Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions, including electron transfer and catalysis. Specialized multiprotein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein, on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC(2)D scaffold from Escherichia coli by XAS and M & ouml;ssbauer, UV-visible absorption, and EPR spectroscopies. Interestingly, we propose that SufBC(2)D harbors two iron-sulfur-containing species, a [2Fe-2S] cluster and an as-yet unidentified species. Mutagenesis and biochemistry were used to propose amino acid ligands for the [2Fe-2S] cluster, supporting the hypothesis that both SufB and SufD are involved in the Fe-S cluster ligation. The [2Fe-2S] cluster can be transferred to ferredoxin in agreement with the SufBC(2)D scaffold function. These results are discussed in the context of Fe-S cluster biogenesis.

ABSTRACTIron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions including electron transfer and catalysis. Specialized multi-protein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS, Mössbauer, UV-visible absorption and EPR spectroscopy. Interestingly, we propose that SufBC2D harbors two types of Fe-S cluster, a [2Fe-2S] cluster with an unprecedented usual coordination and a previously unreported [3Fe-3S] cluster. These data combined with mutagenesis and biochemistry allow to propose ligands for these clusters. These results support the hypothesis that both SufB and SufD are involved in Fe-S cluster ligation and are discussed in the context of Fe-S cluster biogenesis where both [2Fe-2S] and [4Fe-4S] clusters need to mature cellular Fe-S protein targets.

Fe-S cluster-containing proteins occur in most organisms, wherein they assist in myriad processes from metabolism to DNA repair via gene expression and bioenergetic processes. Here, we used both in vitro and in vivo methods to investigate the capacity of the four Fe-S carriers, NfuA, SufA, ErpA, and IscA, to fulfill their targeting role under oxidative stress. Likewise, Fe-S clusters exhibited varying half-lives, depending on the carriers they were bound to; an NfuA-bound Fe-S cluster was more stable (t(1/2) = 100 min) than those bound to SufA (t(1/2) = 55 min), ErpA (t(1/2) = 54 min), or IscA (t(1/2) = 45 min). Surprisingly, the presence of NfuA further enhanced stability of the ErpA-bound cluster to t(1/2) = 90 min. Using genetic and plasmon surface resonance analyses, we showed that NfuA and ErpA interacted directly with client proteins, whereas IscA or SufA did not. Moreover, NfuA and ErpA interacted with one another. Given all of these observations, we propose an architecture of the Fe-S delivery network in which ErpA is the last factor that delivers cluster directly to most if not all client proteins. NfuA is proposed to assist ErpA under severely unfavorable conditions. A comparison with the strategy employed in yeast and eukaryotes is discussed.

Biosynthesis of ironsulphur (Fe-S) proteins is catalysed by multi-protein systems, ISC and SUF. However, non-ISC, non-SUF Fe-S biosynthesis factors have been described, both in prokaryotes and eukaryotes. Here we report in vitro and in vivo investigations of such a non-ISC, non SUF component, the Nfu proteins. Phylogenomic analysis allowed us to define four subfamilies. Escherichia coli NfuA is within subfamily II. Most members of this subfamily have a Nfu domain fused to a degenerate A-type carrier domain (ATC*) lacking Fe-S cluster co-ordinating Cys ligands. The Nfu domain binds a [4Fe-4S] cluster while the ATC* domain interacts with NuoG (a complex I subunit) and aconitase B (AcnB). In vitro, holo-NfuA promotes maturation of AcnB. In vivo, NfuA is necessary for full activity of complex I under aerobic growth conditions, and of AcnB in the presence of superoxide. NfuA receives Fe-S clusters from IscU/HscBA and SufBCD scaffolds and eventually transfers them to the ATCs IscA and SufA. This study provides significant information on one of the Fe-S biogenesis factors that has been often used as a building block by ISC and/or SUF synthesizing organisms, including bacteria, plants and animals.

Iron/sulfur (Fe/S) proteins are central to the functioning of cells in both prokaryotes and eukaryotes. Here, we show that the yhgI gene, which we renamed nfuA, encodes a two-domain protein that is required for Fe/S biogenesis in Escherichia coli. The N-terminal domain resembles the so-called Fe/S A-type scaffold but, curiously, has lost the functionally important Cys residues. The C-terminal domain shares sequence identity with Nfu proteins. Mossbauer and UV-visible spectroscopic analyses revealed that, upon reconstitution, NfuA binds a [4Fe-4S] cluster. Moreover, NfuA can transfer this cluster to apo-aconitase. Mutagenesis studies indicated that the N-and C-terminal domains are important for NfuA function in vivo. Similarly, the functional importance of Cys residues present in the Nfu-like domain was demonstrated in vivo by introducing Cys-->Ser mutations. In vivo investigations revealed that the nfuA gene is important for E. coli to sustain oxidative stress and iron starvation. Also, combining nfuA with either isc or suf mutations led to additive phenotypic deficiencies, indicating that NfuA is a bona fide new player in Isc-and Suf-dependent Fe/S biogenesis pathways. Taken together, these data demonstrate that NfuA intervenes in the maturation of apoproteins in E. coli, allowing them to acquire Fe/S clusters. By taking into account results from numerous previous transcriptomic studies that had suggested a link between NfuA and protein misfolding, we discuss the possibility that NfuA could act as a scaffold/chaperone for damaged Fe/S proteins.

Understanding the biogenesis of iron–sulfur (Fe–S) proteins is relevant to many fields, including bioenergetics, gene regulation, and cancer research. Several multiprotein complexes assisting Fe–S assembly have been identified in both prokaryotes and eukaryotes. Here, we identify in Escherichia coli an A-type Fe–S protein that we named ErpA. Remarkably, erpA was found essential for growth of E. coli in the presence of oxygen or alternative electron acceptors. It was concluded that isoprenoid biosynthesis was impaired by the erpA mutation. First, the eukaryotic mevalonate-dependent pathway for biosynthesis of isopentenyl diphosphate restored the respiratory defects of an erpA mutant. Second, the erpA mutant contained a greatly reduced amount of ubiquinone and menaquinone. Third, ErpA bound Fe–S clusters and transferred them to apo-IspG, a protein catalyzing isopentenyl diphosphate biosynthesis in E. coli . Surprisingly, the erpA gene maps at a distance from any other Fe–S biogenesis-related gene. ErpA is an A-type Fe–S protein that is characterized by an essential role in cellular metabolism.

The synthesis of 5′-di- and 5′-triphosphate of 8-vinyladenosine to be tested on ribonucleotide reductases requires the modification of known methods. The phosphate group was introduced by treatment with an in situ generated chlorophosphite. Protection of the 2′,3′ diol with acetyl groups suppressed depurination during acid removal of the phosphotriester protecting groups. The di- and triphosphate compounds were obtained by treatment of the activated adenylic acid with phosphate or pyrophosphate anions followed by removal of the acetate protecting groups. Preliminary studies were conducted on Escherichia coli ribonucleotide reductase and have shown that the diphosphate compound is efficiently reduced.

Publisher Summary This chapter describes methods (light absorption and electron paramagnetic resonance (EPR) spectroscopy) for monitoring the various types of tyrosyl radicals in class I RNRs. These radicals are important targets for antiproliferative compounds. Three types of tyrosyl radicals have been observed among a large number of purified reductases. R2 proteins from E. coli, Mycobacterium tuberculosis, and Arabidopsis thaliana may be considered as representatives of each of these classes. The three proteins can be prepared from overexpressing E. coli strains, which have been transformed with the corresponding plasmids: pVNR2,14 pMtbR2,11,12, and pETR2,13 respectively. The presence of a stable tyrosyl radical in class I RNR can be easily concluded from its X-band EPR spectrum at liquid helium temperature, which shows a characteristic doublet centered at g = 2.0. The experimental conditions for recording the EPR spectrum of the various radicals are discussed in the chapter.

The local electrostatic environment plays a critical role in determining the physicochemical properties of reactive radicals in proteins. High-field electron paramagnetic resonance (HF-EPR) spectroscopy has been used to determine the sensitivity of the tyrosyl radical g-values to local electrostatic environment. Site-specific mutants of ribonucleotide reductase from Escherichia coli were used to study the effect of introducing a charge group on the HF-EPR spectrum of the stable tyrosyl (Y122) radical. The changes affected by the mutations were small, but measurable. Mutation of isoleucine-74 to an arginine (I74R) or lysine (I74K) induced disorder in the hyperfine interactions. Similar effects were observed for the mutation of valine-136 to an arginine (V136R) or asparagine (V136N). For five or six mutants studied, the g(x)() component of the g-tensor was distributed. For the isoleucine-74 to lysine (I74K) and leucine-77 to phenylalanine (L77F) mutants, a shift of 1 x 10(-)(4) in g(x)() value was also detected. For the I74K mutant, it is shown that the shift is consistent with the introduction of a charged residue, but cannot be distinguished from changes in the electrostatic effect of the nearby diiron center. For the L77F mutant, the shift is induced by the diiron center. Using existing tyrosyl radical g-tensor measurements, we have developed a simple effective charge model that allows us to rationalize the effect of the local electrostatic environments in a number of proteins.

ion at the specific glycine residue (9). Under exposure to air, protein b stabilizes (2Fe-2S) centers instead, which under anaerobic and reductive conditions are transformed back into active (4Fe-4S) centers (10). The combination of an iron-sulfur center and AdoMet for generating free radicals appears to be a general strategy in biological systems. It is now quite well established that such a chemistry is indeed utilized also in the pyruvate formate-lyase system and in the biotin synthase (11, 12). Even though there is no amino acid sequence homology between these systems it has been suggested that the cysteines of the CXXXCXXC motif common to biotin synthase and the activating components of pyruvate formate-lyase and ribonucleotide reductase (Fig. 1A) provide a specific metal binding site in each of these enzymes (11). The Fe-S enzyme lipoate synthase also contains this motif but has not been shown yet to require AdoMet for activity (13, 14). The lysine aminomutase belongs to this class of enzymes but is not included in Fig. 1 because its amino acid sequence is

Ribonucleotide reductase (RNR) is a key enzyme for DNA synthesis since it provides cells with deoxyribonucleotides, the DNA precursors. Class I alpha 2 beta 2 RNRs contain a dinuclear iron center and an essential tyrosyl radical in the beta 2 component (protein R2). This is also true for the purified protein R2 of Mycobacterium tuberculosis RNR, as shown by iron analysis, light absorption and EPR spectroscopy. EPR spectroscopy at 286 GHz revealed a high g(x) value, suggesting that the radical is not hydrogen bonded, as in other prokaryotic R2s and in contrast with eukaryotic R2s (from Arabidopsis thaliana and mouse). Furthermore, it proved to be very resistant to scavenging by a variety of phenols and thiols and by hydroxyurea, similar to the Escherichia coli radical. By comparison, the plant and mouse radicals are very sensitive to drugs such as resveratrol and 2-thiophenthiol. The radical from M. tuberculosis RNR does not seem to be an appropriate target for new antituberculous agents

Resveratrol, a natural phytoalexin found in grapes, is well known for its presumed role in the prevention of heart disease, associated with red wine consumption. We show here that it is a remarkable inhibitor of ribonucleotide reductase and DNA synthesis in mammalian cells, which might have further applications as an antiproliferative or a cancer chemopreventive agent in humans.

Ribonucleotide reductase is a key enzyme for DNA synthesis. Its small component, named protein R2, contains a tyrosyl radical essential for activity. Consequently, radical scavengers are potential antiproliferative agents. In this study, we show that the reactivity of the tyrosyl radical towards phenols, hydrazines, hydroxyurea, dithionite and ascorbate can be finely tuned by relatively small modifications of its hydrophobic close environment. For example, in this hydrophobic pocket, Leu77-->Phe mutation resulted in a protein with a much higher susceptibility to radical scavenging by hydrophobic agents. This might suggest that the protein is flexible enough to allow small molecules to penetrate in the radical site. When mutations keeping the hydrophobic character are brought further from the radical (for example Ile74-->Phe) the reactivity of the radical is instead very little affected. When a positive charge was introduced (for example Ile74-->Arg or Lys) the protein was more sensitive to negatively charged electron donors such as dithionite. These results allow us to understand how tyrosyl radical sites have been optimized to provide a good stability for the free radical.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta1) and 74 (beta2) kDa. Immunological and structural evidence suggests that beta2 is a degradation product of beta1 and that the active enzyme is a dimer of beta1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100-degrees-C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and an antiferromagnetically coupled diferric center. Recent crystallographic studies [Nordlund, P., Eklund, H., & Sjöberg, B.-M. (1990) Nature 345, 593-598] have shown that both the radical and the diiron site are deeply buried inside the protein and thus strongly support the hypothesis of long-range electron-transfer processes within protein R2. This study shows that monosubstituted hydrazines and hydroxylamines are able to reduce the tyrosyl radical and the ferric ions, under anaerobic conditions. It allows characterization of the site from which those compounds transfer their electrons to the iron/radical center. The efficiency of any given reducing agent is not solely governed by its redox potential but also by its size, its charge, and its hydrophobicity. We suggest, as a possible alternative to the long-range electron-transfer hypothesis, that conformational flexibility of the polypeptide chain might exist in solution and allow small molecules to penetrate the protein and react with the iron/radical center. This study also shows that two reduction mechanisms are possible, depending on which center, the radical or the metal, is reduced first. Full reduction of protein R2 yields reduced R2, characterized by a normal tyrosine residue and a diferrous center. Both the radical and the diferric center are regenerated from reduced R2 by reaction with oxygen, while only the diferric center is formed by reaction with hydrogen peroxide.

The diiron(III) center of the protein R2, a subunit of the ribonucleotide reductase, can be efficiently reduced with diimide to the mixed valence Fe(II)/Fe(III) state, which can be detected EPR spectroscopically. A comparison with the spectra of the mixed valence state of hemerythrin suggests that both Fe atoms are only weakly antiferromagnetically coupled, and are bound via a hydroxo bridge.

Das Dieisen(III)‐Zentrum des Proteins R2, einer Untereinheit der Ribonucleotid‐Reductase, läßt sich mit Diimid effizient zum gemischtvalenten FeIIFeIII‐Zustand reduzieren, der EPR‐spektroskopisch nachgewiesen werden kann. Der Vergleich mit den Spektren des gemischtvalenten Zustands von Hämerythrin legt nahe, daß beide Fe‐Atome nur schwach antiferromagnetisch gekoppelt und über eine Hydroxobrücke verbunden sind.

The active form of protein B2, the small subunit of ribonucleotide reductase from E. Coli, contains a binuclear non heme iron center and a tyrosyl radical. MetB2 is an inactive form that lacks the radical but retains the Fe(III) center. We earlier proposed that the function of the iron center was to catalyze the one-electron oxidation of the tyrosine residue from metB2 by dioxygen. We now report that incubation of metB2 with single oxygen atom donors, hydrogen peroxide, 3-chloroperoxybenzoic acid, monoperoxophtalate and 2-iodosobenzoate, also results in the formation of the tyrosyl radical, as monitored by UV-visible and EPR spectroscopy. A mechanism of reductive activation of dioxygen by the binuclear non heme iron center involving iron-oxo intermediates is proposed.