Characterization of Fe-S Clusters in Proteins by Mössbauer Spectroscopy

Abstract

⁵⁷Fe Mössbauer spectroscopy is unparalleled in the study of Fe-S cluster-containing proteins because of its unique ability to detect all forms of iron. Enrichment of biological samples with the ⁵⁷Fe isotope and manipulation of experimental parameters such as temperature and magnetic field allow for elucidation of the number of Fe-S clusters present in a given protein, their nuclearity, oxidation state, geometry, and ligation environment, as well as any transient states relevant to enzyme chemistry. This chapter is arranged in five sections to help navigate an experimentalist to utilize ⁵⁷Fe Mössbauer spectroscopy for delineating the role and structure of biological Fe-S clusters. The first section lays out the tools and technical considerations for the preparation of ⁵⁷Fe-labeled samples. The choice of experimental parameters and their effects on the Mössbauer spectra are presented in the following two sections. The last two sections provide a theoretical and practical guide on spectral acquisition and analysis relevant to Fe-S centers.

1. Introduction

⁵⁷Fe Mössbauer spectroscopy takes advantage of the recoilless resonant emission and absorption of γ-rays by ⁵⁷Fe nuclei in solids [1–3] and is able to resolve electronic and chemical properties of ⁵⁷Fe nuclei, such as oxidation states, hyperfine interactions, geometry, ligands, etc. [1, 3–5]. ⁵⁷Fe Mössbauer spectroscopy has thus found a wide application in the study of biological Fe-containing cofactors, especially in the field of Fe-S cluster-containing proteins [1, 6–9]. Fe-S clusters have been extensively studied by ⁵⁷Fe Mössbauer, and their spectroscopic parameters can serve as “fingerprints” for identifying their type and redox state [1, 3, 6–8]. Mössbauer spectroscopy is also useful for the quantification of Fe-S clusters, identification of changes in their coordination environment and nuclear configuration, as well as trapping of intermediates or short-lived states [10]. Combined, these factors make ⁵⁷Fe Mössbauer the method of choice for the characterization of Fe-S clusters, yielding invaluable information about the structure and chemistry of these cofactors.

2. Materials

2.1. Basic Mössbauer Spectrometer Setup

1. Mössbauer spectrometer: A typical Mössbauer spectrometer for studies on Fe-S cluster-containing proteins consists of a ⁵⁷Co γ-ray radiation source (10–100 mCi) mounted on a velocity transducer (see below), a cryostat for performing measurements in a temperature range between 1.5 and 220 K, and a γ-ray counting system. A weak permanent magnet (30–80 mT) can be preferentially mounted, which helps with the identification of biological Fe-S clusters. A spectrometer equipped with a superconducting system can be alternatively used to carry out field-dependent measurements at higher magnetic field strengths (i.e., 0.1–10 T).

2.2. Solutions for In Vivo and In Vitro Fe-S Enrichment

1. ⁵⁷Fe solution: 500 mM Fe²⁺ in 2 N H₂SO₄. Dissolve 57 mg of the ⁵⁷Fe metal in 2 mL of 2 N H₂SO₄ (see Note 1). Allow solvation to proceed overnight at room temperature or for 1–2 h at 70 °C. At high temperatures, the solution will bubble due to the release of H₂, so keep the lid loose. Dilute the ⁵⁷Fe stock with water to 125 mM and store indefinitely at −20 °C. Confirm Fe concentration using the ferrozine assay [11]. For in vivo assembly, add the ⁵⁷Fe solution directly to the cell cultures. For reconstitution reactions, prepare and store the ⁵⁷Fe stock solution anaerobically. Prior to addition to the reconstitution mixture, dilute the Fe solution with a buffer compatible with the protein of interest to achieve working concentrations of Fe between 10–50 mM and check the pH with a pH strip. If very acidic, adjust the buffer concentration or re-pH as necessary. Care should be taken to ensure that solutions are strictly O₂-free; otherwise, the ⁵⁷Fe²⁺ will readily oxidize to ⁵⁷Fe³⁺.

2. Na₂S solution: 100 mM sodium sulfide solution. Dissolve 24 mg of solid Na₂S·9H₂O in 1 mL buffer for a 100 mM solution. The pH of the buffer should be kept basic (i.e., above pH 7.5) to prevent the formation of volatile H₂S. Na₂S solutions should always be freshly prepared.

3. Pyridoxal phosphate (PLP) solution: 100 mM pyridoxal-5-′-phosphate solution. Dissolve 24 mg of solid PLP in 1 mL buffer. If very acidic, adjust the buffer concentration or re-pH as necessary.

4. l-cysteine solution: 100 mM cysteine solution. Dissolve 12.1 mg of solid l-cysteine in 1 mL buffer. The l-cysteine solution should always be freshly prepared.

3. Methods

3.1. Preparing the Optimal Biological Mössbauer Sample

The quality of Mössbauer spectra is reflected in the signal-to-noise (S/N) ratio and depends on both the sample (i.e., extent of ⁵⁷Fe labeling, purity, and homogeneity) and spectroscopy-related (experimental and theoretical) factors [3, 4]. When designing an experiment, the aim is to maximize the S/N ratio and minimize acquisition time. In this respect, the most important considerations are usually sample thickness, ⁵⁷Fe concentration, and sample purity.

3.1.1. Sample Size, Thickness, and ⁵⁷Fe Concentration

1. Optimize the thickness and size of a Mössbauer sample. These factors greatly influence the S/N ratio. Optimal samples have a thickness of ~6 mm and volumes ranging between 300 and 500 μL depending on the Mössbauer cell type (see Note 2). Thicker samples (higher effective ⁵⁷Fe concentration) do not increase the S/N ratio further because nonresonant absorption attenuates the Mössbauer effect [3, 12].

2. Optimize the ⁵⁷Fe sample concentration. The natural abundance of ⁵⁷Fe is 2.2%, and samples for Mössbauer spectroscopy need to be isotopically enriched. As a rule of thumb, ⁵⁷Fe concentrations of 1–5 mM [3] provide spectra in relatively short acquisition times (1 mM ⁵⁷Fe typically yields high-quality statistics between 12 and 24 h, Fig. 1). Because spectral quality is also dependent on the spin state (diamagnetic, paramagnetic) and sample purity (distinct Fe species present), longer times or higher isotope concentrations may be needed.

Fig. 1.

Effect of acquisition time on the S/N ratio of Mössbauer spectra and the importance of good statistics. The Mössbauer spectrum shown corresponds to that of the chemically reconstituted MftC [47], a radical-SAM enzyme that contains three [4Fe-4S] clusters, one that binds SAM and two auxiliary clusters. The sample has an Fe concentration of 2.3 mM. The spectra were acquired at 4.2 K and in the presence of a magnetic field applied parallel to the γ beam. Although good statistics can be obtained after a two-hour acquisition time, only after 8 h of accumulation are the broad paramagnetic contributions of adventitious Fe (indicated by arrows) reliably detectable

3.1.2. In Vivo ⁵⁷Fe Enrichment of Fe-S Cluster-Containing Proteins

The endogenous Fe-S cluster biosynthetic machinery of Escherichia (E.) coli is often outpaced by the demand for cluster assembly during heterologous expression. Therefore, a large fraction of the isolated protein is devoid of the cofactor (apo) or incorporates nonnative Fe-S cluster fragments. This limitation can be overcome by stimulating endogenous or exogenous expression of Fe-S biosynthetic pathways.

1. For upregulation of the endogenous de novo Fe-S cluster biosynthetic pathways ISC and SUF [13, 14] in E. coli, use the derivative strains ΔiscR and SufFeScient, which lack the repressor iscR [9, 15] and Fur binding site at the sufA promoter [14, 16], respectively (see also Chapter 4 of this edition).

2. Alternatively, coexpress the protein of interest with the plasmid encoding for the Azotobacter (A.) vinelandii isc operon (pDB1282) or the E. coli suf operon (pGSO164 or pPH151) [17, 18]. Protocols for overexpression of ⁵⁷Fe-labeled Fe-S-containing proteins are summarized in Fig. 2 and elaborated in the literature [19–22]. These can be modified to include the addition of ⁵⁷Fe to final concentrations of 25–125 μM upon induction of protein expression.

Fig. 2.

⁵⁷Fe enrichment of Fe-S clusters in proteins. In vivo assembly of Fe-S clusters with the Mössbauer isotope can be enhanced by coexpression with either the plasmids expressing biological cluster assembly machineries (pDB1282 or pGSO164/pPH151) or in cell strains lacking the repressors IscR or SufR. In both cases, cells should be grown in minimal media and with addition of ⁵⁷Fe and l-cysteine. In vivo methods afford protein loaded with the cofactor after purification. Alternatively, protein can be purified in its apo form and the cofactor assembled in vitro via reconstitution procedures. This can be performed chemically by addition of a reducing agent followed by the slow addition of sulfide and ⁵⁷Fe, or semi-enzymatically by addition of a reducing agent followed by the desulfurase IscS, l-cysteine, and ⁵⁷Fe. Following all reconstitution procedures, it is important to extensively desalt the protein to remove any adventitiously bound iron species

3.1.3. In Vitro ⁵⁷Fe Chemical Reconstitution of Fe-S Clusters

Fe-S clusters can be reconstituted into apoproteins via chemical or enzymatic methods [21–25]. General protocols are outlined below. Reactions should be performed under strictly O₂-free conditions and preferably on ice.

1. Add 20–50 equivalents of thiol-specific reducing agents (e.g., dithiolthreitol (DTT) or β-mercaptoethanol) to the protein (see Notes 3 and 4) and let incubate for 15–20 min to scavenge any trace amounts of O₂ and ensure complete reduction of disulfides.

2. Add ⁵⁷Fe in 4 aliquots over the course of an hour. Protein commonly precipitates upon the addition of iron salts, so the reconstitution mixture should be thoroughly mixed upon every aliquot addition. The ⁵⁷Fe amount added should correspond to that expected for the number and type of Fe-S clusters bound to the protein of interest.

3. Add Na₂S in 4 aliquots over the course of an hour with thorough mixing. The final concentration of Na₂S should be stoichiometric with respect to the Fe added in step 2 (see Notes 5 and 6).

4. Let the reaction proceed for at least 4 h or preferably overnight (~16 h) at 4 °C.

5. Centrifuge the reconstitution mixture to remove any precipitates and desalt by using commercially available desalting columns (e.g., PD-10, NAP-5) (see Note 7).

6. Load the desalted solution on a size-exclusion column (e.g., Hiprep 16/60 Sephracryl HR S-200) equilibrated in the reconstitution buffer. This step is required to remove adventitiously bound iron and soluble inorganic or protein aggregates (see Note 8).

3.1.4. In Vitro ⁵⁷Fe Semi-Enzymatic Reconstitution of Fe-S Clusters with IscS [22, 25, 26]

1. Add 20–50 equivalents of thiol-specific reducing agents (e.g., DTT or β-mercaptoethanol) and let incubate for 15–20 min to scavenge any trace amounts of O₂ and ensure complete reduction of disulfides.

2. Add IscS (typically in a molar ratio IscS: protein of 1:50–200). Estimate IscS concentration based on the PLP-cofactor bound (ε₃₈₈ = 5305 M⁻¹ cm⁻¹) or supplement with PLP at an equimolar concentration of IscS.

3. Add ⁵⁷Fe in 4 aliquots over the course of an hour.

4. Initiate reaction with the addition of l-cysteine and incubate overnight (~16 h) at 4 °C.

5. Follow steps 5 and 6 of Subheading 3.1.3 for the cleanup of the reconstitution reaction. If chemical or semi-enzymatic Fe-S reconstitutions are not successful, there are other avenues that one can resort to (see Note 9).

3.2. Spectroscopic Parameters and Chemical and Electronic Information Obtained from Mössbauer

For a ⁵⁷Fe nucleus in the sample to resonantly absorb the γ irradiation from the source, there must be an overlap between their respective absorption and emission spectra. The emission spectrum of the source and the absorption spectrum of the sample occur at slightly different energies as a result of the different electric and magnetic properties experienced by the respective Mössbauer atoms that perturb or split the ⁵⁷Fe energy levels differently (Fig. 3). The energy of the source γ-photons can be modulated to match that of the sample via the Doppler effect by attaching the source to a velocity transducer [3, 5]. Thus, the x-axis and parameters of a Mössbauer spectrum are cited in a velocity scale (mm/s). The fundamental ⁵⁷Fe Mössbauer spectrum in the absence of magnetic interactions consists of a “quadrupole doublet,” with two resonance lines of equal integrated intensities [3, 4]. The quadrupole doublet is described by the two principal Mössbauer parameters: isomer shift (δ) and quadrupole splitting (ΔE_Q) [1, 2, 5].

Fig. 3.

Experimental setup and chemical and electronic information obtained from ⁵⁷Fe Mössbauer spectroscopy. (a) The ⁵⁷Co source (mounted on a velocity transducer), the sample (housed in a cryostat), and detector (proportional gas flow counter) are all aligned in the spectrometer. (b) Decay scheme of ⁵⁷Co to ⁵⁷Fe emitting the 14.4 keV γ-photon and the relevant transitions in the sample that give rise to a quadrupole doublet. Zero velocity on the velocity scale is calibrated with respect to α-iron (c) Mössbauer transitions in the presence of an external magnetic field for an effective S = 1/2 compound. The number of allowed transitions and their relative intensities depend on the orientation of the external magnetic field with respect to the γ-beam

3.2.1. Isomer Shift (δ)

The isomer shift (δ) is determined by taking the average absorption energy of the quadrupole doublet (centroid) relative to zero velocity (see Note 10). The isomer shift is a measure of the electron density at the nucleus and contains useful chemical information as it is sensitive to oxidation state, spin state, and ligand covalency. In Fe-S clusters, the Fe ions are in their high-spin electronic configuration, and the Mössbauer isomer shifts follow the trends: [1, 6, 7]

1. δ(Fe³⁺) < δ(Fe^2.5+) < δ(Fe²⁺)

2. δ(soft ligands) < δ(ionic ligands)

3. δ(four-coordinate complexes) < δ(six-coordinate complexes)

The isomer shift has a temperature-dependent component (second-order Doppler shift, δ_SOD) that results in slight deviations (i.e., ± 0.01–0.05 mm/s) in the isomer shift values upon increasing the temperature [3, 4, 6]. Comparisons of δ should thus be made from spectra collected at the same temperature.

3.2.2. Quadrupole Splitting (ΔE_Q)

The excited I = 3/2 state has a quadrupole moment that interacts with the electronic distribution surrounding the ⁵⁷Fe nucleus (termed the electric field gradient) [1, 3, 4, 6]. This interaction lifts the degeneracy of the excited state (without shifting the mean of the energy), leading to an energy separation that is defined as the quadrupole splitting, ΔE_Q (Fig. 3). The quadrupole splitting is determined from the spectrum by taking the energy separation between the two resonance lines of the quadrupole doublet (Fig. 3) and provides information on the local symmetry of the electric field gradient of the ⁵⁷Fe nucleus. Similar to the isomer shift, Fe-S clusters, with their mostly thiolate-based coordination, follow typical trends, and a decrease in oxidation state can be followed by an increase in the ΔE_Q. Exceptions in this trend are not uncommon, and often deviations from commonly reported ΔE_Q values are suggestive of the Fe ions being coordinated by more ionic protein-based ligands, binding small molecules and substrates, or having a higher coordination number. Similar to the isomer shift, ΔE_Q can also be slightly temperature-dependent, especially if there are admixtures of excited states in the ground state [1, 6].

3.2.3. Magnetic Hyperfine Interactions (A)

Fe compounds with a paramagnetic ground state (S > 0) exhibit more complex spectra than a quadrupole doublet because the interaction between the unpaired electron and the ⁵⁷Fe nucleus perturbs the nuclear energy levels. In the presence of an external magnetic field for a system with an effective S = 1/2 and as described in detail elsewhere (see Note 11) [1, 3, 4, 6, 27], the excited I = 3/2 state splits into four, and the ground I = 1/2 state splits into two energy levels. This results in a total of four or six allowed transitions depending on whether the applied field is parallel or perpendicular with respect to the γ-beam, Fig. 3. Hamiltonian-based simulations of spectra recorded at different applied magnetic fields provide the hyperfine constants (A), which are tensorial values commonly expressed in units of MHz, Tesla, or Gauss (see Note 12). The magnitude and sign of hyperfine constants inform on the spin coupling scheme in polynuclear Fe-S clusters and their electronic configuration [1, 6]. The observed hyperfine constants between Fe-S clusters of the same nuclearity will be very similar and only deviate if local geometry changes perturb their electronic structure. Hyperfine constants are therefore useful for deducing the electronic structure, especially for Fe-S clusters with unusual ligation or chemistry.

3.3. Experimental Considerations for Recording Mössbauer Spectra of Fe-S Proteins

Although the measurement conditions are dependent on the Fe-S type under interrogation, the experimental approaches for their characterization share common features that are broadly applicable for different Fe-S cluster types.

3.3.1. Temperature

The choice of temperature has two major experimental implications in the Mössbauer spectra: (1) the S/N ratio and (2) the spectral complexity of paramagnetic Fe-S clusters.

1. The probability for recoil-free emission and absorption is known as the f (or Lamb-Mössbauer) factor and is a measure of the Mössbauer effect. The f factor is strongly temperature-dependent because it is inversely proportional to the mean-square displacement of the ⁵⁷Fe nucleus. Thus, lower experimental temperatures will increase the f factor and consequently, the S/N ratio. The f factor can vary after a change in redox state or addition of a ligand, but at liquid helium temperatures (4.2 K), it has been semi-empirically shown to be equal to ~0.8 for all ⁵⁷Fe species, allowing for their reliable quantification (see Note 13) [3, 4].

2. At 4.2 K, electronic spin relaxation is slower than the Larmor precession frequency of the ⁵⁷Fe nucleus (slow relaxation), leading to magnetically split Mössbauer spectra (Fig. 4) [1, 3, 4, 6, 7, 28]. At higher temperatures, typically > 80 K, electronic spin relaxation is faster than the ⁵⁷Fe nucleus Larmor frequency (fast relaxation), and paramagnetic spectra collapse into quadrupole doublets.

Fig. 4.

Mössbauer spectra of the as-purified and dithionite-reduced carbon monoxide dehydrogenase (CODH) from Desulfovibrio vulgaris [28] demonstrating the effects of slow and fast relaxation on diamagnetic and paramagnetic states of Fe-S clusters. (Left) Recorded in the slow relaxation limit (4.2 K) in a ± 8 mm/s velocity scale, and in the presence of a small magnetic field (78 mT) applied parallel to the γ-beam. (Right) Recorded in the fast relaxation limit (120 K) in a ± 4 mm/s velocity scale, and in the absence of an applied magnetic field. The as-isolated form of CODH contains four [4Fe]-containing clusters and one [2Fe-2S] clusters in their 2+ states (all are S = 0, diamagnetic), while the dithionite-reduced form contains the same number of clusters in their one-electron-reduced forms (all are S = 1/2, paramagnetic)

3.3.2. Applied Magnetic Field

The presence and strength of the applied magnetic field have different implications in the Mössbauer spectra of diamagnetic and paramagnetic Fe-S clusters, respectively. At 4.2 K, small applied fields (30–80 mT) will, in most cases, yield magnetically split spectra for paramagnetic Fe-S clusters and quadrupole doublets for diamagnetic Fe-S clusters. Application of high magnetic fields (> 0.1 T) is mostly employed to verify the diamagnetism of the ground state and obtain the complete set of magnetic parameters. However, measurements at strong magnetic fields require specialized Mössbauer cryostats equipped with superconducting magnets and are often not required for the basic characterization of common Fe-S clusters. Instead, the use of a weak permanent magnet is less experimentally demanding and sufficient to lift the degeneracy of nuclear states and help distinguish between paramagnetic and diamagnetic Fe-S clusters. The applied field can also help identify adventitiously bound Fe that is not assembled in Fe-S clusters (Fig. 1, see Note 14).

3.3.3. Velocity Scale

The velocity scale represents the range of energies scanned by the spectrometer. Generally, transitions of ⁵⁷Fe atoms have energies within the ± 12 mm/s range, but those typically observed for Fe-S clusters are within ± 8 mm/s [1, 6, 7, 29]. Larger velocity scales will increase acquisition times and decrease resolution; therefore, velocity scales are chosen on the basis of maintaining spectral resolution and capturing all signals in the minimum amount of time (Fig. 4). Typically, if the type of Fe-S clusters in the sample is unknown or paramagnetic Fe-S clusters are present, scales of ± 8 mm/s (or greater) are initially chosen. A smaller scale (± 4 mm/s) is employed for diamagnetic compounds to obtain the best spectral resolution in the shortest acquisition time.

3.4. Types of Fe-S Clusters, Their Electronic Configurations, and Representative Mössbauer Fingerprints

Fe-S clusters have unique ⁵⁷Fe Mössbauer spectroscopic signatures that allow for their identification as well as characterization of their electronic and geometric structures, irrespective of their oxidation and spin state. The characteristic Mössbauer parameters of the most commonly occurring Fe-S centers are listed in Table 1. Fe-S clusters are assemblies of elemental iron and sulfide and can adopt a vast range of structures of variable complexity (Fig. 5). Only the inorganic part of their core is inserted in their notation and formal charge assignment. Their nomenclature can be simply viewed as [xFe-yS]^z+, in which x is the number of Fe ions, y is the number of sulfide ions, and z is the overall charge accounting only for the Fe and S²⁻ constituents (and not the protein ligands). The individual Fe ions attain the formal valences Fe²⁺ and Fe³⁺ and adopt a high-spin electronic configuration due to coordination to “soft” ligands (e.g., S-, O-, and N-based).

Table 1.

Mössbauer parameters of common Fe-S clusters

Type	Formal valences	S tot	δ (mm/s)a	|ΔEQ| (mm/s)a	A-values (Ax, Ay, Az) (MHz)
[1Fe]3+rubredoxin [66]	Fe3+	5/2	0.32	0.5	−22.6, −21.8, −23.2
[1Fe]3+desulforedoxin [67]	Fe3+	5/2	0.25	0.75	−10.4, −10.4, −10.4
[1Fe]2+rubredoxin [66]	Fe2+	2	0.70	3.25	−27.6, −11.4, −41.3
[1Fe]2+desulforedoxin [67]	Fe2+	2	0.70	3.55	−27.6, −27.6, −9.3
[2Fe-2S]2+4Cys [30]	2Fe3+	0	0.27	0.60
[2Fe-2S]2+NEET [68]	Fe3+ (Cys)	0	0.26	0.47
	Fe3+ (His)		0.30	0.96
[2Fe-2S]2+Rieske [31]	Fe3+ (Cys)	0	0.24	0.52
	Fe3+ (His)		0.32	0.91
[2Fe-2S]1+4Cys [30]	Fe3+	1/2	0.35	0.65	−56, −50, −43
	Fe2+		0.60	2.70	+14, +21, +35
[2Fe-2S]1+NEET [32]	Fe3+	1/2	0.32	1.07	−55.7, −46.7, −41.6
	Fe2+		0.68	3.15	+29.5, +28.5, +9.1
[2Fe-2S]1+Rieske [31]	Fe3+	1/2	0.31	0.63	−55, −50, −43
	Fe2+		0.74	3.05	+11, +14, +33
[2Fe-2S]0 [33]	Fe2+ (Cys)	0	0.70	2.76
	Fe2+ (His)		0.81	2.32
[3Fe-4S]1+cube [36]	3 Fe3+	1/2	0.27	0.63	−45, +20, +6
[3Fe-4S]1+linear [69]	3 Fe3+	5/2	0.3	n.d.	−19.2; -18.2; +13.9b
[3Fe-4S]0 [35]	1 Fe3+	2	0.32	0.52	+13.7, +15.8, +17.3
	2 Fe2.5+		0.46	1.47	−20.5, −20.5, −16.4
[4Fe-4S]3+ [42]	2 Fe3+	1/2	0.29	0.88	+19.2, +22.4, +19.3
	2 Fe2.5+		0.40	1.03	−28.2, −30.6, −32.6
[4Fe-4S]2+ [43]	4 Fe2.5+	0	0.42	1.12c
[4Fe-4S]2+-SAM[39, 40, 41]	2 Fe2.5+	0	0.42–0.48	0.97–1.21
	1 Fe3+-like		0.34–0.40	0.86–1.10
	1 Fe2+-like		0.64–0.80	1.15–1.62
[4Fe-4S]1+ [43]	2 Fe2+	1/2c	0.58	1.89	+26.5, +13.5, +8.6
	2 Fe2.5+		0.49	1.32	−31.7, −32.6, −27.9
[4Fe-4S]0 [70]	3 Fe2+	4	0.65	1.51	−14.2, −8.8, −10.0
	1 Fe2+		0.65	2.19	+6.9, +10.7, +10.8

^aMössbauer parameters for spectra acquired at 4.2 K

^bThese values are represented as A_iso, the average of A_x, A_y, and A_z, for each Fe site

^cHigher spin ground states, such as S = 3/2, 5/2, and 7/2, have been also observed, but are not as common

Fig. 5.

Structures, oxidation states, Fe valences, and spin states of biologically relevant Fe-S clusters. *Higher spin ground states of [4Fe-4S]¹⁺ clusters, such as S = 3/2, 5/2, and 7/2 have been also observed, but are not as common

3.4.1. [1Fe] Centers

The simplest form of an Fe-S cluster occurs in the so-called rubredoxins and consists of a single Fe ion in a tetrahedral geometry bound to the protein polypeptide by the thiols of four cysteine residues (Fig. 5). This is the building block of all higher-order Fe-S cluster structures. The as-isolated form of the rubredoxin-like [Fe] center is in the ferric form (Fe³⁺, S = 5/2), and one-electron reduction results in the formation of the ferrous form (Fe²⁺, S = 2). Both states are paramagnetic, and their spectra have been described in detail elsewhere [66, 67]. Typical Mössbauer parameters are listed in Table 1.

3.4.2. [2Fe-2S] Clusters

[2Fe-2S] clusters have a diamond-like core (Fig. 5) and are bound to the protein polypeptide by four amino acid residues. To date, three formal redox states of [2Fe-2S] clusters have been observed (i.e., 2+, 1+, and 0). On the basis of the chemical nature of their protein ligands, [2Fe-2S] clusters are classified into three subfamilies: Thioredoxin-like clusters (ligated by four cysteine residues), NEET-like clusters (ligated by three cysteines and one histidine), and Rieske clusters (one Fe is ligated to two cysteines and one Fe is ligated to two histidines).

The oxidized [2Fe-2S]²⁺ form consists of an antiferromagnetically (AF) coupled high-spin diferric pair with a diamagnetic (S = 0) ground state. For a [2Fe-2S]²⁺ cluster, two quadrupole doublets are expected (one for each Fe³⁺), which may overlap or differ depending on the local environment of the Fe ions (Fig. 6). Thus, the Mössbauer spectra of thioredoxin-like [2Fe-2S]²⁺ clusters with two chemically equivalent Fe ions exhibit one quadrupole doublet (superposition of two doublets) [30], whereas Mössbauer spectra of NEET-like and Rieske clusters with two Fe ions in unique chemical environments exhibit two distinct quadrupole doublets [31, 32].

Fig. 6.

Representative simulated ⁵⁷Fe Mössbauer spectra of [2Fe-2S] clusters in all three oxidation states and under conditions of fast relaxation (quadrupole doublet spectra) in a ± 4 mm/s velocity scale. In [2Fe-2S] clusters, differences in ligation state are reflected in site differentiation and distinct quadrupole doublets. Black lines represent the total (observed) ⁵⁷Fe Mössbauer spectrum. The individual quadrupole doublets corresponding to the ferric sites are shown with green lines, and those corresponding to the ferrous sites are shown with purple lines

The one-electron-reduced [2Fe-2S]¹⁺ form consists of a ferric and a ferrous Fe ion that is AF-coupled to yield a paramagnetic ground state (S = 1/2) [30]. At low temperatures (slow relaxation) and in the presence of a magnetic field, each Fe ion produces complex spectra due to hyperfine interactions [7]. The Mössbauer spectrum at high temperatures (fast relaxation) collapses into two quadrupole doublets, one for the Fe²⁺ and one for the Fe³⁺ center (see Note 15, Fig. 6).

The two-electron-reduced form, [2Fe-2S]⁰, is not accessible in biological systems but has been demonstrated in Rieske clusters by reduction with a europium salt [33]. The two Fe²⁺ ions are AF-coupled, yielding a diamagnetic (S = 0) ground state (Table 1).

3.4.3. [3Fe-4S] Clusters

[3Fe-4S] clusters typically have a cuboidal structure (Fig. 5, see Note 16). To date, two formal redox states of [3Fe-4S] clusters have been observed (i.e., 1+ and 0). The oxidized [3Fe-4S]¹⁺ state consists of three high-spin Fe³⁺ ions coupled to yield a ground state of S = 1/2 as a result of “spin frustration” [34, 35]. In the slow relaxation limit, the Mössbauer spectrum of a [3Fe-4S]¹⁺ cluster exhibits a complex hyperfine structure [36], whereas in the fast relaxation limit, the spectrum collapses into a single quadrupole doublet (three overlapping ones) with parameters δ ~ 0.3 mm/s and ΔE_Q ~ 0.53 mm/s (Fig. 7). One-electron reduction yields the [3Fe-4S]⁰ state that contains one Fe³⁺ ion and a valence-delocalized “ferrous-ferric” pair (see Note 17) [6, 8, 35, 37]. The valence-delocalized pair of the [3Fe-4S]⁰ cluster can be assigned the “observed” valences Fe^2.5+-Fe^2.5+ (S = 9/2) and is AF-coupled to the third Fe³⁺ ion (S = 5/2), resulting in a S = 2 ground state. At 4.2 K and in the absence of a magnetic field, the Mössbauer spectrum is a combination of two quadrupole doublets with intensity ratios of 2:1 for the Fe^2.5+-Fe^2.5+ pair and Fe³⁺ ion, respectively. The quadrupole doublet of the Fe^2.5+-Fe^2.5+ pair has δ ~ 0.46 mm/s and ΔE_Q ~ 1.47 mm/s, whereas the doublet of the Fe³⁺ site has δ ~ 0.32 mm/s and ΔE_Q ~ 0.52 mm/s (Table 1).

Fig. 7.

Representative simulated Mössbauer spectra of [3Fe-4S] and [4Fe-4S] clusters at their various oxidation states. Spectra are under conditions of fast relaxation and in a ± 4 mm/s velocity scale. Black lines represent the total (observed) spectra. The subspectra from ferric sites are shown in green, ferrous sites shown in purple, and the mixed-valent Fe^2.5+ are shown in light blue. The ferric-like and ferrous-like sites observed in radical SAM clusters upon SAM binding are shown in dark green and red, respectively

3.4.4. [4Fe-4S] Clusters

[4Fe-4S] clusters have four Fe ions arranged in a distorted tetrahedral cubane structure and attain four possible formal redox states (i.e., 3+, 2+, 1+, and 0) (Fig. 5). [4Fe-4S] clusters are classified into high-potential and low-potential centers depending on whether they operate in the 3+/2+ or the 2+/1+ redox couple, respectively. The central redox state for both types, [4Fe-4S]²⁺, consists of two valence-delocalized Fe^2.5+-Fe^2.5+ pairs that are AF-coupled leading to a diamagnetic (S = 0) ground state [1, 6, 7]. The four Fe^2.5+ ions experience similar chemical environments and are thus spectroscopically indistinguishable with parameters δ ~ 0.42 mm/s and ΔE_Q ~ 1.12 mm/s. Therefore, the spectrum of a [4Fe-4S]²⁺ cluster often manifests as a single quadrupole doublet instead of two or four (Table 1, see Note 18).

In contrast to [2Fe-2S] clusters, for [4Fe-4S] clusters it is sometimes challenging (and often impossible) to resolve small changes in the coordination environment. Typically, in [4Fe-4S] clusters in which the fourth Fe has a protein-based ligand other than cysteine, the differences in Mössbauer parameters are too small to be resolved (see Note 19). However, addition of substrates or small molecules can produce distinct features that are not Mössbauer “silent.” Small molecule binding to one of the Fe sites of [4Fe-4S]²⁺ clusters causes a soft “breaking” of the valence delocalization in one of the two mixed-valent pairs as a result of changes in coordination number, symmetry, etc. The observed outcome is a pronounced site differentiation in the Mössbauer spectra with one of the Fe ions having more ferric and the other Fe ion having more ferrous character [38]. The spectra of such [4Fe-4S]²⁺ clusters consist of three quadrupole doublets (instead of one), and the relative contribution of the four Fe ions to the total area of the spectrum is 2:1:1 reflecting the two Fe^2.5+, one Fe²⁺-like, and one Fe³⁺-like sites. The most common example of site-differentiated clusters is that observed in radical S-adenosyl-l-methionine (SAM)-dependent enzymes, in which the bidentate coordination of SAM via the amide and carboxylic groups to one of the Fe sites produces these distinct spectral features (Fig. 7, Table 1) [38–41].

The one-electron-oxidized [4Fe-4S]³⁺ form of high-potential clusters consists of a diferric pair AF-coupled to a valence-delocalized Fe^2.5+-Fe^2.5+ pair, resulting in an S = 1/2 ground state [42]. The presence of the diferric subcore in the [4Fe-4S]³⁺ leads to a decrease in the average isomer shift and quadrupole coupling parameters, which can be seen in the high-temperature spectra as a shift toward lower energies (i.e., velocities) with respect to those of [4Fe-4S]²⁺ clusters (Fig. 7). At 4.2 K and in the presence of a magnetic field, a complex magnetically split spectrum is observed. Characteristic parameters for the Fe³⁺-Fe³⁺ pair are δ ~ 0.29 mm/s and ΔE_Q ~ 0.88 mm/s and δ ~ 0.40 mm/s and ΔE_Q ~ 1.03 mm/s for the mixed-valent Fe^2.5+-Fe^2.5+ pair (Table 1).

The one-electron-reduced [4Fe-4S]¹⁺ form of low-potential clusters consists of one diferrous pair AF-coupled to a valence-delocalized Fe^2.5+-Fe^2.5+ pair, yielding an S = 1/2 ground state (see Note 20). The presence of the diferrous subcore in [4Fe-4S]¹⁺ clusters is seen in the high-temperature spectra as a shift toward higher energies (i.e., velocities) as a result of an increase in the average isomer shift and quadrupole coupling parameters for the diferrous pair (with respect to those of the mixed-valent pair). At 4.2 K and in the presence of a magnetic field, the spectrum is a convolution of two magnetically split species of equal intensity ratios, one for the ferrous pair and one for the mixed-valent pair [43]. Representative isomer shift values are δ ~ 0.58 mm/s for the Fe²⁺-Fe²⁺ pair and δ ~ 0.49 mm/s for the Fe^2.5+-Fe^2.5+ pair (Table 1, see Note 21).

In some proteins, the two-electron-reduced all ferrous [4Fe-4S]⁰ state of low-potential clusters is attainable by treatment with strong reducing agents [44, 45]. In this form, the S = 4 ground state results from AF-coupling of one Fe²⁺ ion to the remaining three Fe²⁺ ions (Table 1). The Mössbauer spectrum consists of two components in a 3:1 intensity ratio with parameters δ = 0.71 mm/s and ΔE_Q = 1.80 mm/s (75%) and δ = 0.71 mm/s and ΔE_Q = 2.60 mm/s (25%), which are representative of high-spin Fe²⁺S₄ sites [29].

3.4.5. Other Clusters

Fe-S clusters are cofactors of extraordinary structural plasticity and can incorporate additional protein-based elements to extend their redox capabilities or adopt more complex structures of higher nuclearities beyond the four classical types discussed in Subheadings 3.4.1–3.4.4. Despite their unusual structures or higher nuclearity, their Mössbauer parameters resemble those of the simpler clusters (see Note 22).

3.5. Applications of Mössbauer Spectroscopy

3.5.1. Determination of Fe-S Cluster Stoichiometry—“Counting” Fe-S Clusters

Mössbauer spectroscopy, when combined with elemental analysis such as quantification of elemental iron and sulfide, allows for the absolute quantification of the Fe-S clusters. The amount of inorganic sulfide can easily be measured via a colorimetric assay by monitoring methylene blue formation and has been described in detail elsewhere [22]. Iron can be quantified by two common methods: the ferrozine assay and inductively coupled plasma atomic emission spectrometry (ICP-AES). Although ICP-AES detects all metal ions independent of oxidation state, quantification using the colorimetric ferrozine assay does not rely on expensive instrument time and results can be quickly obtained (~20 min) [11, 46].

The presence, type, and number of Fe-S clusters in a protein cannot be solely inferred on the basis of amino acid sequence. In this respect, pairing elemental analysis with Mössbauer spectroscopy is an unambiguous method for assigning stoichiometry and type of Fe-S centers in a protein [7]. This approach has been successfully applied for radical-SAM enzymes that often coordinate extra auxiliary clusters in addition to the SAM-binding cluster (e.g., LipA, MftC, etc.) [23, 47]. A typical approach for “counting” the number of Fe-S clusters is outlined below (see Note 23).

1. Estimate the concentration of elemental Fe and sulfide by employing the colorimetric assays referenced above.

2. Ensure that your protein is > 95% homogeneous and pure. Determine protein concentration (see Note 24).

3. The Fe and sulfide should ideally be in 1:1 stoichiometry except in the case of [3Fe-4S] or more complex clusters. Excess Fe with respect to sulfide may suggest that not all Fe is assembled in Fe-S clusters (see Notes 8 and 13).

4. The following formula can be used to calculate the number of clusters per protein:

   $$\#\mathrm{of}\mathrm{clusters}=\left(\right[\mathrm{Fe}]\times p/[protein\left]\right)/n$$

   in which p is the fractional percentage (1 for 100%), n is the cluster nuclearity (e.g., n = 4 for a [4Fe-4S] cluster).

Numerical example 1.

Assume [protein] = 100 μM, [Fe] = 600 μM, and the Mössbauer spectrum is a “clean” quadrupole doublet with parameters characteristic of [4Fe-4S] clusters, which means that the spectrum corresponds to 100% of the absorption. The number of [4Fe-4S] clusters is therefore (600 × 1/100)/4 = 1.5 [4Fe-4S] clusters.

Numerical example 2.

Assume the Mössbauer spectrum consists of two doublets, one with characteristic values for a [2Fe-2S]²⁺ and one for a [4Fe-4S]²⁺ cluster in 1:2 ratio. Assume [protein] = 100 μM, [Fe] = 600 μM. Employing the formula from 4 and assuming a fractional percentage for [2Fe-2S] 0.33 and [4Fe-4S] 0.66, we can calculate the following number of clusters per protein:

$$\begin{gathered} \#\text{of}[2\mathrm{F}\mathrm{e}-2\mathrm{S}]\text{cluster}=(600\times 0.33)/100/2 \\ =0.99[2\mathrm{F}\mathrm{e}-2\mathrm{S}]\text{clusters} \end{gathered}$$

and

$$\begin{gathered} \#\text{of}[4\mathrm{F}\mathrm{e}-4\mathrm{S}]\text{clusters}=(600\times 0.66)/100/4 \\ =0.99[4\mathrm{F}\mathrm{e}-4\mathrm{S}]\text{clusters} \end{gathered}$$

Therefore, the protein contains one [2Fe-2S] and one [4Fe-4S] cluster. In these examples, the spectra were free from any adventitiously bound Fe or any other contaminating species, which may complicate analysis and lower the confidence in Fe-S cluster “counting” (see Notes 5 and 8).

3.5.2. Fe-S Cluster Interconversions and Identification of Multiple Cluster Binding Sites

Fe-S clusters in proteins can be modular and adopt different nuclearities upon oxidative, reductive, and reconstitution processes, and such interconversions in their structure can be uniquely identified by Mössbauer spectroscopy [8, 10, 48]. This is particularly important if both forms are EPR-silent and have overlapping spectroscopic signals in UV/VIS or other techniques. Some seminal examples consist of (1) the global transcriptional regulator FNR, in which sensing of O₂ involves the transformation of a [4Fe-4S]²⁺ cluster to a [2Fe-2S]²⁺ cluster [49], and (2) the scaffold proteins IscU and IscA, in which treatment with a reductant leads to conversion of the [2Fe-2S]²⁺ cluster to a [4Fe-4S]²⁺ cluster [24, 26]. Other proteins can coordinate multiple clusters in distinct binding sites. For example, Mössbauer spectroscopy revealed two distinct cluster binding sites in biotin synthase (BS) and CIAPIN1 [50, 51].

3.5.3. Trapping of Reaction “Intermediates” and Selectively Labeling Fe Sites of Interest

Mössbauer can also provide insight into substrate binding, redox-state changes, and Fe-S cluster interconversions occurring within a subsecond timescale. Intermediate states in a reaction pathway are “trapped” in frozen solutions by utilizing freeze-quench techniques. One such example is the elucidation of the role of the auxiliary cluster in LipA, which serves as a two sulfur atom donor and leads to its sacrificial disassembly after substrate turnover [39]. In other cases, one can specifically label the Fe site of interest with ⁵⁷Fe, whereas all other Fe ions are ⁵⁶Fe-labeled, rendering them Mössbauer-silent [38]. This allows for elucidation of the electronic structure of labile Fe sites in Fe-S clusters that participate in chemistry.

3.5.4. Mössbauer on ⁵⁷Fe-Enriched Whole Cells

In some cases, the chemical nature of Fe-S cofactors must be interrogated in vivo to assess the biological relevance of in vitro studies, obviate any artifactual states introduced during purification, or if the protein of interest is insoluble. Under these circumstances, Mössbauer can be performed on intact (whole) cells using the following protocol [52–54].

1. Prepare ⁵⁷Fe-enriched whole cells that express the empty parent vector and the vector containing the gene of interest (see Note 25).

2. Directly transfer the same quantity of cell pellet to the Mössbauer cup for both samples (see Note 26).

3. Record Mössbauer spectra on both samples and subtract the spectrum of the parent vector to obtain the contribution of the protein of interest.

4. Notes

1. ⁵⁷Fe is often obtained as fine metal powder and needs to be dissolved either in HCl or H₂SO₄ to yield the respective ferrous salt. The reaction of elemental Fe with H₂SO₄ produces FeSO₄ and H₂. Fe dissolution should be performed with dilute amounts of acid to prevent the formation of ferric salts and alteration of the pH of the growth media or reaction solutions [55].

2. A typical Mössbauer cell (also referred to as holder or cup) is cylindrical (12.3 mm OD, 10 mm height), made of delrin or nylon, and requires a volume of approximately 500 μL. However, it can also be tapered on the inside to afford the same sample thickness with less volume (300–400 μL). After the transfer of the sample to the cell, the solution should be frozen by slowly submerging the cell into liquid nitrogen to avoid formation of a “peak” at the center of the sample.

3. There is no restriction in the buffer composition of the samples (salt, pH, glycerol, additives).

4. Reconstitutions are typically performed with dilute protein solutions (10–100 μM); however, some are more successful at higher protein concentrations. At high sample concentrations, care should be taken that there is no aggregation leading to the formation of biologically irrelevant states. Centrifuge the sample to remove any precipitated protein or particles. Protein precipitates will affect line shape (in addition to introducing nonrelevant Fe species in the spectra).

5. Assembly of Fe-S clusters is in direct competition with undesirable formation of polysulfide-iron aggregates that often appear as black precipitates. Chemical methods of reconstitution circumvent this issue by gradually adding iron and sulfide. For a slower and more controlled release of sulfide, reconstitution is performed utilizing IscS desulfurase, which produces sulfide from l-cysteine.

6. If there is precipitation upon addition of iron or sulfide, add less concentrated solutions (1–10 mM) or try modifying the order in which the iron and sulfide are added (e.g., add sulfide first or alternate adding iron and sulfide).

7. This step is suggested to achieve a crude cleanup of the sample and remove any particulates that decrease resolution of the next chromatographic step and overall performance of the analytical column.

8. Iron can also be adventitiously bound to the protein of interest and not in the form of Fe-S clusters. Non-Fe-S cluster-bound Fe can exist either in the high-spin ferrous or ferric forms, which often complicate analyses and cluster counting. Typical parameters for adventitious Fe²⁺ are δ ~ 1.15 mm/s and ΔE_Q ~ 3.2 mm/s and for adventitious Fe³⁺ are δ ~ 0.45 mm/s and ΔE_Q ~ 0.8 mm/s [56].

9. Enzymatic transfer of an intact Fe-S cluster by the Fe-S scaffold protein IscU has been successful for [2Fe-2S] and [4Fe-4S] clusters [57, 58]. For more complex cases, such as NifB, reconstitution with preassembled, chemically synthesized [4Fe-4S] clusters has been successful [21, 59, 60].

10. The Mössbauer velocity scale is calibrated relative to the centroid of the magnetically spit spectrum of α-iron, which is considered zero velocity [3].

11. Removal of the external magnetic field often simplifies paramagnetic spectra; however, some Fe compounds will exhibit paramagnetic splitting even in the absence of an external field depending on the strength of the local magnetic field exerted by the unpaired electron density.

12. Conversion factors for hyperfine constant (A) units for the ground state (I = 1/2) of ⁵⁷Fe.

    1. 1 Tesla = 10,000 Gauss (G) or 10 kG
    2. 1 Tesla = 0.7238 MHz

13. The Mössbauer spectrum is a weighted superposition of the individual subspectra of the different Fe sites in the sample. The relative area of each subspectrum is proportional to the relative concentration of the corresponding Fe center. At 4.2 K, Mössbauer spectroscopy can be used more reliably in a quantitative manner due to the similarity of f factors at this temperature.

14. The presence of high-spin Fe³⁺ manifests itself as broad paramagnetic features and lowers the confidence of Fe-S cluster quantifications.

15. The caveat of high-temperature measurements is that they cannot distinguish between an Fe³⁺ in a mononuclear center or an Fe³⁺ in a polynuclear cluster. Therefore, an unequivocal determination of the chemical nature of the distinct Fe species contributing to the spectra demands that spectra are also collected at 4.2 K and in the absence and presence of a magnetic field.

16. “Linear” [3Fe-4S] cluster forms are also possible and are afforded by a rearrangement allowing for the central iron to be ligated to four sulfides (Fig. 5). Linear [3Fe-4S]¹⁺ clusters have an S = 5/2 ground state.

17. Delocalization can be simply viewed as an extended sharing of the extra electron between the two Fe ions, resulting in the observed Fe^2.5+-Fe^2.5+ valences and is a common feature in [3Fe-4S] and [4Fe-4S] clusters. Parameters for the Fe^2.5+ are intermediary between those of Fe³⁺ and Fe²⁺.

18. In practice, each of the Fe ions of the [4Fe-4S]²⁺ experiences a slightly different chemical environment (second sphere interactions, hydrogen bonding, etc.). Thus, the two resonance lines of the single doublet observed are often asymmetric both in intensity and linewidth.

19. Greater resolution and spectral separation may be attained by recording spectra at higher magnetic fields (i.e., 5 T), which may allow for distinguishing the Fe site that has a protein-based ligand other than a cysteine residue. Some examples of such [4Fe-4S] clusters are (1) the distal [4Fe-4S] cluster in [Ni-Fe] hydrogenases in which the fourth Fe is coordinated by a histidine [61] and (2) lipoyl synthase (LipA) in which the fourth Fe is coordinated by an aspartate [62].

20. Higher-spin ground states (i.e., S = 3/2, 5/2, and 7/2) for [4Fe-4S]¹⁺ clusters are possible as a result of perturbations in cluster coordination or distortions in local geometry [63, 64]. If experimentally available, one should carry out measurements at higher magnetic fields (i.e., 5 T) to assess the exact electronic configuration and spin state.

21. In the absence of a magnetic field, a quadrupole doublet spectrum is usually expected for a [4Fe-4S]¹⁺ cluster. Measurements both in the presence and absence of a small applied magnetic field help ascertain the paramagnetism, while taking the difference spectrum allows analysis of only the paramagnetic components.

22. [4Fe-4S] clusters have electronic properties majorly influenced by valence delocalization (double exchange) and exhibit enormous plasticity, such that in the presence of structural changes, the Fe ions rearrange themselves to preserve this electronic configuration. Therefore, [4Fe-4S] clusters incorporating additional metal ions or adopting distorted or higher nuclearity structures greatly maintain the Mössbauer and hyperfine parameters of prototypical [4Fe-4S] clusters.

23. A widely used program for fitting and simulating Mössbauer spectra is the WMOSS software (http://www.wmoss.org), which is freely available. The parameters collected in Table 1 can be employed as an initial set of parameters for analyzing and interpreting Mössbauer data.

24. Fe-S clusters in one or more oxidation states can have distinct optical spectra, originating from S-to-Fe charge transfer transitions that can aid in their identification [65]. Most clusters have a broad absorption peak around ~310 nm that can interfere with the protein absorbance at 280 nm. Thus, concentrations of Fe-S cluster-containing protein samples may be estimated by the Bradford assay or amino acid analysis.

25. Mössbauer can detect all endogenous ⁵⁷Fe-containing proteins in addition to the overexpressed protein of interest.

26. Typically, the whole cell sample should fill less than half of the Mössbauer cup due to the high concentration of ⁵⁷Fe, and high-quality Mössbauer spectra can be recorded within a few hours [54].