Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2]+ clusters, which constitute a fundamental building block of all higher nuclearity Fe–S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2]2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV–visible–NIR absorption, CD and variable-temperature MCD, and protein–film electrochemistry. The results indicate that the [Fe2S2]+ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3–9.0, because of protonation of the coordinated serinate residue. However, freezing high-pH samples results in partial or full conversion from valence-delocalized S = 9/2 to valence-localized S = 1/2 [Fe2S2]+ clusters. MCD saturation magnetization data for valence-delocalized S = 9/2 [Fe2S2]+ centers facilitated determination of transition polarizations and thereby assignments of low-energy MCD bands associated with the Fe–Fe interaction. The assignments provide experimental assessment of the double exchange parameter, B, for valence-delocalized [Fe2S2]+ centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized S = 9/2 [Fe2S2]+ fragments in higher nuclearity Fe–S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe–S clusters in general is discussed in light of these results.

Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe2S2]2+ cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically, after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe3S4]+ cluster. The excited-state electronic properties and ground-state electronic and vibrational properties of the linear [Fe3S4]+ cluster have been characterized using UV–vis absorption/CD/MCD, EPR, Mössbauer, and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe3S4]+ cluster with properties similar to those reported for synthetic linear [Fe3S4]+ clusters and the linear [Fe3S4]+ clusters in purple aconitase. Moreover, the results indicate that the Fe–S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe2S2]2+ clusters, rather than linear [Fe3S4]+ clusters or mixtures of linear [Fe3S4]+ and [Fe2S2]2+ clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe4S4]2+ cluster that is competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe3S4]+ and [Fe4S4]2+ clusters in Grx5 has been assessed by spectroscopic, mutational, and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe3S4]+ clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe4S4]2+ cluster-containing proteins are discussed in light of these results.

Monothiol glutaredoxins (Grxs) are proposed to function in Fe–S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe2S2]2+ cluster, and to accept [Fe2S2]2+ clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe2S2]2+ cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe–S cluster carrier proteins (NifIscA and SufA). The results reveal rapid, unidirectional, intact and quantitative cluster transfer from the [Fe2S2]2+ cluster-bound forms of A. thaliana GrxS14, S. cerevisiae Grx3, and A. vinelandii Grx-nif homodimers to A. vinelandiiNifIscA and from A. thaliana GrxS14 to A. thaliana SufA1. Coupled with in vivo evidence for interaction between monothiol Grxs and A-type Fe–S cluster carrier proteins, the results indicate that these two classes of proteins work together in cellular Fe–S cluster trafficking. However, cluster transfer is reversed in the presence of Fra2, since the [Fe2S2]2+ cluster-bound heterodimeric Grx3–Fra2 complex can be formed by intact [Fe2S2]2+ cluster transfer from NifIscA. The significance of these results for Fe–S cluster biogenesis or repair and the cellular regulation of the Fe–S cluster status are discussed.

Nfu-type proteins are essential in the biogenesis of iron–sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with the deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV–visible absorption and circular dichroism and resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S]2+ and [4Fe-4S]2+ clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S]2+ cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S]2+ cluster donor for adenosine 5′-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.

In the bacterial ISC system for iron–sulfur cluster assembly, IscU acts as a primary scaffold protein, and the molecular co-chaperones HscA and HscB specifically interact with IscU to facilitate ATP-driven cluster transfer. In this work, cluster transfer from Azotobacter vinelandii [Fe2S2]2+ cluster-bound IscU to apo-Grx5, a general purpose monothiol glutaredoxin in A. vinelandii, was monitored by circular dichroism spectroscopy, in the absence and in the presence of HscA/HscB/Mg-ATP. The results indicate a 700-fold enhancement in the rate of [Fe2S2]2+ cluster transfer in the presence of the co-chaperones and Mg-ATP, yielding a second-order rate constant of 20 000 M–1 min–1 at 23 °C. Thus, HscA and HscB are required for efficient ATP-dependent [Fe2S2]2+ cluster transfer from IscU to Grx5. The results support a role for monothiol Grx’s in storing and transporting [Fe2S2]2+ clusters assembled on IscU and illustrate the limitations of interpreting in vitro cluster transfer studies involving [Fe2S2]-IscU in the absence of the dedicated HscA/HscB co-chaperone system.

The ability of Azotobacter vinelandiiNifIscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe–S cluster biogenesis. NifIscA is shown to bind one Fe(III) or one Fe(II) per homodimer and the spectroscopic and redox properties of both the Fe(III)- and Fe(II)-bound forms have been characterized using the UV–visible absorption, circular dichroism, and variable-temperature magnetic circular dichroism, electron paramagnetic resonance, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (S = 3/2) Fe(III) center (E/D = 0.33, D = 3.5 ± 1.5 cm–1) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (S = 2) Fe(II) center (E/D = 0.28, D = 7.6 cm–1) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound NifIscA undergoes reversible redox cycling between the Fe(III)/Fe(II) forms with a midpoint potential of +36 ± 15 mV at pH 7.8 (versus NHE). l-Cysteine is effective in mediating release of free Fe(II) from both the Fe(II)- and Fe(III)-bound forms of NifIscA. Fe(III)-bound NifIscA was also shown to be a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results.

The mechanism of [4Fe-4S] cluster assembly on A-type Fe–S cluster assembly proteins, in general, and the specific role of NifIscA in the maturation of nitrogen fixation proteins are currently unknown. To address these questions, in vitro spectroscopic studies (UV–visible absorption/CD, resonance Raman and Mössbauer) have been used to investigate the mechanism of [4Fe-4S] cluster assembly on Azotobacter vinelandiiNifIscA, and the ability of NifIscA to accept clusters from NifU and to donate clusters to the apo form of the nitrogenase Fe-protein. The results show that NifIscA can rapidly and reversibly cycle between forms containing one [2Fe-2S]2+ and one [4Fe-4S]2+ cluster per homodimer via DTT-induced two-electron reductive coupling of two [2Fe-2S]2+ clusters and O2-induced [4Fe-4S]2+ oxidative cleavage. This unique type of cluster interconversion in response to cellular redox status and oxygen levels is likely to be important for the specific role of A-type proteins in the maturation of [4Fe-4S] cluster-containing proteins under aerobic growth or oxidative stress conditions. Only the [4Fe-4S]2+-NifIscA was competent for rapid activation of apo-nitrogenase Fe protein under anaerobic conditions. Apo-NifIscA was shown to accept clusters from [4Fe-4S] cluster-bound NifU via rapid intact cluster transfer, indicating a potential role as a cluster carrier for delivery of clusters assembled on NifU. Overall the results support the proposal that A-type proteins can function as carrier proteins for clusters assembled on U-type proteins and suggest that they are likely to supply [2Fe-2S] clusters rather than [4Fe-4S] for the maturation of [4Fe-4S] cluster-containing proteins under aerobic or oxidative stress growth conditions.

Fumarate and nitrate reduction (FNR) regulatory proteins are O2-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions [4Fe-4S]2+-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S]2+ cluster undergoes a rapid conversion to a [2Fe-2S]2+ cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between [4Fe-4S]2+ and [2Fe-2S]2+ clusters in Escherichia coli FNR. Selective 34S labeling of the bridging sulfides in the [4Fe-4S]2+ cluster-bound form of FNR facilitated identification of resonantly enhanced Cys32S-34S stretching modes in the resonance Raman spectrum of the O2-exposed [2Fe-2S]2+ cluster-bound form of FNR. This result indicates O2-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S]2+ cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O2 exposure of [4Fe-4S]2+-FNR. The [4Fe-4S]2+ cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S]2+ cluster by anaerobic incubation with DTT and Fe2+ ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O2-exposed forms of O2-sensitive [4Fe-4S] clusters in radical S-adenosylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O2 sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters.

Monothiol glutaredoxins (Grxs) are proposed to function in Fe–S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe2S2]2+ cluster, and to accept [Fe2S2]2+ clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe2S2]2+ cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe–S cluster carrier proteins (NifIscA and SufA). The results reveal rapid, unidirectional, intact and quantitative cluster transfer from the [Fe2S2]2+ cluster-bound forms of A. thaliana GrxS14, S. cerevisiae Grx3, and A. vinelandii Grx-nif homodimers to A. vinelandiiNifIscA and from A. thaliana GrxS14 to A. thaliana SufA1. Coupled with in vivo evidence for interaction between monothiol Grxs and A-type Fe–S cluster carrier proteins, the results indicate that these two classes of proteins work together in cellular Fe–S cluster trafficking. However, cluster transfer is reversed in the presence of Fra2, since the [Fe2S2]2+ cluster-bound heterodimeric Grx3–Fra2 complex can be formed by intact [Fe2S2]2+ cluster transfer from NifIscA. The significance of these results for Fe–S cluster biogenesis or repair and the cellular regulation of the Fe–S cluster status are discussed.