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we will briefly overview the process of the biogenesis of Fe-S clusters, essentially to frame a later discussion on possible structural requirements of the recipient apo-proteins for interactions with the Fe-S scaffolding proteins and transfer of the newly formed Fe-S cluster.
Rather than attempt to extensively cover all studies addressing folding and stability of Fe-S proteins, we will rather overview the challenging aspects and pitfalls of using this proteins as models for folding studies. In the last part of the chapter we will use our own work on di cluster ferredoxins as a case study.
Iron-sulfur proteins are a multifaceted class of proteins containing iron-sulfur clusters (Fe-S) as a prosthetic group. These proteins are ubiquitously found within all life domains and are involved in a plethora of essential biological processes and cellular pathways, such as respiration and photosynthesis, as well as DNA and RNA metabolism in addition to intervene in the regulation of iron homeostasis and gene expression. This functional versatility can be well correlated to the structural diversity and intrinsic chemical properties of the clusters. In fact, these amazing complexes of iron and cysteinate sulfur can present distinct polynuclear combining, up to eight irons (Figura com os varios centros ?), as well as the ability to interconvert and undergo ligand exchange reactions. Fe-S clusters are in great majority bound to the proteins by thiolate ligation, and accordingly cysteinyl sulfur is often the most frequently implemented ligand of Fe-S active sites. Besides the diverseness of the clusters, the evolution of multiple suitable folds hosting the clusters has also undoubtedly contributed to expand and/or optimize these proteins functions. In this respect, and despite the prominent biological importance of these class of proteins, the study of Fe-S clusters assembly in the cell and the underlying clusters mediation and contribution to a given protein fold and stability as well as in the folding pathway as yet started to emerge. Here we will overview the state of the art concerning the interplay between FeS clusters and protein folding and stability.
Biogenesis of iron-sulfur proteins: an overview
Iron-sulfur clusters are inorganic structures known to form spontaneously from the chemical assembly of ferric iron, thiol, and sulfide in the exclusion of oxidants. In a clear contrast to this intrinsic feature, cluster biosynthesis in all living cells does not occur spontaneously and requires surprisingly complex biochemical assembly systems. The need for assisted, rather than spontaneous, Fe-S cluster assembly and insertion into apoproteins is most likely the result of exigent anaerobic conditions, and the toxicity resulting from the high concentrations of iron and sulfide ions required for efficient chemical reconstitution in side the cell. Furthermore, the protein-mediated assembly of Fe-S clusters is likely to be more tightly regulated and efficient under biological control; in addition, protein mediation is likely to assure an increased specificity in the transfer of the formed Fe-S centre to the target peptide.
The biosynthesis of Fe-S clusters in eukaryotes and prokaryotes relies essentially on the general concept that a multistep process assemblies transient Fe-S clusters on scaffold proteins (1). These, so-called class of scaffold proteins function as the direct receptor of sulfur and iron deposition where clusters are formed and transfer into the recipient apoproteins (2, 3). The underlying overall mechanism and the molecular doers involved in this complex process, has however only started to be unveiled. In a brief summary, the first step of the process is believed to start with the association of a cysteine desulphurase with a scaffold protein. This transient complex ultimately results in the transfer of sulfur from the persulfide groups of cysteine desulphurases to the conserved cysteines residues present in scaffold proteins (4-6). Regarding the source of iron no clear consensus is yet available, although frataxin and frataxin homolog have been suggested to function as the iron donor (7-10). Presently, the molecular biochemistry of clusters assembly in scaffold proteins remains poorly understood as well as the number and the precise type of clusters that can be formed in each scaffold class of proteins. Nevertheless, it is established that redox proteins must be involved in the process as a source of electrons that provide the reducing equivalents necessary for cluster assembly (11-13). In addition it is also suggested that clusters assembly is likely to initially form [2Fe-2S] clusters as the basic building block for further clusters stoichiometry constructions and that these assemblies strongly depend on redox conditions (14). Based on in vitro studies the cluster transfer mechanism implies the initial formation of a transient complex between the cluster donor (scaffold protein) and the acceptor (the apo-protein target) within which a direct transfer occurs, leading to the apoform of the scaffold protein and the holo form of the target protein (15, 16). In this process, chaperone proteins have been considered to assist cluster transfer to target proteins (17, 18), yet little is known about the recognizing process between scaffold proteins and apo-states. In this respect, it has been demonstrated an apparent lack of specificity between the protein donor and acceptor (16). In fact, there is no acceptor specificity for a given donor, since the latter is in general able to deliver its cluster to different targets with comparable rates, leading to the formation of both [2Fe-2S] and ( complexes (19). As well, a given acceptor protein was proven to be able to accept clusters from different scaffold proteins; for example, ferredoxin can obtain its cluster in vitro from different donor proteins, (20, 21). To our view this strongly suggests that the protein-protein interaction between the cluster donor and acceptor has to be set through non specific interactions. Eventually, it can be speculated that the abundant hydrophobic residues that frequently surround the clusters and scaffold proteins may play a role in establishing non-specific hydrophobic interactions.
The interplay between protein folding and iron-sulfur cluster binding
A large diversity of proteins bind Fe-S clusters: a recent analysis has identified nearly 50 distinct structural folds which are able to accommodate Fe-S clusters (22). These folds are found in a variety of Fe-S proteins, from large multidomain complexes to small proteins. To some extent, in all cases the protein conformation and stability is affected by Fe-S cluster binding. However, the crosstalk between the folding of a Fe-S protein and binding of its cluster(s) may be rather diverse. Binding of the Fe-S cluster to its recipient apo-protein will shape the protein structure and local conformation, stabilising the protein as a result of the metal-protein interactions. In return the polypeptide chain accommodates the cluster and generates a protective ligand framework against the oxidative degradation of the oxygen-sensitive centre. However there are also many cases of Fe-S folds in which the clusters are harboured in less protective environments. What then could determine these differences?
Evolutionarily, one can speculate that nature may have taken advantage of this successful interplay, which has led to the ferredoxin type proteins, whose fold is considered to be primordial. In fact, for small Fe-S proteins preferentially engaged in electron transport purposes, the stability of the Fe-S clusters is essential for biological function and therefore the clusters are generally hosted in a relatively rigid and protective fold. This is well illustrated in several studies in which sets of hydrophobic residues have been shown to be critical for Fe-S cluster stability and function in rubredoxins, HiPIP and Ferredoxins (23) (24) (25). However, throughout evolution the functions of Fe-S proteins has expanded as a result of nature having recruited or generated protein folds with different characteristics, where the Fe-S sites are either labile or located at rather exposed domains or within subunit interfaces. This is illustrated for example in the crystal structure of SoxR (26) and CnfU (27) proteins, that evidenced a completely surface-exposed [2Fe-2S] cluster, as well as in the NMR structural characterization of poplar glutaredoxin C1 protein, which evidenced a bridging iron-sulfur cluster at the active site (28). Interestingly, proteins with unwrapped clusters seem to be mostly related with regulatory or scaffolding functions.
Folding and stability of small iron-sulfur proteins
Small iron sulfur proteins such as rubredoxins and ferredoxins are a priori good models for folding and stability studies considering their sizes, available structural information and the fact that they are usually very well characterized proteins, also in respect to the properties of the Fe-S clusters themselves, which are amenable for different spectroscopic methods. This is rather advantageous in experimental design as it allows combining methodologies that monitor changes in protein structure (such as far-UV CD, Trp emission and FT-IR) with those that report modifications at the Fe-S cluster itself (such as EPR, Resonance Raman and visible absorption or CD). This allows for a broad coverage of protein folding and unfolding events, which can be monitored under different perspectives, as illustrated in Figure 1 which shows a number of different spectroscopic fingerprints of a di-cluster ferredoxin that can be effectively used to monitor the folding status of the protein. In recent years we have been implementing this multi-technique approach to study the conformational properties and folding of rubredoxin (29),
Rieske proteins (30, 31) and di-cluster ferredoxins (32, 33).
One major pitfall in folding studies using Fe-S proteins arises from the fact that unfolding is in general irreversible, as a result of the disintegration of the Fe-S centers, which are required for the protein to refold into its native conformation, and are unable to self assemble from the individual components in solution without the assistance of the Fe-S biogenesis proteins. By itself this is indicative that Fe-S clusters are key structural and stabilizing elements that act as relevant nucleation points for the folded protein state. Nevertheless, thermodynamic information can be extracted from thermal transitions by studying the dependence of the apparent melting temperature (Tm) with the heating rate (ï®ï€©ï€ which allows to eliminate the artifacts of the kinetics of the irreversible transition, that become negligible at high heating rates. Therefore, the y-axis intersect of a Tm vs. 1/ï€ ï® plot gives the melting temperature at an infinite heating rate, i.e. at equilibrium. This formalism has been originally developed to deal with protein aggregation events during calorimetric analysis (34) and has subsequently been effectively used to analyze the thermodynamics of thermal unfolding of several Fe-S proteins (29, 35). In fact this methodology also allows to infer what might be the source of irreversibility, when the effect of varying the heating rate is monitored using a combination of different techniques. This is exemplified by studies on the thermal unfolding transition of a rubredoxin, which was monitored by differential scanning calorimetry and visible spectroscopy at increasing heating rates. In this case, while the Tm values determined by calorimetry almost did not change, those determined by visible absorption which monitored directly the iron site degradation were very affected, thus clearly suggesting that the Fe-S site is the source of the irreversibility of the reaction (29). One other approach to overcome this limitation is the characterization of unfolding pathways which may elicit the involvement of intermediates or highlight the influence of a particular interaction in the protein or Fe-S cluster, therefore providing an insight into the reverse process. Also, a direct comparison between related proteins may disclose different unfolding mechanisms, such as those observed between the rubredoxin from M. jannaschii, which undergoes thermal denaturation via a simple two step mechanism with concomitant loss of tertiary structure, hydrophobic collapse and disintegration of the iron-sulfur centre (36), and that of P. furiosus, which has a complex kinetic behavior comprising at least three intermediate steps (37).
Di-cluster ferredoxins as a case study
In recent years we have been extensively studying the family of seven iron di-cluster ferredoxins from thermoacidophilic archaea as working models for folding and stability studies in Fe-S proteins. These are small (~11.6 kDa) acidic (pI ~3.5) proteins which contain a [3Fe-4S]1+/0 (cluster I) and a [4Fe-4S]2+/1+ centre (cluster II) within a (ï¢ï¡ï¢)2 core fold and a N-terminal extension (~30 amino acids) which comprise a His/Asp Zn2+ site (38). These are highly abundant cytosolic proteins, which has allowed in cell EPR studies to establish the biological relevance of [3Fe-4S]1+/0 centres (39), in opposition to the possibility that they could have arisen from oxidative damage of a ï›4Fe-4Sï2+/1+ cluster (40, 41). The crystal structure of the Sulfolobus tokodaii ferredoxin elicited the presence of an as yet unknown zinc site (42). However, this structure was obtained from an oxidative corruption of the native form, with the tetranuclear centre converted into a trinuclear form originating two ï›3Fe-4Sï1+/0 centres (42), which prompted several studies attempting to explain the discrepancy between the native and the oxidative damaged form (43, 44). This aspect has been recently clarified with the publication of the at 2.0 Å resolution ferredoxin from Acidianus ambivalens ferredoxin (AaFd), in its physiological form harbouring the intact clusters (45). Unless otherwise noted, the latter has been our working model which is described in the following sections.
Ferredoxin hyperstability and unfolding pathways
The proteomes from thermophilic consist of naturally thermally resistant proteins, but early studies have shown that thermophilic di-cluster Ferredoxins proteins were extremely resistant to denaturation: incubation at 70°C during 72 h has no denaturing effect (38) and incubation at neutral pH with 8M GuHCl also does not result in protein unfolding (46). These are unique properties considering the fact that these proteins harbor cuboid inorganic structures whose relative stability is unclear and are know to easily undergo degradative conversions in many other Fe-S. Therefore, the di-cluster ferredoxin model as a tool aimed at characterizing the source of protein hyperstability and discriminate between the relative contributions arising from the chemical properties of the polypeptide chain and those of the Fe-S clusters and zinc site. Since ferredoxin was not unfolding at pH 7.0, even in the presence of 8 M GuHCl, more extreme pH values (4 < pH > 10) had to be screened in order to perturb electrostatic interactions and therefore decrease the melting transition to bellow the boiling point of water (46, 47).
For these reasons, the initial mechanistic studies on ferredoxin unfolding were carried out at alkaline conditions (pH 10) and it has been suggested that ferredoxin unfolding would involve the degradation of the Fe-S clusters via a linear three iron sulfur centre intermediate (48) on the basis of the appearance of visible bands at 520nm and 610 nm (48), identical to the one observed when an identical structure is formed in purple aconitase (49). The latter is formed when inactive [3Fe4S] aconitase is exposed to pH > 9 or is partly denatured with urea (49). A purple compound, for which there is no known biological function, is then generated with a characteristic set of spectroscopic signatures at 520 and 610 nm, which and can be re-converted to the [4Fe4S] active form, upon lowering the pH and incubation with an iron salt and a thiol. This observation in di-cluster ferredoxins was later generalized to other iron-sulfur proteins (50, 51), even those containing centres with lower nuclearities (52, 53); unlike in aconitase, the reconversion back to the original clusters was not taking place. However, subsequent kinetic and spectroscopic analysis aimed at analyzing if one of the clusters had a preponderant role in the formation of this hypothetical intermediate have established that shortly after initial protein unfolding, iron release proceeds monophasically at a rate comparable to that of cluster degradation, and that no typical EPR features of linear three-iron sulfur centres are observed (54). Further, it was observed that EDTA prevents formation of the transient bands and that sulfide significantly enhances its intensity and lifetime, even after protein unfolding. Altogether these observations showed suggested that such intermediate was an artefact arising from iron sulfides which were produced when the Fe-S clusters were disassembled at pH 10, and the misleading spectroscopic fingerprint was even shown to be generated upon alkaline unfolding of other Fe containing proteins (55). On the other hand, ferredoxin unfolding under acid conditions (pH 2) is monophasic and cluster disruption and protein unfolding are simultaneous events.
Effects of electrostatic interactions and metal centres on ferredoxin stability
What are then the factors underlying the extraordinary stability of this ferredoxin? Clearly electrostatic interactions are playing a role, and so are the Fe-S clusters and eventually the His/Asp zinc site as well. Can we determined the relative contributions of ionic interactions over iron-sulfur clusters to the stability of this ferredoxin? With this goal in mind we have carried out a study in which the protein conformational changes and stability of Fe-S clusters were evaluated upon poising the protein at different pH values. In the pH 5-8 interval, the protein has a very high apparent melting temperature (Tm ~120°C), which nevertheless decreases towards pH extremes. In particular, acidification triggers events in two steps: down to the isoelectric point (pH 3.5) the Fe-S clusters remain unchanged, the secondary structure content increases and the single Trp becomes more solvent shielded, denoting structural packing resulting from protonation, presumably of Asp and Glu residues. Interestingly, this pH change has a minor affect on the Fe-S clusters, as shown by the fact that the absorption 410nm band and chemical shifts of the ï¢-CH2 protons from the clusters ligand cysteines, remain essentially unaffected (25). The relative stabilizing contribution of the clusters becomes evident when stabilizing ionic interactions are switched off as a result of poising the protein at pH 3.5, at an overall null charge: under these conditions, the Fe-S clusters disassemble at Tm = 72°C, whereas the protein unfolds at Tm = 52°C. This stabilizing effect is also evident under other buffer conditions as the presence of EDTA lowers the observed melting temperature in thermal ramp experiments and the midpoint denaturant concentration in equilibrium chemical unfolding experiments (54). Further pH drop from 3.5 to 1.5 resulted in a marked increase in the ANS fluorescence emission that is concomitant with the disintegration of Fe-S clusters. In fact, at pH 1.5 the spectroscopic signature of the Fe-S clusters is absent and a ~20 fold increased in the ANS emission, in comparison to that of the native state at pH 7 is observed. These results clearly indicate that the acidification below pH 3.5 is enough to disintegrate the Fe-S clusters, making hydrophobic regions simultaneously accessible. In fact, the disintegration of the Fe-S clusters triggers a structural rearrangement of the apo-state, leading to a conformation with poor tertiary contacts, substantial secondary structure and strong ANS binding, which are structural characteristics of molten globule states (see below).
Monitoring Ferredoxin unfolding at different levels of metalloprotein organization
In order to analyze globally the conformational and Fe-S cluster changes occurring during ferredoxin unfolding we have combined a battery of biophysical methods that were used to monitor ferredoxin unfolding at different levels of the organization of this metalloprotein (33). However, upon acidification down to pH 2.5 the protein folding and its iron-sulfur centers remain intact, although destabilized, making them amenable to thermal unfolding studies with Tm transitions well below the boiling point of water
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