Characterization of NocL involved in thiopeptide nocathiacin I biosynthesis: a [4Fe-4S] cluster and the catalysis of a radical S-adenosylmethionine enzyme

The radical S-adenosylmethionine (AdoMet) enzyme superfamily is remarkable at catalyzing chemically diverse and complex reactions. We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. We now report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis. EPR and UV-visible absorbance spectroscopic analyses demonstrated the interaction between L-Trp and the [4Fe-4S] cluster of NocL, leading to the assumption of nonspecific interaction of [4Fe-4S] cluster with other nucleophiles via the unique Fe site. This notion is supported by the finding of the heterogeneity in the [4Fe-4S] cluster of NocL in the absence of AdoMet, which was revealed by the EPR study at very low temperature. Furthermore, a free radical was observed by EPR during the catalysis, which is in good agreement with the hypothesis of a glycyl radical intermediate. Combined with the mutational analysis, these studies provide new insights into the function of the [4Fe-4S] cluster of radical AdoMet enzymes as well as the mechanism of the radical-mediated complex carbon chain rearrangement catalyzed by MIA synthase.     

Atomic resolution structures of oxidized [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus in two crystal forms: systematic distortion of [4Fe-4S] cluster in the protein

Diffraction data of two crystal forms (forms I and II) of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus have been collected to 0.92 A and 1.00 A resolutions, respectively, at 100 K using synchrotron radiation. Anisotropic temperature factors were introduced for all non-hydrogen atoms in the refinement with SHELX-97, in which stereochemical restraints were applied to the protein chain but not to the [4Fe-4S] cluster. The final crystallographic R-factors are 9.8 % for 7.0-0.92 A resolution data of the form I and 11.2 % for the 13.3-1.0 A resolution data of the form II. Many hydrogen atoms as well as multiple conformations for several side-chains have been identified. The present refinement has revised the conformations of several peptide bonds and side-chains assigned previously at 2.3 A resolution; the largest correction was that the main-chain of Pro1 and the side-chain of Lys2 were changed by rotating the C(alpha)-C bond of Lys2. Although the overall structures in the two crystal forms are very similar, conformational differences are observed in the two residues at the middle (Glu29 and Asp30) and the C-terminal residues, which have large temperature factors. The [4Fe-4S] cluster is a distorted cube with non-planar rhombic faces. Slight but significant compression of the four Fe-S bonds along one direction is observed in both crystal forms, and results in the D(2d) symmetry of the cluster. The compressed direction of the cluster relative to the protein is conserved in the two crystal forms and consistent with that in one of the clusters in Clostridium acidurici ferredoxin.     

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.     

Identification of variant molecules of Bacillus thermoproteolyticus ferredoxin: crystal structure reveals bound coenzyme A and an unexpected [3Fe-4S] cluster associated with a canonical [4Fe-4S] ligand motif

During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.     

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.     

IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

Biosynthesis of isoprenoids: crystal structure of the [4Fe-4S] cluster protein IspG

IspG protein serves as the penultimate enzyme of the recently discovered non-mevalonate pathway for the biosynthesis of the universal isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The enzyme catalyzes the reductive ring opening of 2C-methyl-d-erythritol 2,4-cyclodiphosphate, which affords 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. The protein was crystallized under anaerobic conditions, and its three-dimensional structure was determined to a resolution of 2.7 Å. Each subunit of the c₂ symmetric homodimer folds into two domains connected by a short linker sequence. The N-terminal domain (N domain) is an eight-stranded β barrel that belongs to the large TIM-barrel superfamily. The C-terminal domain (C domain) consists of a β sheet that is flanked on both sides by helices. One glutamate and three cysteine residues of the C domain coordinate a [4Fe-4S] cluster. Homodimer formation involves an extended contact area (about 1100 Å²) between helices 8 and 9 of each respective β barrel. Moreover, each C domain contacts the N domain of the partner subunit, but the interface regions are small (about 430 Å²). We propose that the enzyme substrate binds to the positively charged surface area at the C-terminal pole of the β barrel. The C domain carrying the iron-sulfur cluster could then move over to form a closed conformation where the substrate is sandwiched between the N domain and the C domain. This article completes the set of three-dimensional structures of the non-mevalonate pathway enzymes, which are of specific interest as potential targets for tuberculostatic and antimalarial drugs.     

3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.     

The [4Fe-4S] cluster of quinolinate synthase from Escherichia coli: investigation of cluster ligands

Nicotinamide adenine dinucleotide (NAD) derives from quinolinic acid which is synthesized in Escherichia coli from l-aspartate and dihydroxyacetone phosphate through the concerted action of l-aspartate oxidase and the [4Fe-4S] quinolinate synthase (NadA). Here, we addressed the question of the identity of the cluster ligands. We performed in vivo complementation experiments as well as enzymatic, spectroscopic and structural in vitro studies using wild-type vs. Cys-to-Ala mutated NadA proteins. These studies reveal that only three cysteine residues, the conserved Cys113, Cys200 and Cys297, are ligands of the cluster. This result is in contrast to the previous proposal that pointed the three cysteines of the C(291)XXC(294)XXC(297) motif. Interestingly, we demonstrated that Cys291 and Cys294 form a disulfide bridge and are important for activity.     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

Characteristic features of the heterologous functional synthesis in Escherichia coli of a 2[4Fe-4S] ferredoxin

Different strategies have been used to express synthetic genes all encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin (Fd) in Escherichia coli. The polypeptide can be produced as the C-terminal addition to a hybrid Cro::Protein A fusion protein lacking the metallic centers. The incorporation of the [4Fe-4S] clusters into the cleaved apoFd cannot be carried out in the same conditions as those affording holoFd from purified C. pasteurianum apoFd. In contrast, fully functional Fds can be produced from non-fused synthetic genes under the dependence of strong promoters. The yields of recombinant Fd, although sufficient to purify significant quantities of protein, are limited by the very short half-life of the 2[4Fe-4S] Fd in E. coli, irrespective of the expression system used. These features are characteristic of 2[4Fe-4S] Fds when compared with the far more stable recombinant rubredoxin, and probably other small iron-sulfur proteins which have already been produced in high yields. The reasons for the high turnover of 2[4Fe-4S] Fds are discussed.     

Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.     

Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5'-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5'-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 +/- 0.46 eq of iron and 0.670 +/- 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g axially = 2.03 and g radially = 1.93. M?ssbauer spectra of the (57)Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S](2+) clusters, as evidenced by the typical M?ssbauer parameters-isomer shift, delta = 0.47 mm/s, quadrupole splitting of Delta E(Q) = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.     

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]²⁺ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]²⁺ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]²⁺ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]²⁺ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]²⁺ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.     

Pyruvate formate-lyase-activating enzyme: strictly anaerobic isolation yields active enzyme containing a [3Fe-4S](+) cluster

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-l-methionine (AdoMet). Although PFL-AE is an iron-dependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV-visible, EPR, and resonance Raman data support the presence of a [3Fe-4S](+) cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.     

Modulating the midpoint potential of the [4Fe-4S] cluster of the nitrogenase Fe protein

Protein-bound [FeS] clusters function widely in biological electron-transfer reactions, where their midpoint potentials control both the kinetics and thermodynamics of these reactions. The polarity of the protein environment around [FeS] clusters appears to contribute largely to modulating their midpoint potentials, with local protein dipoles and water dipoles largely defining the polarity. The function of the [4Fe-4S] cluster containing Fe protein in nitrogenase catalysis is, at least in part, to serve as the nucleotide-dependent electron donor to the MoFe protein which contains the sites for substrate binding and reduction. The ability of the Fe protein to function in this manner is dependent on its ability to adopt the appropriate conformation for productive interaction with the MoFe protein and on its ability to change redox potentials to provide the driving force required for electron transfer. Phenylalanine at position 135 is located near the [4Fe-4S] cluster of nitrogenase Fe protein and has been suggested by amino acid substitution studies to participate in defining both the midpoint potential and the nucleotide-induced changes in the [4Fe-4S] cluster. In the present study, the crystal structure of the Azotobacter vinelandii nitrogenase Fe protein variant having phenylalanine at position 135 substituted by tryptophan has been determined by X-ray diffraction methods and refined to 2.4 A resolution. A comparison of available Fe protein structures not only provides a structural basis for the more positive midpoint potential observed in the tryptophan substituted variant but also suggests a possible general mechanism by which the midpoint potential could be controlled by nucleotide interactions and nitrogenase complex formation.     

Reversible inactivation of E. coli endonuclease III via modification of its [4Fe-4S] cluster by nitric oxide

Endonuclease III, a highly conserved enzyme initiating the base excision repair of oxidized DNA bases, hosts a [4Fe-4S] cluster. Unlike many other iron-sulfur clusters, the [4Fe-4S] cluster of endonuclease III is stable and resistant to both oxidation and reduction. Here we show that the [4Fe-4S] cluster of the E. coli endonuclease III can be readily modified by nitric oxide forming the protein-bound dinitrosyl iron complex in vitro and in vivo. Modification of the [4Fe-4S] cluster completely inhibits the DNA glycosylase activity of the endonuclease III. Remarkably, the enzymatic activity is restored when the [4Fe-4S] cluster is re-assembled in the endonuclease III dinitrosyl iron complex with L-cysteine, cysteine desulfurase (IscS) and ferrous iron in vitro. Furthermore, the nitric oxide-modified [4Fe-4S] cluster in endonuclease III is efficiently repaired in aerobically growing E. coli cells, and this repair does not require new protein synthesis. These results suggest that the E. coli endonuclease III can be reversibly inactivated by nitric oxide via modification of its [4Fe-4S] cluster.     

Possible direct involvement of the active-site [4Fe-4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

The GcpE enzyme converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the DOXP pathway for isoprene biosynthesis. Purification of the enzyme under exclusion of air leads to a preparation that contains solely [4Fe-4S] clusters. Kinetic studies showed that in the presence of the artificial reductant dithionite and MEcPP a new transient iron-sulfur-based signal is detected in electron paramagnetic resonance (EPR) spectroscopy. Similarity of this EPR signal to that detected in ferredoxin:thioredoxin reductase indicates that during the reaction an intermediate is directly bound to the active-site cluster.