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Iron–sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status. Fe/S clusters are synthesized by the mitochondrial iron-sulfur cluster assembly (ISC) machinery. Here the authors combine crystallography and small angle X-ray scattering measurements to structurally characterize the core ISC complex and give functional insights into eukaryotic Fe/S cluster synthesis.

Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chemistry; the remainder of the reaction mechanism, especially the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resolution crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-containing 8-mer peptide substrate and 1 eq AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissociates from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron–sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.

Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target.

Significance The isolation of an active formate hydrogenlyase is a breakthrough in understanding the molecular basis of bacterial hydrogen production. For over 100 years, Escherichia coli has been known to evolve H ₂ when cultured under fermentative conditions. Glucose is metabolized to formate, which is then oxidized to CO ₂ with the concomitant reduction of protons to H ₂ by a single complex called formate hydrogenlyase, which had been genetically, but never biochemically, characterized. In this study, innovative molecular biology and electrochemical experiments reveal a hydrogenase enzyme with the unique ability to sustain H ₂ production even under high partial pressures of H ₂. Harnessing bacterial H ₂ production offers the prospect of a source of fully renewable H ₂ energy, freed from any dependence on fossil fuel. Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H ₂ even at high partial pressures of H ₂. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich’s ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich’s ataxia therapies. The mechanism of iron-sulfur (Fe-S) cluster biosynthesis is not fully understood. Here, the authors develop a physiologically relevant in vitro model of Fe-S cluster assembly, allowing them to elucidate the sequence of Fe-S cluster synthesis along with the respective roles of ferredoxin-2 and frataxin.

The NEET family is a relatively new class of three related [2Fe-2S] proteins (CISD1–3), important in human health and disease. While there has been growing interest in the homodimeric gene products of CISD1 (mitoNEET) and CISD2 (NAF-1), the importance of the inner mitochondrial CISD3 protein has only recently been recognized in cancer. The CISD3 gene encodes for a monomeric protein that contains two [2Fe-2S] CDGSH motifs, which we term mitochondrial inner NEET protein (MiNT). It folds with a pseudosymmetrical fold that provides a hydrophobic motif on one side and a relatively hydrophilic surface on the diametrically opposed surface. Interestingly, as shown by molecular dynamics simulation, the protein displays distinct asymmetrical backbone motions, unlike its homodimeric counterparts that face the cytosolic side of the outer mitochondrial membrane/endoplasmic reticulum (ER). However, like its counterparts, our biological studies indicate that knockdown of MiNT leads to increased accumulation of mitochondrial labile iron, as well as increased mitochondrial reactive oxygen production. Taken together, our study suggests that the MiNT protein functions in the same pathway as its homodimeric counterparts (mitoNEET and NAF-1), and could be a key player in this pathway within the mitochondria. As such, it represents a target for anticancer or antidiabetic drug development.

▪ Abstract  Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. Owing to their remarkable structural plasticity and versatile chemical/electronic features [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Three different types of [Fe-S] cluster biosynthetic systems have been discovered, and all of them are mechanistically unified by the requirement for a cysteine desulfurase and the participation of an [Fe-S] cluster scaffolding protein. Important mechanistic questions related to [Fe-S] cluster biosynthesis involve the molecular details of how [Fe-S] clusters are assembled on scaffold proteins, how [Fe-S] clusters are transferred from scaffolds to target proteins, how various accessory proteins participate in [Fe-S] protein maturation, and how the biosynthetic process is regulated.

The notion that animals can detect the Earth’s magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor ( Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields. A polymeric protein complex consisting of a newly identified magnetoreceptor protein and known magnetoreception-related photoreceptor cryptochromes exhibits spontaneous alignment in magnetic fields.

Heme biosynthesis and iron-sulfur cluster (ISC) biogenesis are two major mammalian metabolic pathways that require iron. It has long been known that these two pathways interconnect, but the previously described interactions do not fully explain why heme biosynthesis depends on intact ISC biogenesis. Herein we identify a previously unrecognized connection between these two pathways through our discovery that human aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme biosynthesis, is an Fe-S protein. We find that several highly conserved cysteines and an Ala306-Phe307-Arg308 motif of human ALAD are important for [Fe 4 S 4 ] cluster acquisition and coordination. The enzymatic activity of human ALAD is greatly reduced upon loss of its Fe-S cluster, which results in reduced heme biosynthesis in human cells. As ALAD provides an early Fe-S-dependent checkpoint in the heme biosynthetic pathway, our findings help explain why heme biosynthesis depends on intact ISC biogenesis. Heme biosynthesis depends on iron-sulfur (Fe-S) cluster biogenesis but the molecular connection between these pathways is not fully understood. Here, the authors show that the heme biosynthesis enzyme ALAD contains an Fe-S cluster, disruption of which reduces ALAD activity and heme production in human cells.

Iron-sulfur (FeS) protein biogenesis in eukaryotes begins with the de novo assembly of [2Fe-2S] clusters by the mitochondrial core iron-sulfur cluster assembly (ISC) complex. This complex comprises the scaffold protein ISCU2, the cysteine desulfurase subcomplex NFS1-ISD11-ACP1, the allosteric activator frataxin (FXN) and the electron donor ferredoxin-2 (FDX2). The structural interaction of FDX2 with the complex remains unclear. Here, we present cryo-EM structures of the human FDX2-bound core ISC complex showing that FDX2 and FXN compete for overlapping binding sites. FDX2 binds in either a ‘distal’ conformation, where its helix F interacts electrostatically with an arginine patch of NFS1, or a ‘proximal’ conformation, where this interaction tightens and the FDX2-specific C terminus binds to NFS1, facilitating the movement of the [2Fe-2S] cluster of FDX2 closer to the ISCU2 FeS cluster assembly site for rapid electron transfer. Structure-based mutational studies verify the contact areas of FDX2 within the core ISC complex. De novo [2Fe-2S] cluster synthesis by the mitochondrial ISC complex requires electrons from ferredoxin-2 (FDX2). The authors map FDX2 binding to the core ISC complex, showing that FDX2 can bind in two possible conformations in a manner competitive with frataxin.