Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin,a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.     

Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron−sulfur clusters of human mitoNEET

The outer mitochondrial membrane protein mitoNEET is a cellular target of the antidiabetic drug pioglitazone. Binding of pioglitazone stabilizes the protein against [2Fe-2S] cluster release. Here, we report that reduced nicotinamide adenine dinucleotide phosphate (NADPH) can bind to homodimeric mitoNEET, influencing the stability of the [2Fe-2S] cluster that is bound within a loop region (Y71?H87) in each subunit. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry experiments demonstrated that NADPH binds weakly to mitoNEET(44?108), a soluble domain of mitoNEET containing residues 44?108. Visible?UV absorption measurements revealed the destabilizing effect of NADP binding on the [2Fe-2S] clusters. Disruption of the three-dimensional structure of mitoNEET(44?108) as a result of decomposition of the iron?sulfur clusters was observed by NMR and circular dichroism experiments. Binding of NADPH facilitated release of the iron?sulfur clusters from the protein at pH≤7.0. Residues K55 and H58 of each subunit of mitoNEET were shown to be involved in NADPH binding. NADPH binding may perturb the interactions of K55 and H58 from one subunit with H87′ and R73′, respectively, from the other subunit, thereby interfering with [2Fe-2S] cluster binding. This may account for the destabilization effect of NADPH binding on the [2Fe-2S] clusters.     

ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.     

In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.     

Formation and properties of [4Fe-4S] clusters on the IscU scaffold protein

Rapid and quantitative reductive coupling of two [2Fe-2S]2+ clusters to form a single [4Fe-4S]2+ cluster on the homodimeric IscU Fe-S cluster scaffold protein has been demonstrated by UV-visible absorption, M?ssbauer, and resonance Raman spectroscopies, using dithionite as the electron donor. Partial reductive coupling was also observed using reduced Isc ferredoxin, which raises the possibility that Isc ferredoxin is the physiological reductant. The results suggest that reductive coupling of adjacent [2Fe-2S]2+ clusters assembled on IscU provides a general mechanism for the final step in the biosynthesis of [4Fe-4S]2+ clusters. The [4Fe-4S]2+ center on IscU can be reduced to a S = 1/2[4Fe-4S]+ cluster (g parallel = 2.06 and g perpendicular = 1.92), but the low midpoint potential (< -570 mV) and instability of the reduced cluster argue against any physiological relevance for the reduced cluster. On exposure to O2, the [4Fe-4S]2+ cluster on IscU degrades via a semistable [2Fe-2S]2+ cluster with properties analogous to those of the [2Fe-2S]2+ center in [2Fe-2S]2+ IscU. It is suggested that the ability of IscU to accommodate either [2Fe-2S]2+ or [4Fe-4S]2+ clusters in response to cellular redox status and/or oxygen levels may provide an effective way to populate appropriately cluster-loaded forms of IscU for maturation of different types of [Fe-S] proteins.     

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]²⁺ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]²⁺ clusters, two [2Fe-2S]²⁺ clusters are converted into a [4Fe-4S]²⁺ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]²⁺ cluster from ISCA1-ISCA2 complex. A structural view of the protein–protein recognition events occurring along the [4Fe-4S]²⁺ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]²⁺ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]²⁺ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]²⁺ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]²⁺ cluster transfer.     

Subunit D of RNA polymerase from Methanosarcina acetivorans contains two oxygen-labile [4Fe-4S] clusters: implications for oxidant-dependent regulation of transcription

Subunit D of multisubunit RNA polymerase from many species of archaea is predicted to bind one to two iron-sulfur (Fe-S) clusters, the function of which is unknown. A survey of encoded subunit D in the genomes of sequenced archaea revealed six distinct groups based on the number of complete or partial [4Fe-4S] cluster motifs within domain 3. Only subunit D from strictly anaerobic archaea, including all members of the Methanosarcinales, are predicted to bind two [4Fe-4S] clusters. We report herein the purification and characterization of Methanosarcina acetivorans subunit D in complex with subunit L. Expression of subunit D and subunit L in Escherichia coli resulted in the purification of a D-L heterodimer with only partial [4Fe-4S] cluster content. Reconstitution in vitro with iron and sulfide revealed that the M. acetivorans D-L heterodimer is capable of binding two redox-active [4Fe-4S] clusters. M. acetivorans subunit D deleted of domain 3 (DΔD3) was still capable of co-purifying with subunit L but was devoid of [4Fe-4S] clusters. Affinity purification of subunit D or subunit DΔD3 from M. acetivorans resulted in the co-purification of endogenous subunit L with each tagged subunit D. Overall, these results suggest that domain 3 of subunit D is required for [4Fe-4S] cluster binding, but the [4Fe-4S] clusters and domain 3 are not required for the formation of the D-L heterodimer. However, exposure of two [4Fe-4S] cluster-containing D-L heterodimer to oxygen resulted in loss of the [4Fe-4S] clusters and subsequent protein aggregation, indicating that the [4Fe-4S] clusters influence the stability of the D-L heterodimer and therefore have the potential to regulate the assembly and/or activity of RNA polymerase in an oxidant-dependent manner.     

Repair of nitric oxide-modified ferredoxin [2Fe-2S] cluster by cysteine desulfurase (IscS)

Iron-sulfur proteins are among the sensitive targets of the nitric oxide cytotoxicity. When Escherichia coli cells are exposed to nitric oxide, iron-sulfur clusters are modified forming protein-bound dinitrosyl iron complexes. Such modified protein dinitrosyl iron complexes are stable in vitro but are efficiently repaired in aerobically growing E. coli cells even without any new protein synthesis. Here we show that cysteine desulfurase encoded by the gene iscS of E. coli can directly convert the ferredoxin dinitrosyl iron complex to the ferredoxin [2Fe-2S] cluster in the presence of L-cysteine in vitro. A reassembly of the [2Fe-2S] cluster in the ferredoxin dinitrosyl iron complex does not require any addition of iron or other protein components. Furthermore, a complete removal of the dinitrosyl iron complex from ferredoxin prevents reassembly of the [2Fe-2S] cluster in the protein. The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin [2Fe-2S] cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins.     

The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe-4S] protein

BoxA is the reductase component of the benzoyl-coenzyme A (CoA) oxidizing epoxidase enzyme system BoxAB. The enzyme catalyzes the key step of an hitherto unknown aerobic, CoA-dependent pathway of benzoate metabolism, which is the epoxidation of benzoyl-CoA to the non-aromatic 2,3-epoxybenzoyl-CoA. The function of BoxA is the transfer of two electrons from NADPH to the epoxidase component BoxB. We could show recently that BoxB is a diiron enzyme, whereas here we demonstrate that BoxA harbors an FAD and two [4Fe-4S] clusters per protein monomer. The characterization of BoxA was hampered by severe oxygen sensitivity; the cubane [4Fe-4S] clusters degrade already with traces of oxygen. Interestingly, the adventitiously formed [3Fe-4S] centers could be reconstituted in vitro by adding Fe(II) and sulfide to retrieve the native cubane centers. BoxA is the first example of a reductase of this type that has an FAD and two bacterial ferredoxin-type [4Fe-4S] clusters. In other cases within the catalytically versatile family of diiron enzymes, the related reductases have plant-type ferredoxin or Rieske-type [2Fe-2S] centers only.     

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.     

Bacterial ApbC can bind and effectively transfer iron-sulfur clusters

The metabolism of iron-sulfur ([Fe-S]) clusters requires a complex set of machinery that is still being defined. Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. ApbC is a 40.8 kDa homodimeric ATPase and as purified contains little iron and no acid-labile sulfide. An [Fe-S] cluster was reconstituted on ApbC, generating a protein that bound 2 mol of Fe and 2 mol of S (2-) per ApbC monomer and had a UV-visible absorption spectrum similar to known [4Fe-4S] cluster proteins. Holo-ApbC could rapidly and effectively activate Saccharomyces cerevisiae apo-isopropylmalate isolomerase (Leu1) in vitro, a process known to require the transfer of a [4Fe-4S] cluster. Maximum activation was achieved with 2 mol of ApbC per 1 mol of apo-Leu1. This article describes the first biochemical activity of ApbC in the context of [Fe-S] cluster metabolism. The data herein support a model in which ApbC coordinates an [4Fe-4S] cluster across its dimer interface and can transfer this cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function recently deduced for its eukaryotic homologues.     

FdC1, a novel ferredoxin protein capable of alternative electron partitioning, increases in conditions of acceptor limitation at photosystem I

In higher plants, [2Fe-2S] ferredoxin (Fd) proteins are the unique electron acceptors from photosystem I (PSI). Fds are soluble, and distribute electrons to many enzymes, including Fd:NADP(H) reductase (FNR), for the photoreduction of NADP(+). In addition to well studied [2Fe-2S] Fd proteins, higher plants also possess genes for significantly different, as yet uncharacterized Fd proteins, with extended C termini (FdCs). Whether these FdC proteins function as photosynthetic electron transfer proteins is not known. We examined whether these proteins play a role as alternative electron acceptors at PSI, using quantitative RT-PCR to follow how their expression changes in response to acceptor limitation at PSI, in mutant Arabidopsis plants lacking 90-95% of photosynthetic [2Fe-2S] Fd. Expression of the gene encoding one FdC protein, FdC1, was identified as being strongly up-regulated. We confirmed that this protein was chloroplast localized and increased in abundance on PSI acceptor limitation. We purified the recombinant FdC1 protein, which exhibited a UV-visible spectrum consistent with a [2Fe-2S] cluster, confirmed by EPR analysis. Measurements of electron transfer show that FdC1 is capable of accepting electrons from PSI, but cannot support photoreduction of NADP(+). Whereas FdC1 was capable of electron transfer with FNR, redox potentiometry showed that it had a more positive redox potential than photosynthetic Fds by around 220 mV. These results indicate that FdC1 electron donation to FNR is prevented because it is thermodynamically unfavorable. Based on our data, we speculate that FdC1 has a specific function in conditions of acceptor limitation at PSI, and channels electrons away from NADP(+) photoreduction.     

X-ray absorption spectroscopic analysis of reductive [2Fe-2S] cluster degradation in hyperthermophilic archaeal succinate:caldariellaquinone oxidoreductase subunits

The biological [2Fe-2S] clusters play important roles in electron transfer and cellular signaling for a variety of organisms from archaea, bacteria to eukarya. The two recombinant hyperthermophilic archaeal [2Fe-2S] cluster-binding proteins, SdhC and the N-terminal domain fragment of SdhB, of Sulfolobus tokodaii respiratory complex II overproduced in Escherichia coli are thermostable as isolated, but moderately sensitive to reduction with excess dithionite. We used iron K-edge X-ray absorption spectroscopy to monitor the structural changes of their Fe sites in the irreversible [2Fe-2S] cluster degradation process. Regardless of the differences in the cluster-ligating cysteine motifs and the XAS-detectable [2Fe-2S](2+) cluster environments, a complete reductive breakdown of the [2Fe-2S] clusters resulted in the appearance of a new Fourier transform (FT) peak at approximately 3.3 A with a concomitant loss of the Fe-Fe interaction at ca. 2.7 A for both proteins. On the basis of the unambiguous assignment of the 3.3 A FT peak, our results suggest that a biological [2Fe-2S] cluster breakdown under reducing conditions generally releases Fe(2+) from the polypeptide chain into the aqueous solution, and the Fe(2+) might then be recruited as a secondary ferrous iron source for de novo biosynthesis and/or regulation of iron-binding enzymes in the cellular system.     

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apoferredoxin in an ATP-dependent reaction

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.     

DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

Antioxidant activity of sugar-lysine Maillard reaction products in cell free and cell culture systems

The presence of a linear [3Fe-4S] cluster in a protein was first observed in beef-heart aconitase. Here, we report the formation of linear [3Fe-4S] clusters upon guanidine hydrochloride (GuHCl)-induced unfolding of Aquifex aeolicus [2Fe-2S] ferredoxins (Fd) (AaeFd1, AaeFd4, and AaeFd5) at alkaline conditions (pH 10, 20 degrees C). We find the mechanism of linear [3Fe-4S] cluster formation to depend critically on the speed of polypeptide unfolding. In similarity to seven-iron Fds, polypeptide unfolding determines the rate by which linear [3Fe-4S] clusters form in AaeFd4 and AaeFd5. In contrast, in a disulfide-lacking variant of AaeFd1, which unfolds faster than AaeFd4 and AaeFd5, the polypeptides unfold first and the majority of clusters decompose. Next, unfolded polypeptides retaining intact clusters scavenge iron and sulfur to form linear [3Fe-4S] clusters in a bimolecular reaction. Wild-type AaeFd1 unfolds slower than the speed of linear-cluster decomposition, and the linear species is never populated. Linear [3Fe-4S] clusters may be intermediates during folding of iron-sulfur proteins.     

Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, M?ssbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.     

Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-2S] clusters

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.