Iron-sulfur cluster biosynthesis: characterization of iron nucleation sites for assembly of the [2Fe-2S]2+ cluster core in IscU proteins

ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to a target apoprotein. The intermediate [2Fe-2S] IscU-bound cluster is formed by delivery of iron and sulfur to the apo-IscU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor is not yet established. In this report we characterize iron-binding sites on IscU that appear to nucleate [2Fe-2S] cluster assembly. This iron-bound form of IscU is shown to be viable for subsequent IscS-mediated assembly of holo-IscU. Following on recent reports, we demonstrate the persulfide form of IscU to be a dead-end complex that is incapable of forming holoprotein after addition of ferrous or ferric ion. The latter observation reflects the low binding affinity of persulfido IscU for iron ion.     

Glutathione complexed Fe-S centers

Glutathione (γ-glutamyl-cysteinyl-glycine, GSH) is a major thiol-containing peptide with cellular levels of up to 10 mM. (1) Several recent reports have demonstrated glutaredoxins (Grx) to form [Fe(2)S(2)] cluster-bridged dimers, where glutathione provides two exogenous thiol ligands, and have implicated such species in cellular iron sulfur cluster biosynthesis. We report the finding that glutathione alone can coordinate and stabilize an [Fe(2)S(2)] cluster under physiological conditions, with optical, redox, M?ssbauer, and NMR characteristics that are consistent with a [Fe(2)S(2)](GS)(4) composition. The Fe-S assembly protein ISU catalyzes formation of [Fe(2)S(2)](GS)(4) from iron and sulfide ions in the presence of glutathione, and the [Fe(2)S(2)] core undergoes reversible exchange between apo ISU and free glutathione.     

Characterization of [2Fe–2S]‐Cluster‐Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

Many iron–sulfur proteins involved in cluster trafficking form [2Fe–2S]‐cluster‐bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe–2S]‐cluster‐bridged proteins and their transient cluster‐transfer intermediates. The use of this mechanistic and protein‐characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA‐like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster‐transfer reactions between the holo‐dimers and apo‐ferredoxin (FDX2) are monitored, and intermediate [2Fe–2S] species, such as (FDX2:GLRX5:[2Fe–2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe–2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron–sulfur‐cluster‐bridged protein complexes and transient iron–sulfur‐cluster transfer intermediates.     

Mechanism of glutaredoxin-ISU [2Fe-2S] cluster exchange

Exchange of [2Fe-2S] centers between Grx2 and the cluster scaffold protein ISU, and characterization of two mutually exclusive Grx2 binding sites on ISU by isothermal titration calorimetry supports a direct link for Grx and glutathione involvement in ISU promoted Fe-S cluster biosynthesis.     

Characterization of [2Fe–2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods

Many iron–sulfur proteins involved in cluster trafficking form [2Fe–2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe–2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA-like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe–2S] species, such as (FDX2:GLRX5:[2Fe–2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe–2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron–sulfur-cluster-bridged protein complexes and transient iron–sulfur-cluster transfer intermediates.     

Probing the electronic structure of [2Fe-2S] clusters with three coordinate iron sites by use of photoelectron spectroscopy

Five series of [2Fe-2S] complexes, [Fe(2)S(2)Cl(2)(-)(x)(CN)(x)](-), [Fe(2)S(2)(SEt)(2)(-)(x)Cl(x)](-), [Fe(2)S(2)(SEt)(2)(-)(x)(CN)(x)](-), [Fe(2)S(2)Cl(2)(-)(x)(OAc)(x)](-) (OAc = acetate), and [Fe(2)S(2)(SEt)(2)(-)(x)(OPr)(x)](-) (OPr = propionate) (x = 0-2), were produced by collision-induced dissociation of the corresponding [4Fe-4S] complexes, and their electronic structures were studied by photoelectron spectroscopy. All the [2Fe-2S] complexes contain a [Fe(2)S(2)](+) core similar to that in reduced [2Fe] ferredoxins but with different coordination geometries. For the first three series, which only involve tricoordinated Fe sites, a linear relationship between the measured binding energies and the substitution number (x) was observed, revealing the independent ligand contributions to the total electron binding energies. The effect of the ligand increases in the order SEt --> Cl --> CN, conforming to their electron-withdrawing ability in the same order. The carboxylate ligands in the [Fe(2)S(2)Cl(2)(-)(x)(OAc)(x)](-) and [Fe(2)S(2)(SEt)(2)(-)(x)(OPr)(x)](-) complexes were observed to act as bidentate ligands, giving rise to tetracoordinated iron sites. This is different from their monodentate coordination behavior in the [4Fe-4S] cubane complexes, reflecting the high reactivity of the unsatisfied three-coordinate iron site in the [2Fe-2S] complexes. The [2Fe-2S] complexes with tetracoordinated iron sites exhibit lower electron binding energies, that is, higher reductive activity than the all tricoordinate planar clusters. The electronic structures of all the [2Fe-2S] complexes were shown to conform to the "inverted energy level scheme".     

Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation

The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp(2)-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one "radical SAM" [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5'-deoxyadenosyl 5'-radical, required for substrate-dependent methyl synthase activity.     

Secondary structure analysis of peptides with relevance to iron–sulfur cluster nesting

Peptides coordinated to iron–sulfur clusters, referred to as maquettes, represent a synthetic strategy for constructing biomimetic models of iron–sulfur metalloproteins. These maquettes have been successfully employed as building blocks of engineered heme-containing proteins with electron-transfer functionality; however, they have yet to be explored in reactivity studies. The concept of iron–sulfur nesting in peptides is a leading hypothesis in Origins-of-Life research as a plausible path to bridge the discontinuity between prebiotic chemical transformations and extant enzyme catalysis. Based on past biomimetic and biochemical research, we put forward a mechanism of maquette reconstitution that guides our development of computational tools and methodologies. In this study, we examined a key feature of the first stage of maquette formation, which is the secondary structure of aqueous peptide models using molecular dynamics simulations based on the AMBER99SB empirical force field. We compared and contrasted S…S distances, [2Fe-2S] and [4Fe-4S] nests, and peptide conformations via Ramachandran plots for dissolved Cys and Gly amino acids, the CGGCGGC 7-mer, and the GGCGGGCGGCGGW 16-mer peptide. Analytical tools were developed for following the evolution of secondary structural features related to [Fe-S] cluster nesting along 100 ns trajectories. Simulations demonstrated the omnipresence of peptide nests for preformed [2Fe-2S] clusters; however, [4Fe-4S] cluster nests were observed only for the 16-mer peptide with lifetimes of a few nanoseconds. The origin of the [4Fe-4S] nest and its stability was linked to a “kinked-ribbon” peptide conformation. Our computational approach lays the foundation for transitioning into subsequent stages of maquette reconstitution, those being the formation of iron ion/iron–sulfur coordinated peptides. © 2018 Wiley Periodicals, Inc.     

Inactivation of iron responsive element-binding capacity and aconitase function of iron regulatory protein-1 of skin cells by ultraviolet A

The ultraviolet-A (UVA) component of sunlight produces in cutaneous cells a highly toxic oxidative stress mediated by redox cycling reactions of Fe ions. A tight regulation of cell iron uptake and storage by iron regulatory proteins (IRP) of keratinocytes and fibroblasts avoids these damaging reactions. We report here that about 40 J/cm2 of UVA are required to inactivate half of the binding capacity of apo-IRP-1 to iron responsive elements (IRE) of RNA whereas 15 J/cm2 already inhibit half of the holo-IRP-1 aconitase activity. No increase in the holo-IRP-1 activity is observed during the apo-IRP-1 photoinactivation suggesting that UVA does not trigger a shift between these two forms. As opposed to holo-IRP-1, which contains a 4Fe-4S cluster, apo-IRP-1 has no UVA chromophore. Thus it should be inactivated indirectly by reactive oxygen species generated by the UVA-induced endogenous photo-oxidative stress. The apo-IRP-1 photoinactivation is weakly prevented by the lipophilic oxyradical scavenger vitamin E but not by the hydrophilic azide anion, a singlet oxygen quencher or by diethyldithiocarbamate, a superoxide dismutase inhibitor. However, full protection against photoinactivation of the apo form is observed after incubation with N-acetylcysteine but the latter only partially protects the aconitase function of the holo-IRP-1 from photoinactivation. The marked difference in the kinetics of photoinactivation of the apo and holo forms, the light dose-independent effect of the sulfhydril group reagent, 2-mercaptoethanol and the partial protection brought by the ferric ion complexing agent desferrioxamine suggest that the photochemistry of the 4Fe-4S cluster of the holo form plays little, if any, role in the photoinactivation of the apo-IRP-1/IRE interaction. It is concluded that the apo/holo equilibrium is irreversibly destroyed by UVA irradiation.     

Chemical activity of iron in [2Fe-2S]-protein centers and FeS2(100) surfaces

Iron atoms bonded to sulfur play an important role in proteins, heterogeneous catalysts, and gas sensors. First-principles density functional calculations were used to investigate the structure and chemical activity of a unique [2Fe-2S] center in the split-Soret cytochrome c (Ssc) from Desulfovibrio desulfuricans. In agreement with a previously proposed structural model [Abreu et al., J. Biol. Inorg. Chem. 2003, 8, 360], it is found that the [2Fe-2S] cluster is located in a surface pocket of the Ssc and bonded to only three cysteines. The [2Fe-2S] center in the Ssc is nonplanar and somewhat distorted with respect to canonical [2Fe-2S] centers seen in proteins where the iron-sulfur unit is bonded to four cysteines. In the Ssc, the lack of one Fe-cysteine bond is partially compensated by the separation between the cysteines that minimizes electrostatic repulsion among these ligands. The unique structure of the [2Fe-2S] center in the Ssc makes the center more chemically active than canonical [2Fe-2S] centers in proteins, (RS)(4)[2Fe-2S] inorganic complexes, and an FeS2(100) surface. A [2Fe-2S] center in the Ssc interacts efficiently with electron acceptors (O2, NO, CO) and poorly with a Lewis base such as H2O. The interaction with molecular oxygen is so strong that eventually oxidatively destroys the [2Fe-2S] unit. The bonding energy of the ligands to the [2Fe-2S] centers and FeS2(100) surface increases following the sequence: H2O < CO < NO < O2. The higher the electron affinity of the ligand, the larger its bonding energy. A relatively large positive charge on the Fe cations in FeS2(100) makes this sulfide surface less reactive toward O2, CO, and NO than the [2Fe-2S] centers in proteins and inorganic complexes.     

Reactions of synthetic [2Fe-2S] and [4Fe-4S] clusters with nitric oxide and nitrosothiols

The interaction of nitric oxide (NO) with iron-sulfur cluster proteins results in degradation and breakdown of the cluster to generate dinitrosyl iron complexes (DNICs). In some cases the formation of DNICs from such cluster systems can lead to activation of a regulatory pathway or the loss of enzyme activity. In order to understand the basic chemistry underlying these processes, we have investigated the reactions of NO with synthetic [2Fe-2S] and [4Fe-4S] clusters. Reaction of excess NO(g) with solutions of [Fe2S2(SR)4](2-) (R = Ph, p-tolyl (4-MeC6H4), or 1/2 (CH2)2-o-C6H4) cleanly affords the respective DNIC, [Fe(NO)2(SR)2](-), with concomitant reductive elimination of the bridging sulfide ligands as elemental sulfur. The structure of (Et4N)[Fe(NO)2(S-p-tolyl)2] was verified by X-ray crystallography. Reactions of the [4Fe-4S] clusters, [Fe4S4(SR)4](2-) (R = Ph, CH2Ph, (t)Bu, or 1/2 (CH2)-m-C6H4) proceed in the absence of added thiolate to yield Roussin's black salt, [Fe4S3(NO)7](-). In contrast, (Et4N)2[Fe4S4(SPh)4] reacts with NO(g) in the presence of 4 equiv of (Et4N)(SPh) to yield the expected DNIC. For all reactions, we could reproduce the chemistry effected by NO(g) with the use of trityl-S-nitrosothiol (Ph3CSNO) as the nitric oxide source. These results demonstrate possible pathways for the reaction of iron-sulfur clusters with nitric oxide in biological systems and highlight the importance of thiolate-to-iron ratios in stabilizing DNICs.     

Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins

The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.     

Synthesis of new [8Fe-7S] clusters: a topological link between the core structures of P-cluster, FeMo-co, and FeFe-co of nitrogenases

A coordinatively unsaturated dinuclear iron(II) complex of bulky thiolates, [(TipS)Fe(micro-SDmp)]2 (1; Tip = 2,4,6-(i)Pr(3)C(6)H(2), Dmp = 2,6-(mesityl)(2)C(6)H(3)), was synthesized from stepwise reactions of Fe{N(SiMe(3))2}2 with 1 equiv of HSDmp and then with 1 equiv of HSTip. Complex 1 was found to react with elemental sulfur (S8) in toluene to generate a new class of [8Fe-7S] cluster, [(DmpS)Fe(4)S(3)]2(micro-SDmp)2(micro-STip)(micro(6)-S) (2). The cluster 2 was also produced from one-pot reactions of Fe{N(SiMe(3))2}2 + HSDmp + HSTip + S8 (8:6:10:7/8) and Fe3{N(SiMe(3))2}2(micro-STip)4 + HSDmp + S8 (8/3:16/3:7/8), where another [8Fe-7S] cluster, [(TipS)Fe(4)S(3)]2(micro-SDmp)2{micro-N(SiMe(3))2}(micro(6)-S) (3), was also found as a minor byproduct. In either of the clusters, two Fe(4)S(3) incomplete cubane units are connected by three anionic ligands, namely three thiolate S atoms for 2 or two thiolate S atoms and one amide N atom for 3, and one hexa-coordinate S atom resides at the center of the [8Fe-7S] core. They have a common Fe(II)(5)Fe(III)3 oxidation states, and an S = 1/2 ground spin state was indicated by rhombic EPR signals at 10 K with g = 2.19, 2.07, and 1.96 for 2 and g = 2.13, 2.06, and 1.93 for 3. The structural relevance of clusters 2 and 3 to P-cluster, FeMo-co, and FeFe-co of nitrogenases is discussed.     

Inner-sphere reorganization energy of iron-sulfur clusters studied with theoretical methods

Models of several types of iron-sulfur clusters (e.g., Fe(4)S(4)(SCH(3))(4)(2-/3-/4-)) have been studied with the density functional B3LYP method and medium-sized basis sets. In a vacuum, the inner-sphere reorganization energies are 40, 76, 40, 62, 43, and 42 kJ/mol for the rubredoxin, [2Fe-2S] ferredoxin, Rieske, [4Fe-4S] ferredoxin, high-potential iron protein, and desulfoferrodoxin models, respectively. The first two types of clusters were also studied in the protein, where the reorganization energy was approximately halved. This change is caused by the numerous NH.S(Cys) hydrogen bonds to the negatively charged iron-sulfur cluster, giving rise to a polar local environment. The reorganization energy of the iron-sulfur clusters is low because the iron ions retain the same geometry and coordination number in both oxidation states. Cysteine ligands give approximately the same reorganization energy as imidazole, but they have the advantage of stabilizing a lower coordination number and giving more covalent bonds and therefore more effective electron-transfer paths.     

Evidence from Mössbauer spectroscopy for distinct [2Fe-2S](2+) and [4Fe-4S](2+) cluster binding sites in biotin synthase from Escherichia coli

Biotin synthase is an AdoMet-dependent radical enzyme that catalyzes the insertion of an FeS cluster-derived sulfur atom into dethiobiotin. The dimeric enzyme is purified containing one [2Fe-2S]2+ cluster per monomer, but it is most active when reconstituted with an additional [4Fe-4S]2+ cluster per monomer. Using M?ssbauer spectroscopy coupled with differential reconstitution of each cluster with 57Fe, we show that the reconstituted enzyme has approximately 1:1 [2Fe-2S]2+ and [4Fe-4S]2+ clusters and that the [4Fe-4S]2+ cluster is assembled at an alternate site not previously occupied by the [2Fe-2S]2+ cluster. These data suggest that biotin synthase is evolved to simultaneously accommodate two different clusters with unique roles in catalysis.     

A [4Fe-4S] cluster dimer bridged by bis(2,2':6',2'-terpyridine-4'-thiolato)iron(II)

The use of 2,2':6',2'-terpyridine-4'-thiol (tpySH) was explored as a bridging ligand for the formation of stable assemblies containing both [4Fe-4S] clusters and single metal ions. Reaction of tpySH (2 equiv) with (NH4)2Fe(SO4)(2).6H2O generated the homoleptic complex [Fe(tpySH)2](2+), which was isolated as its PF6(-) salt. The compound could be fully deprotonated to yield neutral [Fe(tpyS)2], and the absorption spectrum is highly dependent on the protonation state. Reaction of [Fe(tpySH)2](PF6)2 with the new 3:1 site-differentiated cluster (n-Bu4N)2[Fe4S4(TriS)(SEt)] yielded the first metal-bridged [4Fe-4S] cluster dimer, (n-Bu4N)2[{Fe4S4(TriS)(mu-Stpy)}2Fe]. Electrochemical studies indicate that the [4Fe-4S] clusters in the dimer act as independent redox units, while UV-vis spectroscopy provides strong evidence for a thioquinonoid electron distribution in the bridging tpyS(-) ligand. TpySH thus acts as a directional bridging ligand between [4Fe-4S] clusters and single metal ions, thereby opening the way to the synthesis of larger, more complex assemblies.     

Structure and Properties of [Fe(4)S(4){2,6-bis(acylamino)benzenethiolato-S}(4)](2)(-) and [Fe(2)S(2){2,6-bis(acylamino)benzenethiolato-S}(4)](2)(-): Protection of the Fe-S Bond by Double NH.S Hydrogen Bonds

Iron-sulfur clusters containing a singly or doubly NH.S hydrogen-bonded arenethiolate ligand, [Fe(4)S(4)(S-2-RCONHC(6)H(4))(4)](2)(-) (R = CH(3), t-Bu, CF(3)), [Fe(4)S(4){S-2,6-(RCONH)(2)C(6)H(3)}(4)](2)(-), [Fe(2)S(2)(S-2-RCONHC(6)H(4))(4)](2)(-) (R = CH(3), t-Bu, CF(3)), and [Fe(2)S(2){S-2,6-(RCONH)(2)C(6)H(3)}(4)](2)(-), were synthesized as models of bacterial [4Fe-4S] and plant-type [2Fe-2S] ferredoxins. The X-ray structures and IR spectra of (PPh(4))(2)[Fe(4)S(4){S-2,6-(CH(3)CONH)(2)C(6)H(3)}(4)].2CH(3)CN and (NEt(4))(2)[Fe(2)S(2){S-2,6-(t-BuCONH)(2)C(6)H(3)}(4)] indicate that the two amide NH groups at the o,o'-positions are directed to the thiolate sulfur atom and form double NH.S hydrogen bonds. The NH.S hydrogen bond contributes to the positive shift of the redox potential of not only (Fe(4)S(4))(+)/(Fe(4)S(4))(2+) but also (Fe(4)S(4))(2+)/(Fe(4)S(4))(3+) in the [4Fe-4S] clusters as well as (Fe(2)S(2))(2+)/(Fe(2)S(2))(3+) in the [2Fe-2S] clusters. The doubly NH.S hydrogen-bonded thiolate ligand effectively prevents the ligand exchange reaction by benzenethiol because the two amide NH groups stabilize the thiolate by protection from dissociation.     

The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine

The combination of resonance Raman, electron paramagnetic resonance and M?ssbauer spectroscopies has been used to investigate the effect of S-adenosyl-l-methionine (SAM) on the spectroscopic properties of the [4Fe-4S]2+ cluster in biotin synthase. The results indicate that SAM interacts directly at a unique iron site of the [4Fe-4S]2+ cluster in BioB and support the hypothesis of a common inner-sphere mechanism for the reductive cleavage of SAM in the radical SAM family of Fe-S enzymes.