Iron–sulfur cluster biosynthesis in photosynthetic organisms

Iron–sulfur (Fe/S) cluster containing proteins are widely distributed in nature and are involved in numerous processes including electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression. The knowledge about the biogenesis of Fe/S clusters, and the assembly and maturation of Fe/S cluster containing proteins is still limited, especially in photosynthetic organisms. In most organisms analyzed so far the biogenesis of Fe/S clusters involves more than one machinery. The additional compartment in photoautotrophic organisms, the plastids, presents an additional challenge for the regulation of Fe/S cluster biogenesis. The requirement for Fe/S proteins in multiple chloroplast processes argues that Fe/S cluster assembly is an essential part of plastid functionality. This review focuses on the interesting and unique aspects of Fe/S cluster biogenesis in photosynthetic organisms and compares them to what is known in other organisms.     

Metabolic regulation of citrate and iron by aconitases: role of iron–sulfur cluster biogenesis

Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate the Fe–S cluster switch and modulate intracellular citrate flux.     

Iron–sulfur protein folds,iron–sulfur chemistry,and evolution

An inventory of unique local protein folds around Fe–S clusters has been derived from the analysis of protein structure databases. Nearly 50 such folds have been identified, and over 90% of them harbor low-potential [2Fe–2S]^2+,+ or [4Fe–4S]^2+,+ clusters. In contrast, high-potential Fe–S clusters, notwithstanding their structural diversity, occur in only three different protein folds. These observations suggest that the extant population of Fe–S protein folds has to a large extent been shaped in the reducing iron- and sulfur-rich environment that is believed to have predominated on this planet until approximately two billion years ago. High-potential active sites are then surmised to be rarer because they emerged later, in a more oxidizing biosphere, in conditions where iron and sulfide had become poorly available, Fe–S clusters were less stable, and in addition faced competition from heme iron and copper active sites. Among the low-potential Fe–S active sites, protein folds hosting [4Fe–4S]^2+,+ clusters outnumber those with [2Fe–2S]^2+,+ ones by a factor of 3 at least. This is in keeping with the higher chemical stability and versatility of the tetranuclear clusters, compared with the binuclear ones. It is therefore suggested that, at least while novel Fe–S sites are evolving within proteins, the intrinsic chemical stability of the inorganic moiety may be more important than the stabilizing effect of the polypeptide chain. The discovery rate of novel Fe–S-containing protein folds underwent a sharp increase around 1995, and has remained stable to this day. The current trend suggests that the mapping of the Fe–S fold space is not near completion, in agreement with predictions made for protein folds in general. Altogether, the data collected and analyzed here suggest that the extant structural landscape of Fe–S proteins has been shaped to a large extent by primeval geochemical conditions on one hand, and iron–sulfur chemistry on the other.     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

Di-iron proteins of the Ric family are involved in iron–sulfur cluster repair

A key element in eukaryotic immune defenses against invading microbes is the production of reactive oxygen and nitrogen species. One of the main targets of these species are the iron–sulfur clusters, which are essential prosthetic groups that confer to proteins the ability to perform crucial roles in biological processes. Microbes have developed sophisticated systems to eliminate nitrosative and oxidative species and promote the repair of the damages inflicted. The Ric (Repair of Iron Centers) proteins constitute a novel family of microbial di-iron proteins with a widespread distribution among microbes, including Gram-positive and Gram-negative bacteria, protozoa and fungi. The Ric proteins are encoded by genes that are up-regulated by nitric oxide and hydrogen peroxide. Recent studies have shown that the active di-iron center is involved in the restoration of Fe–S clusters damaged by exposure to nitric oxide and hydrogen peroxide.     

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

Human Nfu is an iron–sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe–2S](GS)₄ cluster complex, and extraction of the Nfu-bound iron–sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] cluster trafficking and delivery.     

The SufBCD protein complex is the scaffold for iron–sulfur cluster assembly in Thermus thermophiles HB8

Iron–sulfur (Fe–S) clusters are the oldest and most versatile inorganic cofactors that are required to sustain fundamental life processes. Bacteria have three systems of [Fe–S] cluster biogenesis, designated ISC, NIF, and SUF. In contrast, the Thermus thermophiles HB8 has only one system, formed mostly by SUF homologs that contain six proteins: SufA, SufB, SufC, SufD, SufS and SufE. The kinetics of SufC ATPase was studied using a linked enzyme assay method. In the presence of SufB, SufD or SufBD complexes, the activity of SufC was enhanced. The cysteine desulfurase activity of SufS was also stimulated by the presence of the SufBCD complex. The results obtained through enzymology revealed that aconitase activity was activated by [Fe–S] clusters reconstituted on the SufBCD complex. Consolidated results from spectral and enzymatic analysis suggest that the SufBCD complex is a novel type of Fe–S scaffold system that can assemble Fe/S clusters de novo.     

A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe–4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe–4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe–4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe–4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (> 100 mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.     

Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron–sulfur cluster

Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (³⁹³CX₂CX₃CX₁₄C) that was found in only six other proteobacteria (CX₂CX₃CX_11–14C). The cysteine motif is likely to coordinate an unprecedented [Fe–S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe–4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe–4S] cluster centered at g = 2.02. The [3Fe–4S] signal was observed independently of the [4Fe–4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe–4S] and [3Fe–4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe–4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.     

The bound iron–sulfur clusters of Type-I homodimeric reaction centers

The hallmark of a Type-I photosynthetic reaction center (RC) is the presence of three [4Fe–4S]^2+/1+ clusters, named F_X, F_A, and F_B that act as terminal electron acceptors. Their function is to increase the distance, and hence the lifetime, of the initial charge-separated state so that diffusion-mediated processes, such as the reduction of ferredoxin, can occur. Type-I homodimeric RCs, such as those found in heliobacteria, green-sulfur bacteria, and Candidatus Chloracidobacterium thermophilum, are less well understood than Photosystem I, the prototypical Type-I heterodimeric RC found in cyanobacteria and plants. Here, we review recent progress that has been made in elucidating the spectroscopic and biochemical properties of the bound Fe/S clusters and their cognate proteins in homodimeric Type-I RCs. In Heliobacterium modesticaldum, the identification and characterization of two loosely bound polypeptides, PshBI and PshBII that harbor the F_A and F_B clusters threatens to break the long-accepted assumption that Type-I RCs harbor one tightly bound F_A/F_B-containing protein. Additionally, the detection of the F_X cluster in S = 1/2 and S = 3/2 ground spin states has resolved the long-standing issue of its missing EPR spectrum. In Chlorobaculum tepidum, the focus is on the biochemical properties of the unusual extrinsic Fe/S protein, PscB, which is readily dissociable from the RC core. The C-terminal domain of PscB is constructed as a bacterial ferredoxin, harboring the F_A and F_B clusters, but the N-terminal domain contains a number of PxxP motifs and is rich in Lys, Pro, and Ala residues, features characteristic of proteins that interact with SH3 domains. Little is known about Candidatus Chloracidobacterium thermophilum except that the photosynthetic RC is predicted to be a Type-I homodimer with an F_X-binding site. These findings are placed in a context that promises to unify the acceptor side of homodimeric Type-I RCs in prokaryotic phototrophs.     

The F<Subscript>X</Subscript> iron–sulfur cluster serves as the terminal bound electron acceptor in heliobacterial reaction centers

Phototrophs of the family Heliobacteriaceae contain the simplest known Type I reaction center (RC), consisting of a homodimeric (PshA)₂ core devoid of bound cytochromes and antenna proteins. Unlike plant and cyanobacterial Photosystem I in which the F_A/F_B protein, PsaC, is tightly bound to P₇₀₀–F_X cores, the RCs of Heliobacterium modesticaldum contain two F_A/F_B proteins, PshBI and PshBII, which are loosely bound to P₈₀₀–F_X cores. These two 2[4Fe–4S] ferredoxins have been proposed to function as mobile redox proteins, reducing downstream metabolic partners much in the same manner as does [2Fe–2S] ferredoxin or flavodoxin (Fld) in PS I. Using P₈₀₀–F_X cores devoid of PshBI and PshBII, we show that iron–sulfur cluster F_X directly reduces Fld without the involvement of F_A or F_B (Fld is used as a proxy for soluble redox proteins even though a gene encoding Fld is not identified in the H. modesticaldum genome). The reduction of Fld is suppressed by the addition of PshBI or PshBII, an effect explained by competition for the electron on F_X. In contrast, P₇₀₀–F_X cores require the presence of the PsaC, and hence, the F_A/F_B clusters for Fld (or ferredoxin) reduction. Thus, in H. modesticaldum, the interpolypeptide F_X cluster serves as the terminal bound electron acceptor. This finding implies that the homodimeric (PshA)₂ cores should be capable of donating electrons to a wide variety of yet-to-be characterized soluble redox partners.     

Insertion mutants in Drosophila melanogaster Hsc20 halt larval growth and lead to reduced iron–sulfur cluster enzyme activities and impaired iron homeostasis

Despite the prominence of iron–sulfur cluster (ISC) proteins in bioenergetics, intermediary metabolism, and redox regulation of cellular, mitochondrial, and nuclear processes, these proteins have been given scarce attention in Drosophila. Moreover, biosynthesis and delivery of ISCs to target proteins requires a highly regulated molecular network that spans different cellular compartments. The only Drosophila ISC biosynthetic protein studied to date is frataxin, in attempts to model Friedreich’s ataxia, a disease arising from reduced expression of the human frataxin homologue. One of several proteins involved in ISC biogenesis is heat shock protein cognate 20 (Hsc20). Here we characterize two piggyBac insertion mutants in Drosophila Hsc20 that display larval growth arrest and deficiencies in aconitase and succinate dehydrogenase activities, but not in isocitrate dehydrogenase activity; phenotypes also observed with ubiquitous frataxin RNA interference. Furthermore, a disruption of iron homeostasis in the mutant flies was evidenced by an apparent reduction in induction of intestinal ferritin with ferric iron accumulating in a subcellular pattern reminiscent of mitochondria. These phenotypes were specific to intestinal cell types that regulate ferritin expression, but were notably absent in the iron cells where ferritin is constitutively expressed and apparently translated independently of iron regulatory protein 1A. Hsc20 mutant flies represent an independent tool to disrupt ISC biogenesis in vivo without using the RNA interference machinery.     

Reactive oxygen species regulates expression of iron–sulfur cluster assembly protein IscS of Leishmania donovani

The cysteine desulfurase, IscS, is a highly conserved and essential component of the mitochondrial iron–sulfur cluster (ISC) system that serves as a sulfur donor for Fe–S clusters biogenesis. Fe–S clusters are versatile and labile cofactors of proteins that orchestrate a wide array of essential metabolic processes, such as energy generation and ribosome biogenesis. However, no information regarding the role of IscS or its regulation is available in Leishmania, an evolving pathogen model with rapidly developing drug resistance. In this study, we characterized LdIscS to investigate the ISC system in AmpB-sensitive vs resistant isolates of L. donovani and to understand its regulation. We observed an upregulated Fe–S protein activity in AmpB-resistant isolates but, in contrast to our expectations, LdIscS expression was upregulated in the sensitive strain. However, further investigations showed that LdIscS expression is positively correlated with ROS level and negatively correlated with Fe–S protein activity, independent of strain sensitivity. Thus, our results suggested that LdIscS expression is regulated by ROS level with Fe–S clusters/proteins acting as ROS sensors. Moreover, the direct evidence of a mechanism, in support of our results, is provided by dose-dependent induction of LdIscS-GFP as well as endogenous LdIscS in L. donovani promastigotes by three different ROS inducers: H₂O₂, menadione, and Amphotericin B. We postulate that LdIscS is upregulated for de novo synthesis or repair of ROS damaged Fe–S clusters. Our results reveal a novel mechanism for regulation of IscS expression that may help parasite survival under oxidative stress conditions encountered during infection of macrophages and suggest a cross talk between two seemingly unrelated metabolic pathways, the ISC system and redox metabolism in L. donovani.     

Interplay between formation of photosynthetic complexes and expression of genes for iron–sulfur cluster assembly in Rhodobacter sphaeroides?

Photosynthesis Research - Formation of photosynthetic complexes leads to a higher demand for Fe–S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium...     

Iron–sulfur cluster scaffold (ISCU) gene is duplicated in salmonid fish and tissue and temperature dependent expressed in rainbow trout

The iron–sulfur cluster protein ISCU is a scaffold protein tasked with the building and mediation of iron–sulfur [Fe–S]-clusters. These are crucial for [Fe–S]-enzymes, which are involved in essential biological cell processes like metabolism or ion transport. Analysis of ISCU in rainbow trout (Oncorhynchus mykiss) and maraena whitefish (Coregonus maraena) revealed the existence of two gene variants in each of the two salmonids. This study presents the characterization of the duplicated ISCU cDNA sequences in both species as well as the comparative functional analysis of the genes in healthy and affected fish of two rainbow trout strains differing in trait robustness under regional aquaculture conditions. Coding sequences of trout ISCUA and ISCUB genes are spanning over five exons. Open reading frames (ORF) of trout (ISCUA: 495 bp, ISCUB: 498 bp) and whitefish (ISCUA and ISCUB: 495 bp) genes encode for evolutionary highly conserved proteins and share 72% sequence similarity with human ISCU.     

The chloroplast NifS-like protein of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> is required for iron–sulfur cluster formation in ferredoxin

Plastids are known to be able to synthesize their own iron–sulfur clusters, but the biochemical machinery responsible for this process is not known. In this study it is investigated whether CpNifS, the chloroplastic NifS-like cysteine desulfurase of Arabidopsis thaliana (L.) Heynh. is responsible for the release of sulfur from cysteine for the biogenesis of iron–sulfur (Fe–S) clusters in chloroplasts. Using an in vitro reconstitution assay it was found that purified CpNifS was sufficient for Fe–S cluster formation in ferredoxin in the presence of cysteine and a ferrous iron salt. Antibody-depletion experiments using stromal extract showed that CpNifS is also essential for the Fe–S cluster formation activity of chloroplast stroma. The activity of CpNifS in the stroma was 50- to 80-fold higher than that of purified CpNifS on a per-protein basis, indicating that other stromal factors cooperate in Fe–S cluster formation. When stromal extract was separated on a gel-filtration column, most of the CpNifS eluted as a dimer of 86 kDa, but a minor fraction of the stromal CpNifS eluted at a molecular weight of approx. 600 kDa, suggesting the presence of a multi-protein complex. The possible nature of the interacting proteins is discussed.     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.