Fe/S cluster biogenesis involves a complex machinery comprising several mitochondrial and cytosolic proteins. Fe/S clus- ter biosynthesis is closely intertwined with iron trafficking in the cell. Defects in Fe/S cluster elaboration result in severe diseases such as Friedreich ataxia. Deciphering this machinery is a challenge for the scientific community. Because iron is a key player, Fe-Mössbauer spectroscopy is especially appropriate for the characterization of Fe species and monitoring the iron distribution. This minireview intends to illustrate how Mössbauer spectroscopy contributes to unravel steps in Fe/S cluster biogenesis. Studies were performed on isolated proteins that may be present in multiple protein complexes. Since a few decades, Mössbauer spectroscopy was also performed on whole cells or on isolated compartments such as mitochondria and vacuoles, affording an overview of the iron trafficking. Graphical abstract This minireview aims at presenting selected applications of Fe-Mössbauer spectroscopy to Fe/S cluster biogenesis Keywords Mössbauer spectroscopy · Fe/S biogenesis · Iron trafficking · Iron–sulfur cluster Abbreviations SAM S-adenosyl-l -methionine The original version of this article was revised due to a GSH Glutathione retrospective Open Access order. ENDOR Electron-nuclear double resonance EPR Electron paramagnetic resonance * Geneviève Blondin MCD Magnetic circular dichroism email@example.com DTT Dithiothreitol Univ. Grenoble Alpes, CEA, CNRS, LCBM UMR 5249, ICP-MS Inductively coupled plasma mass spectrometry pmb, 38000 Grenoble, France LCBM/pmb, CEA Bât C5, 17 Rue des Martyrs, 38054 Grenoble Cedex 9, France Vol.:(0123456789) 1 3 636 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 and co-workers , Mössbauer was instrumental in char- Introduction acterizing the [2Fe-2S] cluster of the IscR promoter as being ligated by three cysteines and one histidine, with Mössbauer spectroscopy is a powerful probe for the inves- the histidine bound to the ferrous center. IscR has been tigation of proteins and enzymes with iron active sites. found to promote Fe/S cluster biosynthesis through the Accordingly, it has been used extensively for the study Isc pathway in aerobic conditions . The (Cys) (His) of Fe/S clusters [1–3]. This spectroscopy relies on the 3 1 ligation for [2Fe-2S] clusters has been linked to a role resonant absorption of γ photons by Fe nuclei [4, 5]. for the proteins as redox sensors [12–14]: In the oxidized Mössbauer spectroscopy allows the determination of the form, the cluster is less tightly bound, allowing for trans- nuclearity of the clusters, the identification of oxidation fer to other proteins of the machinery. This is the case states of the iron ions, and provides information on their for the mammalian outer mitochondrial membrane protein coordination spheres. It also reveals the spin states of the mitoNEET, which plays a role in Fe-S cluster repair, where iron sites, and their detailed electronic structures (mag- cluster transfer is triggered by oxidative stress conditions netic couplings and electron delocalization) can be estab- . The oxidized and reduced forms of mitoNEET are lished [6–8]. Another main advantage of this spectroscopy readily distinguished by their Mössbauer spectra (Fig. 1) resides in its capability to quantify the different forms of 2+ . While the diamagnetic, oxidized [2Fe-2S] form iron within a sample, the absorption of each type of iron yields two quadrupole doublets (blue lines in Fig. 1), the ions being proportional to its concentration. For all these S = 1/2, reduced [2Fe-2S] form is split by Zeeman effect reasons, Mössbauer spectroscopy is perfectly suited to on nuclear spin states (red lines in Fig. 1). investigate the iron–sulfur cluster assembly machinery. In the oxidized form, the cluster yields two inequivalent Because the Fe isotope is present at only 2% in natural doublets in a 1:1 ratio, each one corresponding to one iron abundance, full enrichment is often required when inves- center, with isomer shifts of 0.26 and 0.30 mm/s. The differ - tigating proteins. Isolated proteins can be purified as-is ence of 0.04 mm/s is evidence of the different coordination or reconstituted with a Fe source. When purified as-is, environments, the coordinated histidine favoring a higher the cells are grown on minimal media, and then provided isomer shift compared to cysteine coordination. As a com- abundant Fe at the time of induction of the overexpressed parison, the Mössbauer spectrum of mammalian ferroche- genes. Purification must be done anaerobically, to avoid air 2+ latase, which has a [2Fe-2S] cluster with (Cys) coordi- oxidation of Fe/S clusters. Alternatively, the apo protein nation, is a perfectly symmetric doublet , while the two can be purified aerobically and then reconstituted anaero - bically with a Fe salt. Reconstitution of Fe/S clusters makes the purification process easier, and sometimes the proteins can simply not be purified with their clusters. This review aims at presenting a selection of results relevant to Fe/S biogenesis, where Mössbauer spectros- copy was crucial in elucidating or at least improving the knowledge of the mechanism of Fe/S cluster assembly. In vitro studies will be first described, dealing either with single proteins or with multiple protein complexes. Pos- sible protein–protein interactions in vivo may lead to sub- tle changes in the Fe/S cluster environments, resulting in variations between what is observed in vitro and what is in fact present in the cells. This was highlighted in the case of a SAM-dependent enzyme in a 2005 paper by Broderick and co-workers . As a consequence, Mössbauer studies performed on whole cells are very informative and some recent works will be presented in this minireview. Evidencing histidine coordination Fig. 1 Characterization by Mössbauer spectroscopy of oxidized (top) and dithionite-reduced (bottom) human mitoNEET recorded at 4.2 K Iron sites in Fe/S clusters are generally tetrahedral with in a magnetic field of 600 G applied parallel to the direction of the four sulfur-coordinating atoms. These can be the thiolate γ-rays. The solid and dashed blue lines represent the contributions of 2+ function of cysteine side chains or bridging sulfide ions. [2Fe-2S] clusters, and the solid and dashed red lines represent the contributions of [2Fe-2S] clusters. From  However, there are exceptions. In a 2012 study by Münck 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 637 2+ iron sites of the [2Fe-2S] cluster of Rieske proteins, with co-workers have used Mössbauer spectroscopy to monitor (Cys) (His) ligation, have a difference in isomer shifts of the NifS-mediated assembly of Fe/S clusters on NifU , 2 2 0.08 mm/s . A difference of 0.05 mm/s, similar to IscR and the IscS-mediated fixation of Fe/S clusters on IscU and mitoNEET, is observed for the scaffold protein IscU in . In the latter study, they have evidenced the sequential 2+ 2+ E. coli, which can bind a [2Fe-2S] cluster coordinated by fixation of one [2Fe-2S] cluster per IscU dimer, then two 2+ 2+ three cysteines and a solvent-exposed coordination site . [2Fe-2S] clusters per IscU dimer, then one [4Fe-4S] In the reduced [2Fe-2S] form, the Mössbauer spectrum cluster per IscU dimer. Subsequently, by isolating the frac- 2+ analyzed with an S = 1/2 spin Hamiltonian (Fig. 1, red lines) tion that contained two [2Fe-2S] clusters per IscU dimer, yields for the Fe(II) site an isomer shift that is a decreas- they proved that dithionite is able to reductively couple 2+ 2+ ing function of the number of coordinated cysteines : the two [2Fe-2S] clusters into one [4Fe-4S] cluster, 0.62 mm/s for A. aeolicus FdI (2 cysteines), ~ 0.70 mm/s and that reduced Isc Ferredoxin is also a capable reduct- for human mitoNEET or E. coli IscR (1 cysteine and 1 his- ant for this purpose . An added difficulty in the last tidine), and 0.75 mm/s for Rieske proteins (2 histidines). experiment is that, in the process of reductive coupling, In other instances, a cysteine to histidine change in the the [2Fe-2S] cluster of reduced IscFdx is oxidized into a 2+ 2+ coordination of [2Fe-2S] clusters impacts the function of [2Fe-2S] cluster, the signal of which interferes with that 2+ proteins directly related to the iron trafficking in the cell. For from IscU–[2Fe-2S] . Mössbauer spectroscopy comes example, it seems to play a role in controlling the location of in particularly handy here: Since only the Fe isotope the Aft1 transcription factor. Under iron-replete conditions, is detected, it is possible to “light up” a given fraction of Aft1 is located in the cytosol. Under iron-depleted conditions the iron ions using Fe-enriched iron, and “switch off” that may originate from a defect in mitochondrial Fe/S bio- another fraction using natural iron for that fraction. Owing genesis, Aft1 moves to the nucleus and activates iron regulon to the low natural abundance of Fe, the non-enriched genes . The two Fe repressors of activation, Fra1 and Fra2, fraction is effectively Mössbauer-silent when mixed with and the two glutaredoxins Grx3 and Grx4 have been identi- a comparable amount of enriched iron. In this case, Huynh fied as key proteins in this process [20–22]. Both Grx3 and and co-workers used Fe-enriched IscU and non-enriched 2+ Grx4 form homodimers that are bridged by a [2Fe-2S] clus- IscFdx. Before reduction (Fig. 2a), the spectrum shows the 2+ ter. Mössbauer spectroscopy clearly established full cysteine exclusive signal of 2 × [2Fe-2S] IscU. After addition of coordination, two from the CGFS active site of Grx3/4, and 1.06 reducing equivalents of IscFdx (Fig. 2b) the signal 2+ two from reduced glutathione (GHS) molecules . Similar of [4Fe-4S] IscU is detected (blue line), but 61% of the 2+ coordinations have been observed in other glutaredoxins [24, initial [2Fe-2S] stay unreacted (red line). By contrast, 25]. It was demonstrated that the Grx3/4 homodimers were dithionite reduction (Fig. 2c) leads to 77% transformation 2+ 2+ unable to regulate Aft1 when lacking the [2Fe-2S] cluster to [4Fe-4S] and barely any unreacted [2Fe-2S] . The . Moreover, both Grx3 and Grx4 strongly interact with difference is attributed to the redox potential of IscFdx, Fra2, as substantiated by the isolation of Fra2/Grx3 and Fra2/ which is higher than that of dithionite, and would be simi- Grx4 heterodimers. In these heterodimers, the bound [2Fe- lar to the apparent redox potential of the reductive cou- 2+ 2S] is coordinated by a single histidine, as evidenced by pling. This is interpreted by the authors as a way for the Isc 2+ Mössbauer spectroscopy and Q-band ENDOR . The his- machinery to modulate the balance between the [2Fe-2S] 2+ tidine has later been identified as histidine 103 from Fra2 . and [4Fe-4S] cluster-bound forms of IscU. A similar heterodimer has been evidenced with the human In an accompanying paper , the same authors Grx3 and BolA2 proteins , the latter being the closest showed that only the [4Fe-4S]-loaded form of IscU is human analogue of the yeast Fra2 protein. The C-terminal competent for the activation of A. vinelandii apo-acon- Grx-domain of Grx3 was demonstrated to be critical for both itase. Because it was previously demonstrated that holo- the interaction of Grx3 with Fra2 and for the inhibition of Fdx can be formed from [2Fe-2S]-IscU and apo-Fdx , Aft1 activity in vivo. This has led proposing the [2Fe-2S] they propose that cluster binding on IscU induces con- Fra2-Grx3/4 complex as a candidate for Aft1 regulation and formational changes that are responsible for the specific as a novel Fe/S cluster binding regulatory complex . interaction of [4Fe-4S]-IscU with target [4Fe-4S] proteins and [2Fe-2S]-IscU with target [2Fe-2S] proteins. More recently, a similar flexibility was reported for Arabidopsis [2Fe‑2S] and [4Fe‑4S] clusters: conversions thaliana Nfu2 . Although the studies are very con- and transfers to apo proteins vincing, direct transfer of an intact [4Fe-4S] cluster from holo-IscU to target proteins has so far only been proved Mössbauer spectroscopy allows the discrimination for A. vinelandii, and there is no consensus on the fact that between [2Fe-2S] and [4Fe-4S] clusters, even when both [4Fe-4S]-IscU may be the physiologically competent form are in the diamagnetic forms with a 2+ charge. Huynh and of IscU. On the contrary, several clues point toward the 1 × 1 3 638 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 ground state, indicating a five-coordinate ligation with two or three cysteinate ligands and respectively three or two non- cysteinate ligands. Both the Fe(III) and Fe(II) forms are able to release Fe(II) in the presence of l -cysteine, but no direct transfer of iron to NifU-1 could be evidenced. Hence, a role as a specific iron donor is not a favored hypothesis. Other proposed roles for A-type proteins are: Downstream carriers for clusters assembled on U-type proteins, or metallochaper- ones that facilitate in situ assembly of [4Fe-4S] clusters on target proteins from [2Fe-2S] clusters assembled on U-type proteins. In a paper published back to back with the one mentioned above, the authors show cycling between a form Nif 2+ of IscA containing one [2Fe-2S] cluster per homodimer, 2+ and one containing one [4Fe-4S] cluster per homodimer 2+ 2+ . [2Fe-2S] to [4Fe-4S] conversion is induced by 2+ 2+ DTT addition, and [4Fe-4S] to [2Fe-2S] conversion is Nif induced by O exposure. The authors conclude that IscA may be able to directly transfer a [4Fe-4S] to target proteins in anaerobic conditions, whereas in aerobic conditions both 2+ the Fe(III)-bound and the [2Fe-2S] -bound forms may play a role in Fe/S protein maturation. This supports the find- ings made on other A-type proteins such as ErcA, which is able to assemble either a [2Fe-2S] or a [4Fe-4S] cluster, and 2+ transfer a [4Fe-4S] cluster to IspG, an iron–sulfur enzyme requiring ErpA function in vivo . After incubation of 2+ Fig. 2 Ferredoxin-mediated reductive coupling of [2Fe-2S] clus- [4 Fe–4S] ErpA with apo IspG and subsequent purification, ters on IscU monitored by Mössbauer spectroscopy (4.2 K; 50 mT the Mössbauer spectrum of the purified IspG displayed the 2+ applied field parallel to γ radiation). 2 × [2Fe-2S] IscU as prepared 2+ characteristic features of [4Fe-4S] clusters. 2+ (a), after reduction with 1.06 reducing equivalents of Fdx [2Fe-2S] As mentioned above, reaction with molecular oxygen may cluster (b), and after reduction with 1.28 reducing equivalents of dith- 2+ ionite per IscU [2Fe-2S] cluster (c). From  be deleterious for [4Fe-4S] clusters. It has been observed in both the mitochondrial and cytosolic iron–sulfur cluster assembly machineries, the latter being far less well under- [2Fe-2S]/dimer as the physiological form of IscU proteins stood than the former. The yeast Dre2 protein and the human . Finally, a species-dependent variability of behaviors anamorsin (also called CIAPIN1) analogue are suspected cannot be discarded. to play a critical role in the cytosolic Fe/S biogenesis. Two Aside from U-type proteins, the role of A-type proteins binding domains have been identified, each one containing has also been investigated using Mössbauer spectroscopy. four cysteines. There is a debate on the number and nature of A-type proteins are expressed from the same operon as their the accommodated Fe/S clusters. In combination with other U-type counterparts. But whereas U-type proteins have been techniques, Mössbauer spectroscopy allowed to propose a proved to act as scaffolds in Fe/S biosynthesis [36, 37], the unifying view . When Dre2 is isolated under anaero- 2+ true role of A-type proteins is largely under debate. It has bic conditions, a [2Fe-2S] is observed in the N-terminal 2+ been evidenced with the help of Mössbauer spectroscopy domain, whereas a [4Fe-4S] cluster is present in the C-ter- that A-type proteins in vitro are able to bind [2Fe-2S] clus- minal domain. When isolated under oxidative conditions, 2+ ters as well as [4Fe-4S] clusters, hence a hypothesized role the [4Fe-4S] cluster is partially converted into a [2Fe- 2+ as alternative Fe/S scaffolds. However, this role was chal- 2S] . The same study suggests that the N- and C-terminal lenged by in vitro studies [38, 39]. Another proposed role is clusters are concertedly assembled. Further investigations that of primary iron donors (to which frataxin is also a can- are required to determine the evolution of the Dre2 Fe/S didate). Mononuclear Fe(III) and Fe(II) complexes of Azo- clusters. Nif tobacter vinelandii IscA have been thoroughly character- As highlighted above, the fact that a protein is able to ized by Mössbauer, EPR and MCD spectroscopies . Spin assemble and transfer a [4Fe-4S] cluster in vitro, does Hamiltonian parameters of the Fe(II) form are very similar to not mean that it is an iron–sulfur scaffold in vivo. To sort those of reduced rubredoxins, whereas the Fe(III) form dis- between biologically relevant species and experimental plays an unprecedented S = 3/2 rhombic intermediate-spin artifacts, specific experiments have to be designed. In some 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 639 instances, the common practice of reconstituting proteins times more efficient than the ternary complex in transferring with Fe salts can be replaced by direct purification of its cluster to mitochondrial aconitase, hinting to a double Fe-enriched proteins. This requires growing the cells in role of frataxin in iron import and Fe/S complex export. Fe-minimal media, and then providing a Fe salt at the time In a recent study, these results have been questioned [48, of induction of the overexpressed gene. This technique has 49], as frataxin was proposed to accelerate a rate-limiting been used to record the Mössbauer spectrum of Fe-labeled transfer step in the formation of [2Fe-2S] clusters. It was SufA purified from the SufABCDSE expression system . also suggested that a competitive Fe/S mineralization occurs Coexpression of the whole operon was necessary because in the cytosol. of the tight interplay between proteins in the Fe/S cluster assembling process. The study revealed that as-purified 2+ SufA contains a [2Fe-2S] cluster with complete cysteinyl Studies on whole cells with a single‑induced ligation, which implies that the cluster is located at the dimer protein interface. The mechanism of formation of the Fe/S clusters in SufA was also addressed using Mössbauer spectroscopy. The first whole-cell Mössbauer studies were performed Thanks to the proportionality between Mössbauer absorp- using a labelled Fe-siderophore for iron uptake [50–52]. tion and Fe content, the ec ffi iency of Fe/S cluster reconsti - More recently, studies on cells with a single overexpressed tution can be assessed. In a study comparing the mechanism protein participating in Fe/S cluster assembly were reported. where Fe is incorporated first and the mechanism where S is These experiments are usually performed by overexpress- incorporated first , it was concluded that (unlike IscA) ing the protein in E. coli. Identification of the Fe/S cluster SufA binds Fe(II) in an unspecific manner, mainly through form of the protein of interest is performed by comparing noncysteinyl ligands, and that Fe/S cluster assembly is more the spectra recorded on cells with and without the induced efficient if sulfuration precedes iron addition. protein. Typical spectra recorded at low temperature in a weak external magnetic field on E. coli cells lacking the Multiple protein complexes Another method for studying the interplay between proteins of the same operon is to purify them together, and then add Fe(II), l -cysteine and cysteine desulfurase to reconstitute Fe/S clusters in protein complexes. This has been done, for instance with the SufBC D complex, which is able to 2+ assemble a [4Fe-4S] cluster displaying in Mössbauer a symmetrical quadrupole doublet with identical parameters to the cluster assembled on SufB only . It was, therefore, postulated that the species that is competent in Fe/S cluster 2+ biosynthesis is the SufBC D complex binding a [4Fe-4S] cluster exclusively through the conserved cysteines of SufB. A similar approach was used in a 2012 study that aimed at shedding light on the role of frataxin in Fe/S cluster biosyn- thesis . Frataxin (FXN) plays an important role in Fe/S cluster biosynthesis, as evidenced by the fact that the Fe/S cluster biosynthesis machinery is disrupted when frataxin is deleted. Although the precise role of frataxin is unknown, it has been proposed as a primary iron transporter. In this study involving murine proteins, it was proved to stabilize the ternary (ISCU/NFS1/ISD11) complex. The Mössbauer spectra of the reconstituted quaternary (ISCU/NFS1/ISD11/ FXN) and ternary (ISCU/NFS1/ISD11) complexes proved unambiguously that both complexes were able to assemble a Fig. 3 4.2 K, 60 mT parallel applied field Mössbauer spectra of the 2+ reconstituted quaternary (ISCU/NFS1/ISD11/FXN) complex (top) [4Fe-4S] cluster. However, both the total iron content per 2+ with 3.5 Fe/Cplx or reconstituted ternary (ISCU/NFS1/ISD11) com- complex and the proportion of iron in the form of [4Fe-4S] plex (bottom) with 2.4 Fe/Cplx. The blue lines represent contribu- clusters were higher for the quaternary complex (Fig. 3). 2+ tions from [4Fe-4S] clusters and the red lines represent contribu- 2+ Additionally, the quaternary complex was found to be 3 tions from [2Fe-2S] clusters. Adapted from  1 3 640 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 overexpressed protein are shown in Fig. 4 and have been located in the − 1/+ 1 mm/s velocity window, like the signal observed in several studies [9, 52–54]. Such spectra extend of the control cells. However, the iron distribution is differ - from − 1 to + 3.5 mm/s. The simulations and the associated ent in cells with an overexpressed iron protein and in cells iron content will be described below. where it is not. This allows the identification of an extra sig- One of the first whole-cell Mössbauer studies related to nal originating from the induced protein. This situation was Fe/S biogenesis involved the transcription factor IscR . met for ISCA1 and ISCA2 proteins. Figure 4 reproduces the The low-temperature low-field Mössbauer spectra recorded Mössbauer spectra recorded at 5.5 K with a weak external on the anaerobically isolated protein revealed a mixture of magnetic field on whole cells with or without overexpressed the oxidized and reduced forms. Measurements were thus ISCA1/2 protein. On both induced samples, one additional performed on whole cells to determine the physiological absorption line is clearly detected at 0.5 mm/s, indicating 2+ form of the cluster in vivo . The spectrum recorded at the presence of a [2Fe-2S] cluster, as determined for the 4.2 K in a 45 mT external magnetic field on cells with over - isolated proteins . Simulations were first performed on expressed wild-type IscR extends from − 4 to + 5 mm/s, the two spectra of control cells. Four doublets were used. thus on a larger velocity range that the spectrum recorded Two correspond to high-spin ferrous ions, one has nuclear 2+ under the same conditions on cells lacking IscR. This parameters close to those determined for [4Fe-4S] clusters strongly suggests the presence of a magnetic cluster associ- and low-spin ferrous heme systems, and the last one corre- ated to IscR. Indeed, features on both edges of the spec- sponds to ferric nanoparticles. Once these nuclear parame- trum of IscR cells evidenced the signature of the reduced ters were known, simulations of the ISCA-overexpressed cell S = 1/2 [2Fe-2S] cluster that accounts for 32% of the iron spectra were performed, allowing only the relative contribu- content. There was no evidence for the presence of either an tions to vary. To reduce the number of unknowns, nuclear 2+ 2+ oxidized [2Fe-2S] cluster or a [4Fe-4S] cluster. These parameters were also fixed for the additional Fe/S cluster experiments demonstrated that IscR accommodates a [2Fe- as parameters of standard oxidized [2Fe-2S] clusters, as 2S] cluster in vivo, which is preponderantly reduced. only these allowed the generation of satisfying simulations. The detection of the formation in vivo of a diamagnetic Accordingly, it was concluded that both ISCA1 and ISCA2, 2+ Fe/S cluster is more difficult because the signal is then when separately overexpressed, harbor a [2Fe-2S] cluster in vivo. In addition, the variations of the different contribu- tions indicate that the ferric nanoparticle pool is the princi- ISCA1 ISCA2 pal source of iron for the generation of these Fe/S clusters. To the best of our knowledge, no Mössbauer studies were performed on cells where more than one protein was overexpressed. 0.5 % 0.5 % Iron trafficking A B Another way to gain more information on the Fe/S biogen- esis in cells is to selectively delete genes that are involved in this process and to look with Mössbauer spectroscopy at the resulting variations in the iron distribution. This strategy has been mainly developed on the budding yeast Saccharomyces 0.5 % 0.5 % cerevisiae. A pioneer work was achieved by Lesuisse and coworkers on mitochondria isolated from S. cerevisiae lack- ing the yfh1 gene . Yfh1 is the yeast frataxin homologue C D of the human frataxin, a key protein in the elaboration of -2 02 4 -2 02 4 –1 –1 Fe/S clusters. A deficiency in frataxin has been evidenced Velocity (mm s ) Velocity (mm s ) for patients suffering from Friedreich’s ataxia, which leads to iron accumulation in their brain and heart tissues [57, 58]. Fig. 4 Mössbauer spectra recorded at 5.5 K using a 60 mT exter- Mössbauer studies on ∆yfh1-mutant cells showed that iron nal magnetic field applied parallel to the γ-beam. The panels a and accumulates in mitochondria as amorphous ferric phosphate b reproduce spectra of whole control cells and panels c and d those of ISCA1 or ISCA2-overexpressing cells. Experimental spectra are nanoparticles (Fig. 5). Accordingly, iron is sequestered and shown with hatched marks and simulations are overlaid as solid black unavailable for Fe/S cluster and heme biogenesis. II lines. Five components were used for simulation: HS Fe (light and 2+ Lindahl and coworkers have performed analogous stud- dark green), [4Fe-4S] clusters and LS ferrous hemes (light blue), III 2+ ies on isolated mitochondria and extended them to the Fe NP (dark blue) and [2Fe-2S] (red). From  1 3 Overexpressing cells Control cells JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 641 investigation of whole yeast cells and isolated vacuoles. As present a large Mössbauer spectrum that stretches from − 10 they had already described elsewhere , Mössbauer spec- and + 10 mm/s (pink line in Fig. 6). The non-heme high- II II troscopy allows the identification and the quantification of spin Fe species (NHHS Fe , green line in Fig. 6) and high- II II groups of iron species when present at higher concentrations spin Fe hemes (HS F e heme, mauve line in Fig. 6) can be than 10–20 µM. Because there is no Mössbauer-silent iron identified by their high-velocity lines centred close to 2.8 III form, this spectroscopy gives a reasonably good overview of and 2.1 mm/s, respectively. The ferric nanoparticles (Fe the iron distribution in the investigated samples. One main NP, blue line in Fig. 6) and the central doublet (CD, red drawback is the necessity of investigating cells enriched in line in Fig. 6) present doublets centred at ~ 0.5 mm/s and Fe isotope, as a minimal amount of it is required to detect a can be discriminated upon their quadrupole splitting. The Mössbauer signal. In combination with other spectroscopies CD doublet accounts for low-spin ferrous heme species (LS II 2+ and techniques, namely electron paramagnetic resonance Fe heme) and for diamagnetic [4Fe-4S] clusters, as these (EPR), UV–visible, electron microscopy and inductively cannot be distinguished. coupled plasma mass spectroscopy (ICP-MS), the con- Ferric nanoparticles have been detected in mitochondria siderable amount of data gathered for more than 10 years [60, 67–69] and in vacuoles [66, 70]. Fe K-edge EXAFS and provided them deep insights into iron metabolism and traf- electron microscopy performed on mitochondria-containing ficking [62, 63]. Practical aspects are detailed in references NP indicate a pentacoordination by oxygen atoms for the III  and . Fe ion (Fig. 5c) [60, 69]. Depending on the batch of vac- Five main groups of iron species were identified, their uoles, the major contribution to the Mössbauer spectra is relative contributions depending on the sample investigated. either analogous to that of mitochondrial ferric nanoparticles III Figure 6 displays together their low-temperature low-field or to that of magnetically noninteracting high-spin Fe ions. Mössbauer spectra scaled to the same area. The correspond- ing nuclear parameters are listed in Table 1 . At 5 or 6 K in a weak external magnetic field (45–60 III III III HS Fe mT), only the non-heme high-spin Fe species (NHHS Fe ) II NHHS Fe CD III Fe NP II Heme HS Fe -12-8-40 48 12 –1 Velocity (mm s ) HO OH Fig. 6 Theoretical Mössbauer spectra at 6 K with a 50 mT external P O magnetic field applied parallel to the γ-beam of the five main com- III Fe O ponents identified in spectra recorded on S. cerevisiae cells, isolated HO mitochondria or isolated vacuoles. They are scaled to the same area and calculated according to published parameters [62, 66] OH OH Table 1 Isomer shift and quadrupole splitting values for the five iron families identified in the yeast S. cerevisiae Fig. 5 Top: Mössbauer spectra of ∆yfh1 yeast mitochondria. Spec- trum a was recorded at 78 K in zero field and spectrum b at 4.2 K II II III III NHHS FeHS Fe CD Fe NPNHHS Fe in a 7 T magnetic field applied parallel to the γ-beam. Experimental hemes spectra are shown with hatched marks and simulations are overlaid as solid lines. Simulation of spectrum b was achieved assuming a dis- δ (mm/s) 1.3 0.91 0.45 0.53 0.54 tribution of the hyperfine field. From . Bottom (c): Coordination ∆E (mm/s) 3.0 2.31 1.15 0.45–0.63 0.39 of the ferric ion in NP according to EXAFS and electron microscopy results  1 3 Transmission 642 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 Based on the Mössbauer signatures of Fe polyphosphate resonant absorption, the gamma-rays are produced by the prepared in different acetate/acetic acid-buffered media, it decay of a parent isotope, which can be packed in a pocket- is proposed that pH plays a critical role . All the studies size source. Therefore, Mössbauer spectrometers require performed evidenced that the accumulation of iron in the no large-size installation. Among all Mössbauer-active iso- cytosol is prevented because of the potential damages that topes, Fe is by far the best suited for spectroscopy, which is II Fe ions can cause. Accordingly, it was proposed that the very fortunate for biologists, and in particular for researchers excess of iron is exported either into vacuoles or into mito- in the field of Fe/S clusters. Let us conclude with a quote chondria, to detoxify the cytosol [71, 72]. from a prominent scientist in the field: “You have to do II Non-heme high-spin F e has been identified in mitochon- Mössbauer. If you are not doing Mössbauer, you are mak- dria, with increased levels as the deficiency in Fe/S clusters ing mistakes” (M. K. Johnson, 2007). and hemes is more pronounced. It was proposed that it serves Acknowledgements This article/publication is based upon work from as an iron pool for the biosynthesis thereof [68, 73]. Similar COST Action CA15133, supported by COST (European Cooperation species were proposed to be present in the cytosol , their in Science and Technology). The authors thank the Agence Nationale amount being more important under iron-deficient growth pour la Recherche (ANR FRATISCA – ANR-14-CE09-0026) and the conditions . Moreover, it is suspected that another non- Labex ARCANE (ANR-11-LABX-0003-01) for their financial support. II heme high-spin F e species is present in vacuoles of cells Open Access This article is distributed under the terms of the Crea- depleted of the vacuolar iron importer CCC1 . tive Commons Attribution 4.0 International License (http://creat iveco II 2+ The signatures of heme HS Fe species and [Fe S ] 4 4 mmons .org/licen ses/by/4.0/), which permits use, duplication, adapta- clusters are detected in mitochondria and in whole cells. tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, Their proportion is maximized under iron-deficient condi- 2+ provide a link to the Creative Commons license and indicate if changes tions . Conversely, the signature of [2Fe-2S] clusters is were made. merely detected because the associated doublet is obscured by that of the ferric nanoparticles (NP). However, they were identified in mitochondria isolated from yeast cells grown under fermenting and iron-deficient conditions . The References 1+ reduced form, namely [2Fe-2S] clusters, was pinpointed in mitochondria of respiring, respiro-fermenting  and of 1. Beinert H, Holm RH, Münck E (1997) Iron–sulfur clusters: nature’s modular, multipurpose structures. Science 277:653–659. iron-deficient fermenting yeast cells . In addition, EPR https ://doi.org/10.1126/scien ce.277.5326.653 measurements suggested the presence of [3Fe-4S] clusters 2. Bill E (2012) Iron–sulfur clusters—new features in enzymes and in mitochondria. However, their amount is low, precluding synthetic models. Hyperfine Interact 205:139–147. https ://doi. their detection by Mössbauer spectroscopy .org/10.1007/s1075 1-011-0411-8 3. Pandelia M-E, Lanz ND, Booker SJ, Krebs C (2015) Mössbauer spectroscopy of Fe/S proteins. Biochim Biophys Acta 1853:1395– 1405. https ://doi.org/10.1016/j.bbamc r.2014.12.005 Concluding remarks 4. Münck E, Stubna A (2003) 2.21—Mössbauer spectroscopy: bioinorganic A2—McCleverty, Jon A. In: Meyer TJ (ed) Compre- hensive coordination chemistry II. Pergamon, Oxford, pp 279–286 We have presented here examples of the use of Mössbauer 5. Gütlich P, Bill E, Trautwein AX (2010) Mössbauer spectroscopy spectroscopy in the field of Fe/S biogenesis. There are many and transition metal chemistry: fundamentals and applications. others, which we have not presented, like the stabilization Springer, Berlin Heidelberg of [2Fe-2S] clusters by full glutathione coordination . 6. Schünemann V, Winkler H (2000) Structure and dynamics of bio- molecules studied by Mössbauer spectroscopy. Rep Prog Phys Such species are suspected to transiently store Fe/S clusters 63:263 and regulate Fe/S biosynthesis . The different pathways 7. Martinho M, Münck E (2010) Fe Mössbauer spectroscopy of Fe/S assembly all involve iron in different states along in chemistry and biology. Physical inorganic chemistry. Wiley, the process. For this reason, Mössbauer spectroscopy is one Hoboken, NJ, USA, pp 39–67 8. Banci L, Ciofi-Baffoni S, Mikolajczyk M et al (2013) Human of the best-suited techniques. It is well appropriate in con- anamorsin binds [2Fe–2S] clusters with unique electronic proper- junction with other techniques, such as EPR or resonance ties. J Biol Inorg Chem 18:883–893. https ://doi.or g/10.1007/s0077 Raman. The advantages of Mössbauer spectroscopy are that 5-013-1033-1 it detects all iron in a sample, regardless of physical state, 9. Yang J, Naik SG, Ortillo DO et al (2009) The iron–sulfur clus- ter of pyruvate formate-lyase activating enzyme in whole cells: spin or oxidation state, and that the signal is proportional to 2+ cluster interconversion and a valence-localized [4Fe-4S] state. the amount of iron in the sample. Other advantages are that Biochemistry 48:9234–9241. https ://doi.org/10.1021/bi901 0286 it is measured in bulk on frozen solutions, so samples are 10. Fleischhacker AS, Stubna A, Hsueh K-L et al (2012) Characteriza- not subject to degradation, and that it is rather inexpensive, tion of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR. Biochemistry 51:4453–4462. https://doi.or g/10.1021/bi300 compared, for example, to synchrotron-based techniques. Although it is a nuclear spectroscopy based on gamma-ray 1 3 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 643 11. Giel JL, Nesbit AD, Mettert EL et al (2013) Regulation of iron– 28. Li H, Mapolelo DT, Randeniya S et al (2012) Human glutaredoxin sulphur cluster homeostasis through transcriptional control of the 3 forms [2Fe-2S]-bridged complexes with human BolA2. Bio- Isc pathway by [2Fe–2S]–IscR in Escherichia coli. Mol Microbiol chemistry 51:1687–1696. https ://doi.org/10.1021/bi201 9089 87:478–492. https ://doi.org/10.1111/mmi.12052 29. Mapolelo DT, Zhang B, Randeniya S et al (2013) Monothiol glu- 12. Bak DW, Elliott SJ (2014) Alternative FeS cluster ligands: tuning taredoxins and A-type proteins: partners in Fe-S cluster traffick - redox potentials and chemistry. Curr Opin Chem Biol 19:50–58. ing. Dalton Trans 42:3107–3115. https://doi.or g/10.1039/C2DT3 https ://doi.org/10.1016/j.cbpa.2013.12.015 2263C 13. Zuris JA, Harir Y, Conlan AR et al (2011) Facile transfer of [2Fe- 30. Smith AD, Jameson GNL, Dos Santos PC et al (2005) NifS- 2S] clusters from the diabetes drug target mitoNEET to an apo- mediated assembly of [4Fe-4S] Clusters in the N- and C-terminal acceptor protein. Proc Natl Acad Sci USA 108:13047–13052. domains of the NifU scaffold protein. Biochemistry 44:12955– https ://doi.org/10.1073/pnas.11099 86108 12969. https ://doi.org/10.1021/bi051 257i 14. Bak DW, Elliott SJ (2013) Conserved hydrogen bonding networks 31. Agar JN, Krebs C, Frazzon J et al (2000) IscU as a scaffold for of MitoNEET tune Fe-S cluster binding and structural stability. iron–sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] Biochemistry 52:4687–4696. https://doi.or g/10.1021/bi400540m and [4Fe-4S] clusters in IscU. Biochemistry 39:7856–7862. https 15. Golinelli-Cohen M-P, Lescop E, Mons C et al (2016) Redox con-://doi.org/10.1021/bi000 931n trol of the human iron–sulfur repair protein MitoNEET activity 32. Unciuleac M-C, Chandramouli K, Naik S et al (2007) In vitro via its iron–sulfur cluster. J Biol Chem 291:7583–7593. https :// activation of apo-aconitase using a [4Fe-4S] cluster-loaded form doi.org/10.1074/jbc.M115.71121 8 of the IscU [Fe-S] cluster scaffolding protein. Biochemistry 16. Ferreira GC, Franco R, Lloyd SG et al (1994) Mammalian fer- 46:6812–6821. https ://doi.org/10.1021/bi602 6665 rochelatase, a new addition to the metalloenzyme family. J Biol 33. Bonomi F, Iametti S, Ta D, Vickery LE (2005) Multiple turnover Chem 269:7062–7065 transfer of [2Fe2S] clusters by the iron–sulfur cluster assembly 17. Fee JA, Findling KL, Yoshida T et al (1984) Purification and scaffold proteins IscU and IscA. J Biol Chem 280:29513–29518 characterization of the Rieske iron–sulfur protein from Thermus 34. Gao H, Subramanian S, Couturier J et al (2013) Arabidopsis thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and ligands. J Biol Chem 259:124–133 is competent for in vitro maturation of chloroplast [2Fe-2S] and 18. Chandramouli K, Unciuleac M-C, Naik S et al (2007) Formation [4Fe-4S] cluster-containing proteins. Biochemistry 52:6633– and properties of [4Fe-4S] clusters on the IscU scaffold protein. 6645. https ://doi.org/10.1021/bi400 7622 Biochemistry 46:6804–6811. https ://doi.org/10.1021/bi602 6659 35. Blanc B, Gerez C, Ollagnier de Choudens S (2015) Assembly of 19. Lill R, Hoffmann B, Molik S et al (2012) The role of mitochondria Fe/S proteins in bacterial systems: biochemistry of the bacterial in cellular iron–sulfur protein biogenesis and iron metabolism. ISC system. Biochim Biophys Acta 1853:1436–1447. https: //doi. Biochim Biophys Acta 1823:1491–1508. https ://doi.or g/10.1016/j.org/10.1016/j.bbamc r.2014.12.009 bbamc r.2012.05.009 36. Mansy SS, Wu G, Surerus KK, Cowan JA (2002) Iron–sulfur clus- 20. Ojeda L, Keller G, Muhlenhoff U et al (2006) Role of glutar - ter biosynthesis: Thermatoga maritima IscU is a structured iron– edoxin-3 and glutaredoxin-4 in the iron regulation of the aft1 tran- sulfur cluster assembly protein. J Biol Chem 277:21397–21404. scriptional activator in Saccharomyces cerevisiae. J Biol Chem https ://doi.org/10.1074/jbc.M2014 39200 281:17661–17669. https ://doi.org/10.1074/jbc.M6021 65200 37. Wu S, Wu G, Surerus KK, Cowan JA (2002) Iron–sulfur cluster 21. Pujol-Carrion N, Belli G, Herrero E et al (2006) Glutaredoxins biosynthesis. Kinetic analysis of [2Fe-2S] cluster transfer from Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxi- holo ISU to apo Fd: role of redox chemistry and a conserved dative stress response in Saccharomyces cerevisiae. J Cell Sci aspartate. Biochemistry 41:8876–8885. https ://doi.org/10.1021/ 119:4554. https ://doi.org/10.1242/jcs.03229 bi025 6781 22. Kumánovics A, Chen OS, Li L et al (2008) Identification of FRA1 38. Ding H, Clark RJ, Ding B (2004) IscA mediates iron delivery for and FRA2 as genes involved in regulating the yeast iron regulon assembly of iron–sulfur clusters in IscU under the limited acces- in response to decreased mitochondrial iron–sulfur cluster synthe- sible free iron conditions. J Biol Chem 279:37499–37504. https sis. J Biol Chem 283:10276–10286. https ://doi.org/10.1074/jbc.://doi.org/10.1074/jbc.M4045 33200 M8011 60200 39. Yang J, Bitoun JP, Ding H (2006) Interplay of IscA and IscU in 23. Li H, Mapolelo DT, Dingra NN et al (2009) The yeast iron regu- biogenesis of iron–sulfur clusters. J Biol Chem 281:27956–27963. latory proteins Grx3/4 and Fra2 form heterodimeric complexes https ://doi.org/10.1074/jbc.M6013 56200 containing a [2Fe-2S] cluster with cysteinyl and histidyl ligation. 40. Mapolelo DT, Zhang B, Naik SG et al (2012) Spectroscopic and Biochemistry 48:9569–9581. https://doi.or g/10.1021/bi901182w functional characterization of iron-bound forms of Azotobac- Nif 24. Bandyopadhyay S, Gama F, Molina-Navarro MM et al (2008) ter vinelandii IscA. Biochemistry 51:8056–8070. https ://doi. Chloroplast monothiol glutaredoxins as scaffold proteins for the org/10.1021/bi300 664j assembly and delivery of [2Fe–2S] clusters. EMBO J 27:1122. 41. Mapolelo DT, Zhang B, Naik SG et al (2012) Spectroscopic and https ://doi.org/10.1038/emboj .2008.50 functional characterization of iron–sulfur cluster-bound forms Nif 25. Lillig CH, Berndt C, Vergnolle O et al (2005) Characterization of Azotobacter vinelandii IscA. Biochemistry 51:8071–8084. of human glutaredoxin 2 as iron–sulfur protein: a possible role as https ://doi.org/10.1021/bi300 6658 redox sensor. Proc Natl Acad Sci USA 102:8168–8173. https :// 42. Loiseau L, Gerez C, Bekker M et al (2007) ErpA, an iron–sulfur doi.org/10.1073/pnas.05007 35102 (Fe–S) protein of the A-type essential for respiratory metabo- 26. Mühlenhoff U, Molik S, Godoy JR et al (2010) Cytosolic mono- lism in Escherichia coli. Proc Natl Acad Sci USA 104:13626– thiol glutaredoxins function in intracellular iron sensing and traf- 13631. https ://doi.org/10.1073/pnas.07058 29104 ficking via their bound iron–sulfur cluster. Cell Metab 12:373– 43. Netz DJ, Genau HM, Weiler BD et al (2016) The conserved 385. https ://doi.org/10.1016/j.cmet.2010.08.001 Dre2 uses essential [2Fe-2S] and [4Fe-4S] clusters for function 27. Li H, Mapolelo DT, Dingra NN et al (2011) Histidine 103 in Fra2 in cytosolic iron–sulfur protein assembly. Biochem J 473:2073– is an iron–sulfur cluster ligand in the [2Fe-2S] Fra2-Grx3 complex 2085. https ://doi.org/10.1042/BCJ20 16041 6 and is required for in vivo iron signaling in yeast. J Biol Chem 44. Gupta V, Sendra M, Naik SG et al (2009) Native Escherichia 286:867–876. https ://doi.org/10.1074/jbc.M110.18417 6 coli SufA, coexpressed with SufBCDSE, purifies as a [2Fe-2S] 1 3 644 JBIC Journal of Biological Inorganic Chemistry (2018) 23:635–644 protein and acts as an Fe-S transporter to Fe-S target enzymes. Saccharomyces cerevisiae. Biochemistry 48:9556–9568. https J Am Chem Soc 131:6149–6153. https://doi.or g/10.1021/ja807 ://doi.org/10.1021/bi901 110n 551e 61. Lindahl PA, Holmes-Hampton GP (2011) Biophysical probes of 45. Sendra M, Ollagnier de Choudens S, Lascoux D et al (2007) iron metabolism in cells and organelles. Curr Opin Chem Biol The SUF iron–sulfur cluster biosynthetic machinery: sulfur 15:342–346. https ://doi.org/10.1016/j.cbpa.2011.01.007 transfer from the SUFS–SUFE complex to SUFA. FEBS Lett 62. Park J, McCormick SP, Chakrabarti M, Lindahl PA (2013) The 581:1362–1368. https ://doi.org/10.1016/j.febsl et.2007.02.058 lack of synchronization between iron uptake and cell growth 46. Wollers S, Layer G, Garcia-Serres R et al (2010) Iron–sulfur leads to iron overload in Saccharomyces cerevisiae during post- (Fe–S) cluster assembly: the SufBCD complex is a new type of exponential growth modes. Biochemistry 52:9413–9425. https Fe-S scaffold with a flavin redox cofactor. J Biol Chem. https: //://doi.org/10.1021/bi401 0304 doi.org/10.1074/jbc.M110.12744 9 63. Wofford JD, Lindahl PA (2015) Mitochondrial iron–sulfur clus - 47. Colin F, Martelli A, Clémancey M et al (2013) Mammalian ter activity and cytosolic iron regulate iron traffic in Saccha- frataxin controls sulfur production and iron entry during de romyces cerevisiae. J Biol Chem. https ://doi.org/10.1074/jbc. novo Fe S cluster assembly. J Am Chem Soc 135:733–740. M115.67666 8 4 4 https ://doi.org/10.1021/ja308 736e 64. Hudder BN, Morales JG, Stubna A et al (2007) Electron para- 48. Fox NG, Chakrabarti M, McCormick SP et al (2015) The human magnetic resonance and Mössbauer spectroscopy of intact iron–sulfur assembly complex catalyzes the synthesis of [2Fe- mitochondria from respiring Saccharomyces cerevisiae. J Biol 2S] clusters on ISCU2 that can be transferred to acceptor mol- Inorg Chem 12:1029–1053. https ://doi.or g/10.1007/s0077 ecules. Biochemistry 54:3871–3879. https ://doi.org/10.1021/ 5-007-0275-1 bi501 4485 65. Lindahl PA, Morales JG, Miao R, Holmes-Hampton G (2009) 49. Fox NG, Das D, Chakrabarti M et al (2015) Frataxin acceler- Chapter 15 isolation of Saccharomyces cerevisiae mitochon- ates [2Fe-2S] Cluster formation on the human Fe–S assembly dria for Mössbauer, epr, and electronic absorption spectroscopic complex. Biochemistry 54:3880–3889. https://doi.or g/10.1021/ analyses. Methods in enzymology. Academic Press, New York, bi501 4497 pp 267–285 50. Matzanke BF, Ecker DJ, Yang T-S et al (1986) Escherichia 66. Cockrell A, McCormick SP, Moore MJ et al (2014) Mössbauer, coli iron enterobactin uptake monitored by Mössbauer spec- EPR, and modeling study of iron trafficking and regulation in troscopy. J Bacteriol 167:674–680. https ://doi.or g/10.1128/ Δccc1 and CCC1-up Saccharomyces cerevisiae. Biochemistry jb.167.2.674-680.1986 53:2926–2940. https ://doi.org/10.1021/bi500 002n 51. Matzanke BF, Bill E, Müller GI et al (1989) In vivo Mössbauer 67. Sutak R, Seguin A, Garcia-Serres R et al (2012) Human mito- spectroscopy of iron uptake and ferrometabolism in Escherichia chondrial ferritin improves respiratory function in yeast mutants coli. Hyperfine Interact 47:311–327. https ://doi.org/10.1007/ deficient in iron–sulfur cluster biogenesis, but is not a functional BF023 51615 homologue of yeast frataxin. MicrobiologyOpen 1:95–104. 52. Matzanke BF, Müller GI, Bill E, Trautwein AX (1989) Iron https ://doi.org/10.1002/mbo3.18 metabolism of Escherichia coli studied by Mössbauer spectros- 68. Holmes-Hampton GP, Miao R, Morales JG et al (2010) A Non- copy and biochemical methods. Eur J Biochem 183:371–379. heme high-spin ferrous pool in mitochondria isolated from https ://doi.org/10.1111/j.1432-1033.1989.tb149 38.x fermenting Saccharomyces cerevisiae. Biochemistry 49:4227– 53. Benda R, Tse Sum Bui B, Schünemann V et al (2002) Iron– 4234. https ://doi.org/10.1021/bi100 1823 sulfur clusters of biotin synthase in vivo: a Mössbauer study. 69. Miao R, Martinho M, Morales JG et al (2008) EPR and Möss- Biochemistry 41:15000–15006. https ://doi.org/10.1021/bi026 bauer spectroscopy of intact mitochondria isolated from Yah1p- 590q depleted Saccharomyces cerevisiae. Biochemistry 47:9888– 54. Cosper MM, Jameson GNL, Eidsness MK et al (2002) Recom- 9899. https ://doi.org/10.1021/bi801 047q 2+ binant Escherichia coli biotin synthase is a [2Fe–2S] protein 70. Cockrell AL, Holmes-Hampton GP, McCormick SP et al (2011) in whole cells. FEBS Lett 529:332–336. https://doi.or g/10.1016/ Mössbauer and EPR study of iron in vacuoles from ferment- S0014 -5793(02)03390 -2 ing Saccharomyces cerevisiae. Biochemistry 50:10275–10283. 55. Beilschmidt LK, Ollagnier de Choudens S, Fournier M et al https ://doi.org/10.1021/bi201 4954 (2017) ISCA1 is essential for mitochondrial Fe S biogenesis 71. Holmes-Hampton GP, Jhurry ND, McCormick SP, Lindahl PA 4 4 in vivo. Nat Commun 8:15124. https:/ /doi.org/10.1038/ncomm (2013) Iron content of Saccharomyces cerevisiae cells grown s1512 4 under iron-deficient and iron-overload conditions. Biochemistry 56. Lesuisse E, Santos R, Matzanke BF et al (2003) Iron use for 52:105–114. https ://doi.org/10.1021/bi301 5339 haeme synthesis is under control of the yeast frataxin hom- 72. Miao R, Holmes-Hampton GP, Lindahl PA (2011) Biophysical ologue (Yfh1). Hum Mol Genet 12:879–889. https ://doi. investigation of the Iron in Aft1-1up and Gal-YAH1 Saccha- org/10.1093/hmg/ddg09 6 romyces cerevisiae. Biochemistry 50:2660–2671. https ://doi. 57. Beilschmidt LK, Puccio HM (2014) Mammalian Fe–S cluster org/10.1021/bi102 015s biogenesis and its implication in disease. Biochimie 100:48–60. 73. Morales JG, Holmes-Hampton GP, Miao R et al (2010) Bio- https ://doi.org/10.1016/j.bioch i.2014.01.009 physical characterization of iron in mitochondria isolated from 58. Martelli A, Puccio H (2014) Dysregulation of cellular iron respiring and fermenting yeast. Biochemistry 49:5436–5444. metabolism in Friedreich ataxia: from primary iron–sulfur clus-https ://doi.org/10.1021/bi100 558z ter deficit to mitochondrial iron accumulation. Front Pharmacol 74. Qi W, Li J, Chain CY et al (2013) Glutathione-complexed iron– 5:130. https ://doi.org/10.3389/fphar .2014.00130 sulfur clusters. Reaction intermediates and evidence for a tem- 59. Seguin A, Sutak R, Bulteau A-L et al (2010) Evidence that plate effect promoting assembly and stability. Chem Commun yeast frataxin is not an iron storage protein in vivo. Biochim 49:6313–6315. https ://doi.org/10.1039/C3CC4 3620A Biophys Acta 1802:531–538. https ://doi.org/10.1016/j.bbadi 75. Qi W, Li J, Chain CY et al (2012) Glutathione complexed s.2010.03.008 Fe-S centers. J Am Chem Soc 134:10745–10748. https ://doi. 60. Miao R, Kim H, Koppolu UMK et al (2009) Biophysical char-org/10.1021/ja302 186j acterization of the iron in mitochondria from Atm1p-depleted 1 3
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