the remnant cuboidal [Fe4S4] or [Fe3S4] cluster is incorporated into a larger cluster by bridging
sulfide or thiolate ligands.
Figure 1. Depictions of common FeS cluster structures: Fe3+ from Clostridium pasteurianum rubredoxin
(pdb 4RXN); [Fe2S2]2+ from Anabaena PCC7119 ferredoxin (pdb 1CZP); [Fe3S4]+ from Azotobacter
vinelandii ferredoxin I (pdb 6FD1); [Fe4S4]2+ from Azotobacter vinelandii nitrogenase Fe protein (pdb
1FP6). Only the side-chain (S-Cβ-Cα) of each cysteine thiolate ligand is shown. Color legend: brown,
iron; yellow, sulfur; cyan, carbon; hydrogen atoms are not shown.

FeS clusters are predominantly found in nature as electron transfer cofactors. The typical range of redox
potentials expected for various cluster types is given in Table 1. Most iron-sulfur clusters have low
midpoint potentials (0 mV to –750 mV) that allow these cofactors to participate in electron transfer
chains. In general, [Fe4S4]2+ clusters undergo reduction at very low potentials in the range –300 to –650
mV, while [Fe2S2]2+ and [Fe3S4]+ clusters are reduced at slightly higher potentials in the range from –100
to –450 mV. A small subset of [Fe4S4]2+ clusters, known as HiPiP clusters, instead undergo oxidation to
an [Fe4S4]3+ cluster with potentials in the range from +120 to +360 mV.
FeS clusters in which one of the coordination sites to an Fe atom remains open can participate in chemical
catalysis, where the FeS cluster usually functions as a Lewis acid. The most thoroughly characterized
example is aconitase, a hydrolase that catalyzes isomerization of citrate to isocitrate via dehydration to
cis-aconitate. The resting enzyme contains an [Fe4S4]2+ cluster coordinated by 3 cysteine thiolate ligands
and 1 hydroxide at the unique Fe site. The substrate also binds to the unique Fe site, where coordination
of the citrate hydroxyl group facilitates Lewis acid catalysis of the dehydration/hydration reaction.
[Fe4S4] and [Fe3S4] clusters are typically degraded in air and aerobic protein purification will often result
in depletion or complete loss of the native FeS cluster. This effect is more rapid if the cluster has an open
coordination site, or if a reductant is present that can convert O2 to the strong oxidant superoxide (O2•–).
Furthermore, once the FeS cluster is degraded, the liberated cysteine thiolate ligands can be irreversibly
oxidized to cysteine sulfenic and sulfinic acids. Since these oxidized cysteine residues will not bind FeS
clusters, the result is loss of cofactor binding and protein function.
Physical Characterization
In a new system where an FeS cluster is observed or suspected, information regarding the cluster type and
redox properties is needed to establish function. Various spectroscopic techniques have been applied to
probe the electronic structure of FeS clusters, and depending on instrument availability, many of these
techniques can be used to establish cluster type and redox status (summarized in Table 1).
Initial characterization of an FeS cluster should begin with a careful analysis of the Fe2+/3+ and S2- to
protein stoichiometry. The UV/visible electronic absorption spectrum can also provide limited
information. A protein containing only an [Fe2S2]2+ cluster is brown, stable to air, and exhibits
characteristic broad maxima near 330, 420, and 460 nm, although the relative intensities of these peaks
can vary widely. Proteins containing either [Fe3S4]+ or [Fe4S4]2+ clusters exhibit very broad spectra with
shoulders at 380 – 430 nm.
Assuming the FeS cluster is stable to reduction or oxidation; electron paramagnetic resonance (EPR)
spectroscopy can provide complementary information. Neither [Fe2S2]2+ nor [Fe4S4]2+ clusters are
detectable with standard perpendicular mode EPR spectrometers. However, reduction of these clusters,
for example with sodium dithionite (Na2S2O4), produces [Fe2S2]+ and [Fe4S4]+ clusters that usually exhibit
characteristic axial EPR spectra with g∥ ≈ 2.02 and g⟘ ≈ 1.94. The [Fe2S2]+ cluster usually undergoes slow
magnetic relaxation, and as a result, the spectrum can be detected at temperatures up to 50 K. In contrast,
the [Fe4S4]+ cluster is fast relaxing and can usually only be detected at <20 K. The as-isolated [Fe3S4]+
cluster exhibits a sharp nearly isotropic EPR signal centered at g ≈ 2.01 that disappears upon reduction.
The HiPiP [Fe4S4]3+ cluster usually exhibits an axial spectrum with g∥ ≈ 2.14 and g⟘ ≈ 2.03. Thus, using a
combination of UV/visible and EPR spectra, one can often establish the major cluster type present.
The preferred method for examining the complete Fe environment within a protein is Mössbauer
spectroscopy, which requires 57Fe enrichment, but can potentially provide a wealth of information
regarding cluster type, oxidation state, and electronic structure of the cluster. A zero- or low-magnetic-
field Mössbauer spectrum appears as one or more overlapping doublets, with each doublet characterized
by the centroid isomer shift (δ) and the quadropole splitting parameter (ΔEQ). Each unique Fe or valence-

delocalized Fe system gives a unique characteristic doublet. The oxidation state of Fe can be assigned
based on the isomer shift, with δ ≈ 0.25 mm/s for Fe3+, δ ≈ 0.70 mm/s for Fe2+, and with intermediate
values for mixed valence systems. The specific cluster type can often be assigned based on the zero-field
quadropole splitting parameter, with [Fe2S2]2+ and [Fe3S4]+ clusters exhibiting narrow splitting (ΔEQ ≈ 0.5
– 1 mm/s) characteristic of high-spin Fe3+, while [Fe3S4]0 and [Fe4S4]2+ exhibit broader splitting (ΔEQ ≈ 1 –
2 mm/s) due to the presence of a valence-delocalized Fe2.5+Fe2.5+ pairs. Detailed information regarding the
electronic structure of FeS clusters can be obtained by analysis of Mössbauer spectra obtained in high
magnetic fields.
Other methods that can provide additional information regarding the electronic structure and ligand
environment of FeS clusters include circular dichroism and magnetic circular dichroism spectroscopy,
pulsed EPR methods including electron-nuclear double resonance (ENDOR) spectroscopy and electron
spin-echo envelope modulation (ESEEM) spectroscopy, Raman or resonance Raman spectroscopy, and x-
ray absorption spectroscopy.
Once the cluster type and oxidation state have been established, the electrochemical midpoint potential
for reduction or oxidation (Em, redox potential) of the cluster can be useful in assigning a redox or non-
redox role for the cluster. Typically this is accomplished by recording UV/visible or EPR spectra of
protein samples while slowly reducing with sodium dithionite or oxidizing with sodium ferricyanide. The
cell potential is determined using microelectrodes, and various organic electron-transfer mediators are
added to facilitate more rapid redox equilibration of the system. Spectral changes as a function of cell
potential can be analyzed using the Nernst equation to obtain the redox potential of the FeS cluster.
Table 1. Typical physical constants and spectroscopic parameters associated with each type of FeS
cluster. The range of values is based on studies of clusters found in bacterial ferredoxins. (n.d., not
determined; n.s., no signal)
Mössbauer parameters
Cluster Type
Em value
(reduction)
(mV)
UV/Visible
absorption
(λmax, nm)
EPR
parameters
(g⟘, g∥ or gav)
δ (mm/s)
ΔEQ (mm/s)
[Fe2S2]2+
–440 to –175
330, 420, 460
n.s.
0.25-0.3
0.5-1.0
[Fe2S2]+
n.d.
550
1.94, 2.02
0.25, 0.5-0.7
0.6, 2-4
[Fe3S4]+
–425 to –130
400 - 430
2.01
0.25-0.3
0.5-0.6
[Fe3S4]0
n.d.
n.d.
n.s.
0.3, 0.45-0.5
0.5, 1.5
[Fe4S4]3+
+120 to +360
310, 380, 450
2.03, 2.14
0.25-0.3, 0.4-0.45
0.8-0.9, 0.9-1.1
[Fe4S4]2+
–650 to –280
380 - 400
n.s.
0.45-0.5
1.0-1.5
[Fe4S4]+
n.d.
n.d.
1.94, 2.02
0.45-0.5, 0.55-0.7
1.0-1.5, 1.5-2
Cross-References
→ Electron transfer cofactors
→ Electron transfer proteins
→ EPR
→ Ferredoxins

→ Mitochondrial electron transfer chains
→ Bacterial electron transfer chains
→ Complex I – NADH-ubiquinone oxidoreductase
→ Redox potential
→ Resonance Raman of cofactors
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