Synthesis, Delivery and Regulation of Eukaryotic Heme and Fees Cluster Cofactors

Archives of Biochemistry and Biophysics 592 (2016) 60e75

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Review article Synthesis, delivery and regulation of eukaryotic heme and FeeS cluster cofactors

Dulmini P. Barupala a, 1, Stephen P. Dzul a, 1, Pamela Jo Riggs-Gelasco b, * Timothy L. Stemmler a, a Departments of Biochemistry and Molecular Biology, and Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201, USA b Department of Chemistry and Biochemistry, College of Charleston, Charleston, SC 29424, USA article info abstract

Article history: In humans, the bulk of iron in the body (over 75%) is directed towards heme- or FeeS cluster cofactor Received 2 September 2015 synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved Received in revised form to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is 13 January 2016 both initiated and ﬁnalized within the mitochondria, while cellular FeeS cluster assembly is controlled Accepted 14 January 2016 by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a Available online 16 January 2016 wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and FeeS cluster Keywords: Iron biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding Heme human disorders related to the breakdown in these essential pathways. FeeS clusters © 2016 Elsevier Inc. All rights reserved. Fe homeostasis Fe-cofactor biosynthesis

Contents

1. The role of iron in biology ...... 61 2. Heme cofactors ...... 61 2.1. Introduction ...... 61 2.2. Heme structure and types of heme in nature ...... 61 2.3. Heme biosynthesis pathway ...... 61 2.3.1. Important steps in heme biosynthesis ...... 62 2.3.2. Regulation of heme biosynthesis ...... 63 2.4. Incorporation of heme into apoprotein recipients ...... 64 2.5. Heme function ...... 65 2.6. Diseases of heme synthesis ...... 67 2.6.1. Porphyrias ...... 67 2.6.2. Additional diseases ...... 67 3. FeeSclusters...... 68 3.1. Introduction ...... 68 3.2. FeeS cluster structure ...... 68 3.3. General FeeS cluster biogenesis pathways ...... 68 3.3.1. Iron sulfur cluster (ISC) pathway ...... 68 3.3.2. Cytosolic ironesulfur assembly (CIA) pathway ...... 69 3.3.3. Sulfur assimilation (SUF) pathway ...... 69

* Corresponding author. Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201. E-mail address: [email protected] (T.L. Stemmler). 1 Authors contributed equally to the writing of this manuscript. http://dx.doi.org/10.1016/j.abb.2016.01.010 0003-9861/© 2016 Elsevier Inc. All rights reserved. D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 61

3.4. FeeS cluster biogenesis regulation ...... 71 3.5. FeeS cluster function ...... 71 3.6. FeeS clusters in human disease ...... 72 3.6.1. Friedreich's ataxia ...... 72 3.6.2. ISCU myopathy ...... 72 3.6.3. GLRX5 sideroblastic anemia ...... 72 3.6.4. Additional diseases ...... 72 4. Summary ...... 72 Acknowledgments ...... 73 References...... 73

1. The role of iron in biology 2. Heme cofactors

Iron's abundance and unique chemical characteristics are often 2.1. Introduction exploited by nature to drive the complex chemistry required by cells to maintain life. Iron is the fourth most abundant element on Among the many metalloporphyrins found in nature, heme is the earth's crust [1], so its high prevalence during early evolution is one of the most abundant. It is utilized in many vital biological certainly a factor for its current ubiquitous presence in nature. The processes including photosynthesis, oxygen transport, biological human body contains 3e4 g of the metal and absorbs 1e2mgofit oxidation and reduction, and many more. Most of the total iron each day [2]. The reactivity and ability of the metal to cycle between content in human body is incorporated into heme-containing the Fe(II), (III) and (IV) oxidation states makes it extremely useful proteins such as hemoglobin, myoglobin, catalases, peroxidases, for driving intricate reactions in biology that include substrate nitric oxide synthases, and cytochromes [9]. The unique structure activation, electron transfer, and oxidationereduction reactions. of heme, which consists of the iron cation coordinated to four ni- Because of this chemical versatility, iron plays a role in nearly every trogen atoms from a tetrapyrrole ring, allows for fine tuning of the biological pathway. While its utility within biology is apparent, the metal's reactivity to carry out the function of the biomolecule to Achilles heel of this essential metal is its tendency to precipitate in which it is attached. Here we discuss the different heme types, their aqueous solutions [3]. Through coordination to biomolecules, biosynthesis and regulation, their function in biology, and finally however, the solubility of the metal can be controlled and its the diseases linked to a loss of heme function. reactivity attenuated. At the elemental level, first coordination sphere ligands stabilize the solubility of the metal while at the 2.2. Heme structure and types of heme in nature same time tune its chemical properties for selective participation in only desired reactions [4]. Ligand variability in this first coordina- Modifications to the basic heme molecule allow for the diverse tion sphere helps control how the metal behaves, however at the array of heme functions found in nature. The parent heme mole- cellular level a complex network of proteins, controlled at the ge- cule, also known as heme b, protoheme IX or protoporphyrin IX, netic level, helps ensure metal availability to specific protein part- serves as the platform in each case. The tetrapyrrole unit of heme ners in the cell [5]. consists of four pyrrole units linked by four methine bridges. A Pathways related to eukaryotic iron homeostasis are shown in nitrogen atom from each pyrrole coordinates the iron atom in the Fig. 1 [6]. Iron is brought into cells either via the transferrin Fe center of the planar tetrapyrrole ring. Distortions from planarity uptake pathway or through direct membrane transporter (ex. the can be critical to heme function and reactivity (examples include DMT1 pathway) [7]. Once in the cell, imported iron can bind to hemoglobin [10] and myeloperoxidase, MPO [11]). Fifth and sixth iron regulatory proteins where it serves to control concentration, ligands to the Fe can be provided by amino acid side chains from the it can be stored within the ferritin complex, it can be exported, or apoprotein or by small inorganic molecules coordinating above and it can be incorporated as cofactors into apoprotein partners. In below the plane of the ring. Eight positions in the tetrapyrrole carry humans, the bulk of iron in the body (over 75%) is directed to- side chain modifications, particularly methyl groups on carbons 2, wards heme- [2] or FeeS cluster cofactor [8] biosynthesis. As- 7, 12, and 18, vinyl groups on carbons 3 and 8, and propionyl groups sembly of the Fe-cofactors follows highly regulated pathways that on carbons 13 and 17 (Fig. 2). Substitutions to the same carbons have evolved to safely build and load these inorganic Fe- with various other side chains create several biologically important containing cofactors into the apoprotein partners while control- heme types found in nature (Fig. 3). Heme A, B, and C are found in a ling metal reactivity. In eukaryotes, heme biosynthesis is initiated wide spectrum of organisms and take part in vital biochemical and completed within the mitochondria, while FeeS cluster as- processes such as respiration, photosynthesis and oxygen trans- sembly is controlled by separate but correlated pathways within port. The additional cofactor types (Heme D, D1, I, M, and O) are both the mitochondria and the cytosol. Iron plays a central role in species-specific and carry out highly specialized functions. Table 1 a wide array of metabolic processes, so it is not surprising that summarizes the structural features, occurrences, and known defects in cofactor assembly lead to human diseases. This review functions of each heme type. details progress towards elucidating the molecular details of cellular heme and FeeS cluster biosynthesis. A focus on both 2.3. Heme biosynthesis pathway regulation and mechanism will be critical for understanding human disorders related to the breakdown in these essential Since heme serves as an essential cofactor to several proteins pathways. involved in central metabolic processes, all organisms have 62 D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75

Fig. 1. Major cellular Fe utilization pathways in humans. Under physiological conditions, most Fe is internalized as the Tf-bound form, which undergoes receptor-mediated endocytosis via binding to TfR1. Decrease in endosomal pH releases Fe(III) from Tf, which is then reduced by the endosomal reductase STEAP3 to Fe(II). Fe(II) is then trans- ported to the cytosol via DMT1 to join the LIP. Fe in LIP can be utilized for storage in ferritin, import to mitochondria for storage and heme and FeeS cluster synthesis, export to the cytosol via ferroportin, cellular Fe regulation via IRPs and incorporation into other Fe proteins in the cytosol. Under non-physiological conditions Fe(III) can enter the cell after being reduced to Fe(II) and imported by DMT1. Tf: Transferrin, TfR1: Transferrin receptor 1, DCYTB: duodenal cytochrome b, DMT1: divalent metal transporter 1, STEAP3: six trans- membrane epithelial antigen of the phosphate 3, LIP: labile iron pool, PCBP: poly (rC)-binding proteins, OMM: outer mitochondrial membrane, IMM: inner mitochondrial membrane, MFRN: Mitoferrins, FTMT: mitochondrial specific ferritin, ABCB7: transporter for unknown source of sulfur XeS from mitochondria to cytoplasm, IRP: iron regulatory proteins, IRE: iron-responsive elements of mRNA, UTR: untranslated region. established a conserved biosynthetic pathway to synthesize the glycine and succinyl-CoA [13e16]. ALAS is found in animals, fungi, cofactor. Atomic detail is available for many of the enzymes non-photosynthetic eukaryotes, and a-proteobacteria (organisms involved in bioassembly of heme, and these enzyme structures that resemble bacterial ancestors of mitochondria). Mammals have have provided key insights into reaction mechanisms. The general two ALAS isoforms; the first isoform (ALAS1) provides house- assembly process consists of four stages: the synthesis of a single keeping functions and a second erythroid specific isoform (ALAS2) pyrrole, the assembly of four pyrroles to make the tetrapyrrole ring, provides for the more robust heme requirements in erythrocytes modification of the side chains, and the insertion of iron into the [17]. Both forms of ALAS are homodimers and require pyridoxal 5’- ring (Fig. 4) [12]. Specific details for this conserved pathway are phosphate (PLP) bound as a Schiff base covalent adduct to a cata- outlined below. lytic lysine. During the joining of glycine and succinyl CoA on the PLP scaffold, both CO2 and Coenzyme A are released as byproducts 2.3.1. Important steps in heme biosynthesis [18]. Upon formation, ALA exits the mitochondria to serve as the The first step in eukaryotic heme biosynthesis is the mito- substrate for the subsequent four-enzymatic conversions that occur fi chondrial formation of 5-aminolevulinic acid (ALA), which is the in the cytosol. Little is known about the speci cs of export, but it precursor that serves as the only source of carbon and nitrogen has been suggested that SLC25A38, a member of the SLC25 family atoms required to build the basic heme unit. Depending on species, of inner mitochondrial membrane transporters, facilitates import of one of two major pathways directs formation of this precursor glycine into the mitochondria in exchange for ALA transfer across leading to production of tetrapyrroles. In the Shemin pathway, the mitochondrial inner membrane [19]. enzyme ALA synthase (ALAS) that resides on the matrix side of the In the second major pathway for ALA synthesis, also known as inner mitochondrial membrane catalyzes the condensation of the C5 pathway, glutamyl-tRNA reductase (GluTR) converts D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 63

Three enzymes associated with the mitochondrial inner membrane complete the terminal steps in the eukaryotic heme biosynthesis. Their arrangement suggests they act as a multiprotein complex that facilitates substrate channeling [35]. Coproporphyri- nogen III entry into the mitochondria is likely mediated by the peripheral-type benzodiazepine receptor (PBR), located on the outer mitochondrial membrane [36]. In the first of the three terminal steps, coproporphyrinogen III undergoes oxidative decarboxylation of its propionyl side chains on two pyrrole rings to form protoporphyrinogen IX. This reaction is completed in higher eukaryotes and in a few bacterial species by the oxygen-dependent coproporphyrinogen III oxidase (CPO), an enzyme located in mitochondrial intermembrane space [37]. CPO requires molecular oxygen as the terminal electron acceptor [38]. In the next step, protoporphyrinogen IX is oxidized to protoporphyrin IX, following the removal of six hydrogen atoms from the porphyrinogen ring, to provide an alternating double bond structure to the macrocycle; this reaction is catalyzed by protoporphyrinogen IX oxidase (PPO) [39]. In eukaryotes, the oxygen- dependent PPO is a homodimer that utilizes molecular oxygen as the terminal electron acceptor and functions as an integral membrane protein located in the inner mitochondrial membrane [40]. The active site of the protein faces the intermembrane space and uses a FAD cofactor for electron transfer. The complete reaction uses three O2 molecules that are reduced to three H2O2 molecules. Fig. 2. Structure of heme. Standard 1e24 IUPAC numbering system is used to number Ferrochelatase (FC) is responsible for the insertion of ferrous the carbon atoms of the tetrapyrrole [183]. iron into protoporphyrin IX to form Fe-protoporphyrin IX or heme, in the next step of heme biosynthesis. FC is an inner mitochondrial membrane protein with its catalytic site located on the mitochon- glutamyl-tRNA to glutamate-1-semialdehyde (GSA) in the first of drial matrix side. Channeling of protoporphyrin IX from PPO to FC the two-step ALA production pathway [20]. The second step in- may occur via direct interaction between the two proteins that volves the conversion of GSA to ALA by the enzyme glutamate-1- allows efficient substrate transfer [40]. FC functions as a homo- semialdehyde-2, 1-amino mutase (GSAM) [21e23]. This second dimer and each monomer contains a 2Fee2S cluster whose func- major pathway is found in plants, archaea and most bacteria. tion is unknown [41,42]. The reaction mechanism involves Once exported to the cytosol, ALA serves as the building block to distortion of the planar porphyrin molecule into a saddle confor- synthesize uroporphyrinogen III following three consecutive steps. mation to facilitate the insertion of ferrous iron [43]. Once syn- The first step involves condensation of two ALA molecules to form thesized, heme can undergo side chain modifications (ex: heme A porphobilinogen (PBG) by porphobilinogen synthase (PBGS), also and heme O synthesis) or covalent attachments (ex: heme C in known as ALA dehydratase (ALAD) [24,25]. PBGS is a homooctamer cytochrome c biogenesis) to produce additional heme types in which each dimer contains one catalytic site [26]. Each active site depending on cellular needs. binds an ALA molecule at two distinct sites and each subunit binds one zinc atom [27]. Of the eight Zn atoms on PBGS, four zinc atoms 2.3.2. Regulation of heme biosynthesis stabilize the enzyme structure while the other four engage in cat- In humans, heme is synthesized at two locations, in erythroid alytic activity [28,29]. Four molecules of porphobilinogen undergo progenitors within bone marrow to provide for developing red polymerization to form 1-hydroxymethylbilane in a reaction cata- cells, and in the liver to provide for numerous heme-containing lyzed by porphobilinogen deaminase (PBGD). PBGD uses a cova- enzymes. Liver responds to various metabolic states in the body, lently attached dipyrromethane cofactor (made of two linked PBG hence heme synthesis in liver is regulated accordingly. Erythroid molecules) to prime the polymerization of four PBG molecules. Six progenitors on the other hand maintain heme production at a PBG molecules form a linear hexapyrrole covalently bound to steady pace to meet the demand of red blood cells. Mechanisms for PBGD, which is then cleaved to yield 1-hydroxymethylbilane and regulation of heme synthesis in these two origins therefore differ. the protein bound cofactor [30]. This unstable tetrapyrrole serves as The first step, catalyzed by two different isoforms of ALAS in the substrate for uroporphyrinogen synthase (UROS), the enzyme liver (ALAS1) and bone marrow (ALAS2), is the rate-limiting step of that synthesizes uroporphyrinogen III. UROS functions as a mono- heme synthesis. Both isoforms are post-transcriptionally regulated mer and completes ring inversion and the subsequent closure of by two different mechanisms. Heme has a negative feedback effect the tetrapyrrole, yielding uroporphyrinogen III [31,32]. Sponta- on ALAS1 transcripts and two alternate splice forms of ALAS1 neous ring closure is also possible, but the resulting product uro- facilitate this regulatory mechanism. One splice form is subjected to porphyrinogen I cannot be converted to heme. Uroporphyrinogen heme-mediated decay [44e46] but the other is resistant to this III also serves as the branching point for the synthesis of chloro- effect and requires translation in order to be regulated by heme- phylls and corrins. In heme biosynthesis, this cyclic intermediate is mediated decay [47,48]. Heme itself blocks translocation of a pre- converted to coproporphyrinogen III, a product that lacks four acid cursor form of ALAS1 from cytoplasm to mitochondrion contrib- functional groups. Subsequent decarboxylation steps are carried uting to its downregulation [49]. Transcription of ALAS1 is out by uroporphyrinogen decarboxylase (UROD) within the cytosol upregulated by the peroxisome proliferator activated coactivator 1a to yield four methyl groups in place of the acetic side chains [33]. (PGC-1a) in correlation to cellular glucose levels [50]. In contrast, UROD is a homodimer with each subunit carrying an active site erythroid-specific transcriptional factors such as GATA1 regulate cleft that faces the other in the dimer interface [34]. ALAS2 transcription by binding to the promoter region [51].In 64 D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75

Fig. 3. Important types of heme found in nature. Side chain differences with respect to the parent heme molecule (heme B) are shown in blue. translational regulation of ALAS2, iron responsive elements (IRE) in significantly elevated compared to ubiquitous tissue and the spe- ALAS2 transcripts can bind iron regulatory proteins (IRP). This cific mechanism resulting in this upregulation has yet to be prevents ALAS2 mRNA translation, allowing heme synthesis in determined [59]. The human CPO gene contains a single active differentiating red cells to be regulated in relation to cellular iron promoter with six Sp1 sites, four GATA binding sites and a novel availability and mitochondrial function [52,53]. regulatory element named CPRE that aids in the elevated expres- In addition, three enzymes within the heme biosynthesis sion in erythroid tissue [60]. The PPO gene contains a GATA1 pathway (PBGS, PBGD, and UROS) are transcriptionally regulated, binding site in its single promoter, suggesting potential erythroid- yet they utilize a dual promoter system that allows erythroid spe- specific regulation [36]. Cis-elements like NF-E2, GATA1 and the cific or non-erythroid specific regulation of a single gene. Alterna- Sp1 binding sites are present in the human FC gene promoter and tive splicing of exons creates two different PBGS transcripts, either they have been found to induce FC expression during erythroid with a housekeeping promoter or with an erythroidespecific pro- differentiation while the GC box maintains housekeeping expres- moter that binds erythroid-specific transcription factors including sion of the gene [61]. Expression of ferrochelatase is also regulated GATA1 [54]. Both PBGS transcripts encode identical proteins by iron availability of the cells, related to the FeeS cluster of FC [62]. because they share the same translational start site despite their It is evident that the heme biogenesis enzymes in the erythroid difference in lengths. On the contrary, alternative splicing of tran- pathway are transcriptionally induced by erythroid-specific transcripts of the PBGD gene produces proteins of different lengths scription factors in coordination with iron uptake. Liver on the [55,56]. Erythroid-specific promoters of PBGD contain several other hand maintains sufficient heme levels by combining syn- specific cis-acting sequences (including GATA1, NF-E2 and CACCC thesis and degradation in response to changes in cellular heme motifs) that are not seen in the housekeeping promoter [57]. pools. Both systems are important however for maintaining cellular Similarly, alternative splicing of UROS gene creates two transcripts. iron homeostasis and they are therefore areas of interest for un- Erythroid-specific promoters of UROS contain eight GATA binding derstanding heme related diseases. sites while the non-erythroid promoter contains NF1, AP1, Oct1, Sp1 and NRF2 binding sites. Both transcripts produce identical proteins 2.4. Incorporation of heme into apoprotein recipients [58]. The remaining enzymes within the pathway have single promoters; regardless, they exhibit erythroid and non-erythroid Although diverse in their function, heme-containing proteins expression differences. The UROD gene carries a promoter of non- share a structurally conserved element, the heme cofactor. Primary erythroid origin but UROD levels in erythroid tissue are factors contributing to the functional diversity of heme proteins are D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 65

Table 1 Comparison of different heme types in nature.

Type Structure remarks Organisms Proteins Function Reference

Heme A C3 ¼ hydroxyethylfarnesyl group Bacteria, archaea, plants and Cytochrome a containing heme- Terminal reaction of aerobic [193] C18 ¼ formyl group animals. Cu oxidases. respiration. Reduction of oxygen Conversion of heme to heme A is Ex: Cytochrome c oxidase/ to water. catalyzed by Heme A synthase. Complex IV in mammalian mitochondria. Heme B Parent heme molecule also In a variety of organisms. Cytochromes b and hemoglobins. Diverse array of functions known as protoheme IX, associated with heme b such as Fe-protoporphyrin IX or heme. electron transport and oxygen Binds to apoproteins non- carrier. covalently. Heme C C3 and C8 ¼ thioether bonds with In a variety of organisms. Cytochromes c Serve as electron carriers to [194] cysteine residues. Ex: bacterial alcohol perform single electron transfers Binds to apoproteins covalently. dehydrogenase, bacterial caa3 in energy transduction processes and cbb3 oxidases, mitochondrial such as photosynthesis, and bacterial bc1 complex, and respiration and N and S cycles. cytochrome cd1 nitrite reductase. Other functions include apoptosis, detoxification of ROS. Heme D C12 ¼ hydroxylated, in a trans In diverse bacteria including Terminal respiratory oxidases. Terminal reaction of aerobic [195e197] conformation to the Azotobacter, Proteus, Acetobacter, Ex: Cytochrome d oxidase. respiration. Reduction of oxygen g-spirolactone. Salmonella, Bacillus, Pseudomonas, to water at low oxygen levels. C13 ¼ hydroxylated, propionyl Haemophilus and E. coli. group forms a g-spirolactone with the hydroxyl group. Heme D1 C3 and C8 ¼ an acetyl group and a In Pseudomonas perfectomarinus, Nitrite reductase that functions in Catalyzes the one-electron [198,199] methyl group on each carbon. Alcaligenes faecalis, Paracoccus dissimilatory nitrogen reduction of nitrite to nitric oxide C2 and C7 ¼ keto groups on each denitrificans, Thiobacillus metabolism. and four-electron reduction of carbon. denitrificans, and Thiosphaera Ex: Cytochrome cd1 nitrite oxygen to water. C13 propionyl side chain has pantotropa. reductase. reduced to incorporate a vinyl group between second and third carbons. Heme I C2 and C12 ¼ hydroxymethyl In mammalian secretory fluids. Peroxidases such as bovine milk Peroxide-driven oxidation of [200] groups, they form ester bonds enzyme lactoperoxidase, halide and pseudohalide ions as a with carboxyl side chains of eosinophil peroxidase and nonspecific antimicrobial defense amino acids. thyroid peroxidase mechanism for the protection of Binds to apoproteins covalently. mucosal surfaces. Heme M C2 and C12 ¼ hydroxymethyl In mammalian neutrophils Myeloperoxidase Peroxide-driven oxidation of [11,200] groups, they form ester bonds chloride and bromide ions as an with carboxyl side chains of antimicrobial defense amino acids. mechanism. C3 ¼ vinyl group on C3 forms a sulfonium ion linkage with the sulfur of a methionyl residue. Heme O C3 ¼ hydroxyethylfarnesyl group. Bacteria such as E. coli. Cytochrome o containing quinol Terminal reaction of aerobic [201,202] oxidases. respiration. Reduction of oxygen

Ex: Cytochrome bo3 to water.

the protein ligands coordinating Fe in the proximal/distal positions convert apocytochrome c to holocytochrome c. These three systems and the covalent linkage of the heme to the biomolecule. Insertion carry out a similar function: to keep both Fe in heme and sulfur in and stabilization of the heme unit within a heme containing pro- cysteine residues of the apoproteins in the reduced state to facili- tein has been studied extensively within cytochrome c. Heme C tate correct covalent attachment. Mechanisms of incorporation of forms two thioether bonds between C3 and C8 vinyl groups of additional heme types into different heme binding proteins can be heme and the cysteine residues of CXXCH motifs in the apoc- quite diverse and species speciﬁc: hence their discussion was ytochrome c. Three systems driving this association have been eliminated from this review but is discussed in additional sources studied extensively (reviewed in [63,64]). System I/CCM (Cyto- [67e70]. chrome C Maturation) is most prevalent in a and g proteobacteria, all plant mitochondria, some protozoal mitochondria and red algae, 2.5. Heme function and typically involves nine assembly proteins including CcmA through CcmH [65]. Although system II/CCS (Cytochrome C Syn- Heme proteins are ubiquitous in nature and perform a wide thesis) was originally studied in green algae Chlamydomonas rein- variety of functions. One abundant class of heme proteins is the hardtii, it can also be found in chloroplasts, most Gram-positive photosynthetic and respiratory cytochromes. Other classes include bacteria, cyanobacteria and some b, d, and ε proteobacteria. Several globins, catalases, peroxidases, cytochrome P450s, oxygenases and of the components in system II vary in different organisms but the others. Here we discuss the versatile chemistry of the heme unit in major components include CcsA, CcsB and CcsX. System III/CCHL several categories of heme proteins. (Cytochrome C Heme Lyase) is mostly restricted to fungal, verte- Peroxidases use H2O2 to oxidize substrates without oxygen brate and invertebrate mitochondria and some protozoal mito- transfer. Their catalytic cycle includes three steps: 1) H2O2 oxidizes chondria. This system employs a cytochrome c heme lyase enzyme, Fe(III) and porphyrin to generate a Fe(IV)oxo porphyrin p cation which is now known as holocytochrome c synthase (HCCS) [66] to radical with water as a product; 2) oxidation of substrate reduces 66 D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75

Fig. 4. Heme biosynthesis pathway. ALA: 5-aminolevulinic acid, ALAS: ALA synthase, PBGS: porphobilinogen synthase, ALAD: ALA dehydratase, PBGD: porphobilinogen deaminase, UROS: uroporphyrinogen synthase, UROD: uroporphyrinogen decarboxylase, PBR: peripheral-type benzodiazepine receptor, CPO: coproporphyrinogen III oxidase, PPO: protoporphyrinogen IX oxidase, FC: Ferrochelatase, IMM: inner mitochondrial membrane, IMS: intermembrane space, OMM: outer mitochondrial membrane. the Fe(IV)oxo porphyrin p cation radical; and 3) a second substrate species [71]. Fe(IV)oxo porphyrin p cation radicals have been pro- reduces Fe(IV) to Fe(III) [71]. Examples from mammalian peroxi- posed as reactive species in catalytic oxygenation reactions. dases include myeloperoxidase (MPO), eosinophil peroxidase Although both peroxidases and cytochromes P450 form Fe(IV)oxo (EPO), and lactoperoxidase (LPO) [72], and these enzymes can intermediates during their catalytic cycles, profound differences in oxidize a wide variety of substrates due to their high reduction protein environments create different products. Cytochromes P450 potentials. The covalent vinyl sulfonium heme linkage in heme M of are mostly known for xenobiotics detoxification in liver, where MPO enables heme distortion and the resulting reduction in elec- drugs and other xenobiotics are hydroxylated and made more tron density in the heme best explains the unusually high reduction soluble, facilitating their conversion to easily eliminated products. potential of MPO [73]. These peroxidases are capable of generating In addition, P450s also participate in the biosynthesis of steroids, oxidants such as hypohalous acids, hypothiocyanous acid, reactive highlighting their importance in metabolism. nitrogen species, singlet oxygen, phenoxyl and hydroxyl radicals, all Additional noteworthy heme enzymes include nitric oxide which are key components in antimicrobial properties exerted by synthase (NOS) and heme oxygenase (HO). NOS catalyzes the the immune system [72]. oxidation of L-arginine to L-citrulline and nitric oxide (NO), a Cytochromes P450 represent another subgroup of heme pro- signaling molecule important for regulation of the cardiovascular teins in high abundance in nature; humans alone carry more than and nervous systems as well as participating in immune responses. fifty P450 enzymes. Despite the immense diversity of P450s in NOS catalysis is similar to P450 mechanism in the first step in u nature, as per published structures, the overall fold in these bio- which L-arginine is converted to N -L-hydroxyarginine except that molecules is basically the same. The typical electron transfer cata- an unusual cofactor, tetrahydrobiopterin or BH4 is required. u lytic cycle of a P450 requires electrons derived from redox protein Oxidation of N -L-hydroxyarginine to L-citrulline in the second step partners such as flavin and FeeS containing proteins. Substrate is proposed to be carried out via peroxy or superperoxy species as binding shifts low-spin hexacoordinate heme to high-spin heme opposed to the Fe(IV)oxo intermediates discussed before. Heme while displacing the axial water ligand, which allows electron oxygenase is responsible for the degradation of free heme, resulting transfer from the redox partner and oxygen binding to heme to in its efficient elimination and the recycling of iron. This conversion form the oxy complex. A second electron transfer step is respon- is carried out in three distinct steps; heme is first converted to sible for generating the heme Fe(III)dihydroperoxy species which aemeso-hydroxyheme, which in turn gets converted to verdoheme finally undergoes heterolytic cleavage to give heme Fe(IV)oxo and subsequently to billiverdin. Studies show it is highly unlikely D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 67 for an Fe(IV)oxo species to serve as an oxidant in the catalytic action and it falls under the category of cutaneous porphyrias. This disease of heme oxygenase since it uses a histidine as an axial ligand [71]. only shows symptoms in the skin, including lesions in areas The complete list of heme proteins is extensive and exceeds the exposed to sun, skin fragility leading to secondary infections, and limits of this review. Instead, we will focus on human disorders that hypertrichosis. Ocular pain and photophobia have also been re- result from loss of heme protein function, many of which are se- ported rarely [79]. The cause of these symptoms is accumulation of vere, and merit detailed discussion. excessive porphyrin in the skin. Tetrapyrroles are highly photo- reactive and hence they absorb energy from the visible region of 2.6. Diseases of heme synthesis electromagnetic spectrum. Excited ring structures reach ground state by transferring energy, which drives peroxidation and Several human diseases are associated with disruption of the oxidation of biological macromolecules such as membrane lipids, heme biosynthetic pathway. Many of these diseases are associated nucleic acids and proteins. Familial PCT, one of the two forms of PCT, with inherited mutations in heme biosynthesis genes, however is an autosomal dominant trait similar to AIP in that only a minority some are caused by environmental factors affecting their enzyme of heterozygotes is affected. In familial PCT, uroporphyrinogen III is products. Along with nine major porphyrias, we will focus on dis- accumulated and excreted in urine due to UROD mutations. Pa- eases associated with disruption of the heme biosynthesis pathway. tients with sporadic PCT, although not associated with UROD mutations, show decreased UROD activity and hepatic Fe overload, 2.6.1. Porphyrias similar to that seen in familial PCT [80]. Hepatoerythropoietic The clinical presentation of porphyria includes skin lesions and porphyria (HEP) is a rare porphyria associated with UROD defi- acute neurovisceral attacks which are related to the accumulation ciency. Symptoms are similar to that seen in PCT (skin lesions, red of specific intermediates in the heme biosynthetic pathway. Nine urine, hypertrichosis and scarring) however they can be much more such diseases have been identified and they are categorized as severe. This rare recessive porphyria often onsets in infancy or hepatic or erythropoietic, pertaining to the organ in which the in- childhood and shows high porphyrin concentrations in erythro- termediates accumulate. However, a more clinical classification of cytes and less than 10% UROD activity [81]. porphyria divides these into three groups: acute, cutaneous and Hereditary coproporphyria (HCP), another acute porphyria, is rare recessive. caused by mutations that destabilize the enzyme CPO. Clinical Even though no disease causing mutations for ALAS1 has ever characteristics are very similar to other acute porphyrias (i.e., AIP) been found in humans, many mutations affecting the function of and show a high coproporphyrinogen III content in urine and feces, the ALAS2 isoform have been identified. Although not commonly as well as increased photosensitivity. Also similar to other acute seen as loss of function mutations in the heme biosynthesis porphyrias, HCP is mostly seen in dominant homozygotes for the pathway, gain of function deletions in ALAS2 are found to be trait, with only some heterozygotes developing the disease [82]. causative of a cutaneous porphyria called X-linked dominant An additional porphyria caused by mutations in a heme erythropoietic protoporphyria (XLDPP). This disease is characterized biosynthesis enzyme includes variegate porphyria (VP), character- by increased ALAS2 activity and excessive protoporphyrin pro- ized by dysfunctional PPO. Symptoms are again very similar to any duction. ALA production is increased such that the final step cata- acute porphyria, showing acute neurovisceral attacks and cuta- lyzed by ferrochelatase is rate-limiting, resulting in the neous photosensitivity. Onset of the symptoms is in adulthood and accumulation of protoporphyrin. Liver damage and photosensi- over 100 PPO gene mutations (including nonsense, missense, tivity are the clinical manifestations of this disease [74]. deletion, insertion and splice mutations) have been identified [83]. Deficiency of ALAD causes a rare recessive porphyria named As in AIP, urinary excretion of ALA and porphobilinogen and fecal ALAD porphyria, for which only less than ten cases have been re- content of protoporphyrinogen IX and coproporphyrinogen III is ported. In this autosomal disorder, loss of ALAD activity in liver and increased during acute attacks. In addition, PPO activity is erythroid precursors leads to excretion of ALA and cop- dramatically reduced (50%) in tissues of VP patients [84]. roporphyrinogen III into the urine. Patients suffer from intermittent The last of the nine porphyrias associated with heme synthesis acute neurovisceral attacks and/or chronic neuropathy, and onset of is another cutaneous porphyria named erythropoietic proto- this disorder ranges from childhood to adulthood [75]. porphyria (EPP). Mutations in the ferrochelatase gene cause a An additional disease related to the first steps in heme biosyn- deficiency in mitochondria leading to accumulation of free proto- thesis is acute intermittent porphyria (AIP). As in all acute por- porphyrin IX, primarily in erythrocytes. Excess protoporphyrin IX is phyrias, acute life-threatening complications occur mostly in the behind the predominant clinical feature of photosensitivity, which adulthood, however in rare cases severe attacks can occur in the begins in childhood [85,86]. childhood. Urinary excretion of ALA and porphobilinogen is increased due to decreased PBGD activity. Activity loss is caused by 2.6.2. Additional diseases mutations in the PBGD gene and over 200 mutations have been Loss of function mutations of ALAS2 is the cause of the disorder identified. This disorder is seen in homozygous dominants for the X-linked sideroblastic anemia (XLSA). A group of point mutations trait and a majority of heterozygotes remain disease-free [76]. alter the protein's ability to bind the PLP cofactor, however other Congenital erythropoietic porphyria (CEP) is another rare reces- mutations affect protein domains outside the cofactor-binding re- sive type of porphyria caused by the deficiency of UROS. This gion. The decrease in heme synthesis efficiency prevents normal autosomal recessive disease that is a result of loss of UROS function erythroblast development in the bone marrow. The resulting leads to the spontaneous formation of uroporphyrinogen I, the overload of iron results in characteristic iron granules surrounding isomer of uroporphyrinogen III that cannot be converted to heme, the nucleus (a sideroblast). This disease is characterized by so it is accumulated and excreted [77]. Clinical features of this microcytic hyperchromic anemia, the presence of mature but pale disease include chronic hemolysis and cutaneous photosensitivity and smaller than normal erythrocytes and Fe overloaded mito- caused by diffusion of uroporphyrinogen I to plasma, and these chondria in erythroblasts in the bone marrow [87]. conditions begin to manifest in early infancy. There have been over ALAD requires Zn for function, and Zn deficiency makes ALAD twenty UROS mutations that cause this disease identified to date susceptible to Pb inhibition. Pb replaces Zn in ALAD and the [78]. inhibited protein leads to high levels of ALA in blood, which is Porphyria cutanea tarda (PCT) is the most common porphyria responsible for neurological manifestations due to its toxicity at 68 D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 high concentrations [88]. A similar disease, named hepatorenal proteins, FeeS proteins are synthesized in their apo-state and tyrosinemia, shows neurotoxicity due to high ALA levels, with obtain their FeeS cluster cofactor from a dedicated FeeS cluster abnormal liver and kidney function. This disease is caused by mu- formation pathway. At present, there are three known general tations in fumarylacetoacetate hydrolase gene, which hydrolyzes pathways for FeeS cluster formation: the iron sulfur cluster (ISC), fumarylacetoacetate to fumarate and acetoacetate during the cytosolic iron sulfur assembly (CIA), and sulfur assimilation (SUF) tyrosine catabolism pathway. In the event of enzyme deficiency, pathways. These three pathways provide FeeS clusters for the fumarylacetoacetate is metabolized to succinylacetone, which is majority of FeeS proteins in almost all organisms. While dedicated structurally similar to ALA, hence it acts as a potent inhibitor for FeeS cluster forming pathways can exist for individual FeeSpro- ALAD [89]. Excess ALA and succinylacetone can be seen in patients' teins, such as the nitrogen fixation (Nif) pathway that provides an urine and blood samples. FeeS cluster for the nitrogenase enzyme in nitrogen-fixing bacteria [94], this review will focus only on the general FeeS cluster pro- 3. FeeS clusters duction pathways.

3.1. Introduction 3.3.1. Iron sulfur cluster (ISC) pathway The most robust and best-characterized pathway for FeeS Iron-sulfur (FeeS) clusters are the second major form of com- cluster biosynthesis is the iron sulfur cluster (ISC) pathway. A plex iron cofactors found in biology. Due to the high abundance of simplified description of this pathway is provided in Fig. 6. The ISC iron and sulfur on the earth's surface, and the easy association of pathway, present in bacteria and in the mitochondria of eukaryotes, these atoms under anaerobic conditions, FeeS clusters likely provides general housekeeping FeeS clusters to a large number of developed early in evolution before the earth's transition to an FeeS proteins. In eukaryotes, this pathway provides FeeS clusters aerobic atmosphere. Consequently, these cofactors are ubiquitous for several key mitochondrial FeeS proteins. ISC was initially in all organisms and play a role in almost every biological pathway. identified in the Azotobacter vinelandii and Escherichia coli bacterial Here we provide an overview of the structure, formation, and species, where ISC genes are arranged in the isc operon. Additional function of FeeS cluster cofactors. early work in eukaryotes (in Saccharomyces cerevisiae and human proteins) revealed a highly homologous system localized to the 3.2. FeeS cluster structure mitochondria. In addition to providing for mitochondrial FeeS proteins, the ISC system provides a component to the cytosolic and In many ways, FeeS clusters are simpler than heme. While heme nuclear FeeS cluster formation pathways, thus ISC is essential for is a mixture of organic (protoporphyrin) and inorganic (iron) the maturation of all cellular FeeS proteins in eukaryotes [95,96]. components, FeeS clusters are strictly inorganic. Iron atoms in ISC serves as a template for understanding general FeeS cluster biological FeeS clusters interact directly with protein residues and production. In human ISC, de novo 2Fee2S synthesis occurs on the sulfur atoms bridge neighboring iron atoms. FeeS clusters exist in a dedicated scaffold protein ISCU [97]. Sulfur for this reaction is variety of configurations depending on their respective number of provided by ISC's dedicated cysteine desulfurase enzyme ISCS via a iron and sulfur atoms. The three most common forms (2Fee2S, persulfide intermediate that gets transferred to ISCU [98] upon 3Fee4S and 4Fee4S) are illustrated in Fig. 5. More complex FeeS formation of an ISCU-ISCS complex. In eukaryotes, ISCS has a clusters have also been observed, including the 7Fee8S and 8Fee8S dedicated protein co-factor ISD11 that is essential for ISCS function clusters identified in ferredoxins from Desulfovibrio africanus [90]. [99]. Electrons for ISCS persulfide release are provided by the The Fe atoms within the FeeS cluster can exist in either ferric or 2Fee2S cluster containing ferredoxin FDX, which in turn gets ferrous forms and cycling between these redox states allows the reduced by the ferredoxin reductase FDXR that uses NADPH as its transfer of electrons for redox reactions. The tendency of the final electron source [100,101]. FDX also interacts with ISCU, oxidized FeS cluster to gain an electron is termed the “reduction providing 2 reducing equivalents for assimilation of two 2Fee2S potential”. By convention, this potential is expressed in comparison clusters on an ISCU dimer to form a single 4Fee4S cluster [97].An to a reference standard hydrogen electrode, which is assigned a additional Fe-binding protein Frataxin interacts with the ISCS-ISCU potential of 0 V. Depending on FeeS cluster type, interactions with complex and regulates ISCS activity [102]. Additionally, there are neighboring amino acids, and solvent accessibility, a single FeeS alternate scaffold proteins (ISCA [103] and NFU1 [104]) that interact cluster can transfer up to two electrons with a reduction potential with ISC proteins and these are believed to be required for the spanning ~1000 mV [91]. This remarkable range of accessible maturation of a specific subset of FeeS proteins. reduction potentials can largely explain the biological utility of the Despite intense study, the physiologic source of iron for ISC re- FeS cluster. mains a subject of debate. Several potential iron donors have been FeeS clusters do not exist freely but are intimately connected to investigated and iron delivery to ISCU has been demonstrated from their apoprotein partner. Free iron will form an insoluble complex several potential sources in vitro within a variety of systems. The when bound to sulfide, so the protein plays a critical role in solu- protein frataxin [105,106] interacts with the ISCU-ISCS complex and bilizing the FeeS unit. FeeS proteins usually bind their corre- could be the source of iron for the pathway [106]. In the bacterial sponding FeeS cofactor via ionic interactions between cysteine system, two additional members of the isc operon have been thiols and iron in the FeeS cofactor. In some cases, FeeS clusters are investigated as potential iron donors, IscX and IscA. The small acidic alternatively ligated via histidine residues. Subsets of 2Fe2S clus- protein IscX binds iron and regulates cysteine desulfurase activity ters, such as those found in Rieske proteins (see section 3.5), are in a manner very similar to Frataxin [107]. The alternative scaffold 19 coordinated by two cysteine and two histidines (Cys2His2) [92] and IscA is also an interesting candidate because of its tight (KD~10 ) a common coordination theme of proteins involved in FeeS cluster binding affinity for mononuclear iron [108] and its capability of biogenesis is Cys3His1 coordination [93]. delivering iron to IscU [109,110]. An additional interesting hy- pothesis is that iron may come from a glutathione-glutaredoxin 3.3. General FeeS cluster biogenesis pathways complex [111]. The lack of conclusive evidence for a specific iron source suggests that in vivo, there could be multiple iron sources or Production of FeeS clusters must be highly regulated to prevent that the mode of iron delivery may be atypical. unwanted reactions of both free iron and sulfur. Similar to heme- A detailed mechanism for FeeS cluster delivery from ISCU to D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 69

Fig. 5. Structure for the 3 most common forms of FeeS Cluster: 2Fee2S, 3Fee4S, and 4Fee4S. downstream targets is currently under investigation. The FeeS almost all cases [125]. CIA is unique among FeeS maturation cluster bound to ISCU is transferred to the glutaredoxin GLRX5 pathways in that it does not obtain reduced sulfur via a dedicated [112,113].Efficient transfer from ISCU to GLRX5 requires involve- cysteine desulfurase. A simplified description of the CIA pathway is ment of the ATPase SSQ1, which binds to both ISCU and GLRX5 provided in Fig. 6. Instead of an ISCS analog, CIA relies on mito- [114]. Binding of SSQ1 to ISCU, along with the interaction of a DnaJ- chondrial export of a sulfur-containing compound, ‘XeS’, via the like co-chaperone JAC1 [115], destabilizes the FeeS cluster on ISCU mitochondrial export protein ABCB7 [126] and the intermembrane facilitating its transfer from ISCU to GLRX5 [116]. GLRX5 is space protein ALR [127]. The identity of XeS is currently unknown, considered the end of the ISC pathway because it is the last com- but may be glutathione-complexed to an FeeS cluster [125].In mon FeeS cluster carrier for all mitochondrial FeeS proteins. human CIA, the primary scaffold for de novo FeeS assembly is a GLRX5 continues the cluster transfer process, however, and it is tetrameric complex formed between CFD1 and NBP35, which binds able to interact with a variety of downstream FeeS proteins a bridging 4Fee4S cluster between the CFD1 and NBP35 subunits [117e119]. The specific recipient depends on the ultimate desti- [128]. Reducing equivalents for this reaction are provided by an nation of the FeeS cluster. For example, 4Fee4S cluster conversion FeeS containing protein CIAPIN1 (similar to FDX in ISC), which in and delivery is facilitated by GLRX5's interaction with two other turn gets reduced by the diflavin reductase NDOR1 (similar to FDXR FeeS proteins, ISCA and IBA57 [120]. in ISC), utilizing reducing equivalents from NADPH [129,130]. Despite being mostly localized to mitochondria [121], ISC is The 4Fee4S clusters from the NBP35-CFD1 complex get trans- required for maturation of all cellular FeeS proteins [96]. The ferred to another FeeS protein IOP1, which binds two 4Fee4S mechanism by which cytosolic FeeS proteins depend on mito- clusters per monomer [131,132]. IOP1, in turn, delivers its FeeS chondrial ISC is actively being investigated. Because FeeS clusters clusters to a multi-component complex called the CIA targeting are not able to cross the inner mitochondrial membrane [122], this complex, which consists of at least three proteins CIA1, CIA2B, and mechanism likely involves the transport of an FeeS cluster pre- MMS19 [133]. The CIA targeting complex is able to interact with a cursor out of the mitochondria and into the cytosol. Recent work variety of recipient FeeS proteins, likely an indication of its function has identified an unknown compound produced by mitochondrial in downstream FeeS cluster delivery. ISCS (named ‘XeS’) that may provide reduced sulfur for cytosolic FeeS cluster formation [123]. 3.3.3. Sulfur assimilation (SUF) pathway Of the three FeeS general cluster formation pathways, the sulfur 3.3.2. Cytosolic ironesulfur assembly (CIA) pathway assimilation (SUF) pathway is probably the most ancient. SUF Recent studies have revealed the involvement of another predominantly exists in prokaryotes, however it is found in specific essential and highly conserved FeeS biosynthetic pathway that is locations in eukarya, including the chloroplasts in some plants present within the cytosol and nucleus of eukaryotes. This pathway [134] and the apicoplasts in some plasmodium species [135], and is called cytosolic iron-sulfur assembly (CIA) [124]. This pathway recently proteins homologous to bacterial SUF were discovered in has been identified in many eukaryotic systems and is essential in the cytosol of a blastocystis species [136]. At present, SUF has been 70 D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75

Fig. 6. Diagram depicting de novo FeeS cluster formation and the main FeeS cluster transfer steps in the ISC, SUF, and CIA systems. Protein names used are from the human system for ISC and CIA, and from the bacterial system for SUF. best characterized in the Gram-negative bacteria E. coli and Erwy- unstable and prone to spontaneous oligomerization. There is also a nia chrysanthemi where its genes are organized into the suf operon paralogue of SufB, named SufD that is able to replace a SufB in the (Fig. 7). SufBC complex, resulting in the SufBCD complex [142]. However, The SUF pathway is similar to ISC in many ways. Like in ISC, SUF SufBC is likely the most active form [143]. The SUF cysteine provides general FeeS cluster formation to accommodate a variety desulfurase SufS functions in a similar manner to ISCS, accepting of FeeS proteins. In fact, SUF and ISC seem to be redundant in sulfur from cysteine via a persulﬁde intermediate. SufS has an Gram-negative bacteria, as the removal of the entire isc or suf essential binding partner SufE, which is required for activity, operon results in no deleterious effects. Simultaneous suf/isc forming the SufSE complex [144]. While it may seem SufE is similar operon deletion, however, is lethal [137]. While ISC and SUF follow to ISD11 from eukaryotic ISC, SufE functions differently from ISD11 the same general mechanism for FeeS cluster formation (Fig. 6), in that it accepts the persulﬁde from SufS and allows the SufS SUF seems to be favored under conditions of oxidative stress and enzyme to complete its turnover [145]. iron limitation [138] and the SUF proteins are correspondingly Details of SUF's downstream cluster delivery are not as well more stable under adverse conditions in vitro [139]. In E. coli, the established as in the ISC pathway. The 4Fee4S cluster formed by SUF pathway centers around two heteromeric complexes called SufBC can be transferred to the A-type carrier protein SufA in vitro SufBC and SufSE. [140,143], but SufBC also may be able to transfer directly to recip- The primary scaffold SufB requires a binding partner SufC for ient apo-proteins. In vivo, SufA is functionally redundant with the activity, forming the SufBC complex in a SufB2C2 arrangement. The ISCA bacterial homologue [146] and possibly acts as an interme- SufBC complex can form a 4Fee4S cluster on SufB that can be diate carrier of the 4Fee4S cluster from SufB, passing it off to transferred to recipient proteins [140]. The exact role of SufC in this downstream apoproteins [147,148]. Another protein involved in process is unknown, but it has ATPase activity that is essential for this process (ErpA) has redundant function with SufA but is FeeS cluster formation on SufB [141]. SufB, on its own is relatively necessary for the development of active FeeS proteins [149]. D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 71

Fig. 7. Illustration of suf and isc operons, with corresponding promoters (Pisc and Psuf), which play a central role in regulation of SUF and ISC at the genetic level in bacteria. Operons depicted are from the E. coli model system. Steps are colored based on the encoded protein's function as follows: red (regulatory), yellow (sulfur delivery), green (primary scaffold), blue (downstream FeeS cluster delivery), cyan (electron transfer), and gray (unknown).

þ Several important details of the SUF pathway remain to be which binds Fe2 under non-stressed conditions when iron levels þ identified. As in ISC, the in vivo source of iron is unknown. Also of are sufficient and oxidative stress is low. With its Fe2 cofactor, Fur interest is SufD's incorporation in the SufBCD complex, which al- binds to the suf promoter at the same site as apo-IscR, inhibiting suf lows SufB to accept iron in vivo [141] and facilitates binding of a expression [159]. When the cell faces iron deficiency, Fur loses its FADH2 cofactor [142]. This cofactor may be able to reduce ferric iron cofactor, dissociates from the suf promoter, thus triggering þ iron, facilitating potential Fe3 sources such as ferritins or ferric transcription of suf. The cell's preference for SUF over ISC in the citrate [150]. While SufA can deliver mononuclear iron to SufBC presence of oxidative stress also reveals the involvement of another in vitro [109], it is currently believed to function downstream of de transcription factor (OxyR), as oxidized OxyR recruits RNA poly- novo FeeS formation (Fig. 6). The source of electrons for SufS merase to the suf promoter. turnover and for FeeS formation remains in question [151]. There are additional regulatory mechanisms for cluster bioassembly beyond the level of gene expression. The small non- 3.4. FeeS cluster biogenesis regulation coding RNA RyhB, for example, is encoded just upstream of the SUF promoter and can bind to the iscRSUA mRNA to prevents its The best-developed model for FeeS biogenesis pathway regu- translation [160]. RyhB expression, however, is constitutively 2þ lation comes from work done in Gram-negative bacterial systems, repressed by Fur-Fe , so RyhB effectively inhibits ISC when con- where both the ISC and SUF pathways are present. This work re- ditions favor SUF [161]. veals a fascinating interplay between ISC and SUF, where the necessary ISC and SUF genes are organized into their respective isc 3.5. FeeS cluster function and suf operons (Fig. 7). While this review has focused on eukaryotic systems, regulatory mechanisms in bacteria may pro- FeeS clusters are versatile biological cofactors found in the most vide insight into how this regulation occurs in eukaryotes. At the fundamental biochemical pathways, including aconitase and suc- center of E. coli FeeS cluster biogenesis regulation is a DNA-binding cinate dehydrogenase of the citric acid cycle and respiratory com- protein IscR, the first member of the isc operon, which directly plexes I-III of the electron transport chain [121]. Nuclear FeeS regulates both the ISC and SUF systems. proteins also have a unique role in DNA damage recognition and Under non-stressed conditions, ISC is favored over SUF for repair. Several forms of DNA polymerase, helicase, glycosylase, and general housekeeping of FeeS cluster biosynthesis [152]. The primase all contain FeeS clusters [125]. Considering their remark- transcriptional regulator IscR can bind a 2Fee2S cluster (forming able range of functions, a thorough summary of various FeeS pro- holo-IscR), obtaining its FeeS cluster from the same ISC machinery teins is well beyond the limited scope of this review. New FeeS utilized by other FeeS proteins [153]. In the holo configuration, IscR proteins continue to be discovered but in many cases, the role of the binds to the isc promoter and prevents binding of RNA polymerase FeeS cluster within the FeeS protein remains unknown, even if the [154]. Thus, holo-IscR acts as a feedback regulator, inhibiting tran- cluster's presence is essential for proper protein function. scription of the entire isc operon when ISC activity is sufficient FeeS clusters can be found in the active site of many essential [155]. IscR is a relatively poor substrate for ISC-mediated FeeS enzymes and usually are involved directly in catalysis. Being stable cluster loading [156] and holo-IscR can only form after the ISC in a variety of redox states, FeeS clusters are best known for their proteins have exhausted their interactions with other apoeFeeS role as electron carriers. FeeS clusters can carry usually one, but proteins. In addition to being a weak ISC substrate, IscR does not sometimes two electrons and are, subsequently, stable in various bind its FeeS cluster tightly and effectively acts as a sensor of reduced states. 2Fee2S clusters, for example, can exist in oxidized þ þ þ þ cellular iron and oxygen conditions [157]. Under high-oxygen or (Fe3 /Fe3 ) or reduced (Fe3 /Fe2 ) forms while 4Fee4S clusters are þ þ þ þ þ þ low-iron conditions, holo-IscR quickly reverts to apo-IscR. Therefore stable in oxidized (Fe3 /Fe3 /Fe3 /Fe2 ), intermediate (Fe3 /Fe3 / þ þ þ þ þ þ under typical aerobic conditions, high oxygen levels cause holo-IscR Fe2 /Fe2 ), and reduced (Fe3 /Fe2 /Fe2 /Fe2 ) forms [162]. The to revert to apo-IscR. Apo-IscR dissociates from the isc promoter and reduction potential of an FeeS cluster is often modulated by in- isc is uninhibited. teractions with nearest neighbor protein residues and by access to In the apo configuration, IscR does not bind to the isc promoter solvent, allowing for a large range of biological functions. Ferre- but instead favors binding to the suf promoter, activating tran- doxins are considered the archetypical FeeS cluster electron car- scription of SUF genes [158]. Appropriate interaction of apo-IscR riers and were the earliest FeeS proteins to be functionally with the suf promoter involves two additional transcription factors: characterized [163]. Ferredoxins are involved in many essential the ferric uptake regulator (Fur) and the peroxide responsive biochemical pathways, transferring electrons for cellular respira- regulator (OxyR). Suf expression is constitutively repressed by Fur, tion, photosynthesis, and nitrogen fixation [164]. A ferredoxin is 72 D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 even involved in the ISC iron sulfur cluster biogenesis pathway, as dehydrogenase and aconitase of the citric acid cycle [184]. Symp- discussed previously (see section 3.3.1) [165]. toms are exacerbated in cells that are metabolically active, such as But FeeS cluster-mediated electron transfer is not limited to the myocytes of skeletal muscle during exercise, and patients with ferredoxins. In fact, one of the most the fundamental electron IM experience exercise intolerance [185]. Prolonged activity can transfer processes, the electron transport chain (ETC), utilizes lead to tachycardia, tachypnea, and muscle pain [186]. Unlike FRDA, numerous FeeS clusters. Respiratory complexes I, II, and III of the IM is not progressive and most cases have a normal life expectancy. ETC all contain FeeS clusters. Respiratory complex I uses a network of 8 FeeS clusters for step-wise electron transfer [166]. Similarly, 3.6.3. GLRX5 sideroblastic anemia complex II contains an FeeS protein component called SDHB with a GLRX5 Sideroblastic Anemia (GSA), a disease caused by mutated 2Fee2S, 3Fee4S, and 4Fee4S cluster [167]. Lastly, complex III uti- GLRX5, has only been identified in a single patient to date. While lizes a unique FeeS cluster called a “Rieske center” [168]. The Rieske GSA is exceedingly rare, this particular case study has revealed a center is a 2Fee2S cluster where one of the iron atoms is coordi- unique mechanism linking FeeS cluster production to general iron nated by histidines instead of cysteines, resulting in a Cys2His2 homeostasis [52,187]. GLRX5, involved in the last step of the ISC coordination [169]. pathway, directs FeeS cluster delivery from ISCU to downstream FeeS clusters can also be involved in non-redox reactions. The targets. One target is the iron-responsive protein IRP1, an FeeS 4Fee4S cluster in aconitase, for example, catalyzes a hydra- protein activated when its FeeS cluster is absent [187]. Defective tionedehydration reaction, ligating directly to the citrate substrate GLRX5, therefore, leads to constitutively active IRP1. IRP1 regulates [170]. In some cases, FeeS clusters appear to only serve a structural several proteins involved in iron homeostasis [188], including those function and not participate in chemistry directly, as is the case in involved in heme production. In particular, apo-IRP1 inhibits endonuclease III [171]. expression of the final enzyme in heme synthesis, aminolevulinate synthase (ALAS2). Defective GLRX5, therefore, leads to insufficient 3.6. FeeS clusters in human disease heme and impaired erythropoiesis, resulting in anemia. Iron that would be directed towards heme production accumulates in the Unlike in Gram-negative bacteria, where ISC/SUF redundancy cytosol of erythroblasts, creating the characteristic ringed- allows for removal of an entire pathway, in humans the absence or sideroblasts [151]. mutation of a single component is often incompatible with life. In select cases there are human diseases that have been linked to 3.6.4. Additional diseases defective FeeS cluster biogenesis pathways. Below we describe Succinate Dehydrogenase (SDH) subunit B, the FeeS cluster several diseases directly linked to dysfunctional FeeS cluster containing protein of the succinate dehydrogenase complex, is a formation. known tumor suppressor. Succinate, the substrate for SDH, stabi- lizes hypoxia-inducible factor (HIF), which regulates key processes 3.6.1. Friedreich's ataxia in cell division and blood vessel growth under hypoxic conditions. With an incidence of 1 in 50,000 [172,173], and a carrier prev- Mutations in SDHB or in fact any of the other main subunits of SDH alence of 1 in 100 [174], Friedreich's ataxia (FRDA) is by far the most (SDHA, SDHC, and SDHD) cause susceptibility to tumor formations prevalent disease linked to defective FeeS cluster formation. FRDA known as paragangliomas or phaeochromocytomas in a disorder is an autosomal recessive genetic disease caused by a GAA- called Hereditary Paraganglioma-Pheochromocytoma [189], stem- trinucleotide repeat expansion in an intron of the frataxin gene, a ming from the accumulation of succinate and stabilization of HIF. protein involved in the ISC pathway [175]. This trinucleotide repeat SDHAF2, an assembly protein that flavinates SDH, is also implicated expansion leads to under-expression of the frataxin gene and in paragangliomas. Recently, two additional FeeS assembly pro- subsequently, low levels of frataxin [176]. These insufficient fra- teins, SDHAF1 and SDHAF3, were found to stabilize FeeS cluster taxin levels are responsible for the pathophysiology of FRDA, but assembly in SDH [190]. The later two proteins have LYR-motifs the precise role of frataxin is still unknown [177]. Frataxin may (Leu-Tyr-Arg) common to proteins involved in FeeS cluster as- deliver iron to the ISC pathway [178], be an allosteric activator of sembly. SDHAF1 deficiency is known to cause leukoencephalopathy the ISCU-ISCS complex [102], or may have a combination of roles. [191]. FRDA tissues demonstrate increased mitochondrial iron deposits Lastly, mutations in either NFU1 or BOLA3, two different genes [179] which leads to increased oxidative stress and cell death in involved in FeeS cluster biogenesis, leads to multiple mitochondrial metabolically active tissues such as cardiomyocytes and neurons of dysfunction syndrome, a condition characterized by defects in the dorsal root ganglia. FRDA presents early in adolescence with Complexes I, II, and III and pyruvate/a-ketoglutarate de- progressive ataxia, or difficulty coordinating movement, sensory hydrogenases. NFU1 is thought to be an alternative to ISCU as a loss, weakness, and dysarthria. FRDA patients are usually wheel- scaffold for FeeS assembly. Both BOLA3 and NFU1 appear to be chair bound in their teens with a significantly reduced quality of life involved in lipoate synthesis, possibly related to a role in assem- and life expectancy [180]. Median age of survival is 35 years with bling FeeS clusters in lipoic acid synthase (LIAS), thus providing an cardiac dysfunction usually being the cause of death [181]. explanation for the reduced PDH and aKGDH activities characteristic of this syndrome. The impaired energy production results in 3.6.2. ISCU myopathy lactic acidosis, encephalopathy [192] and early death. ISCU myopathy (IM) is an additional condition related to a defect in FeeS cluster biogenesis. It is the 2nd most common disorder 4. Summary linked to defective FeeS cluster synthesis but is much less common than FRDA with only 25 known cases. To date, all patients identified The utilization of complex Fe cofactors in biology requires a with IM have come from families of Swedish ancestry [121]. Similar tightly controlled process of cofactor assembly and of delivery to to FRDA, the IM phenotype is inherited in an autosomal recessive the correct apoprotein partner. Numerous ailments, some of which pattern. IM is caused by a splicing defect during ISCU post- are outlined in this review, result when there is a breakdown in the transcriptional processing that leads to defective ISCU protein assembly process or in delivery of the cofactor. The development of [182,183]. Loss of ISCU leads to lower ISC activity and a resulting treatment strategies for these disorders will require a more deficiency of essential FeeS proteins, including succinate advanced molecular understanding of each protein malfunction D.P. Barupala et al. / Archives of Biochemistry and Biophysics 592 (2016) 60e75 73 outlined above. 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