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Bacterial iron detoxification at the molecular level

2020; Elsevier BV; Volume: 295; Issue: 51 Linguagem: Inglês

10.1074/jbc.rev120.007746

1083-351X

Justin M. Bradley, Dimitri A. Svistunenko, Michael T. Wilson, Andrew M. Hemmings, Geoffrey R. Moore, Nick E. Le Brun,

Trace Elements in Health

Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable “free” iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron. Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable “free” iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron. A great deal of the biological importance of iron stems from facile redox transformations between the Fe2+ and Fe3+ oxidation states that underpin its function as a cofactor in many enzymes. Iron-containing proteins are grouped into three main classes. Iron-sulfur clusters are thought to represent the oldest class of iron-containing cofactors. They typically consist of 2–4 iron ions (although occasionally more) but occasionally also contain a heterometal, such as nickel or molybdenum, linked by inorganic sulfide and covalently attached to the protein via the thiol groups of cysteine residues. These versatile cofactors are involved in many processes, including respiration, photosynthesis, nitrogen fixation, hydrogen evolution, and the associated electron transfer chains (1Beinert H. Holm R.H. Münck E. Iron-sulfur clusters: nature's modular, multipurpose structures.Science. 1997; 277 (9235882): 653-65910.1126/science.277.5326.653Crossref PubMed Scopus (1348) Google Scholar). The simplest iron-containing cofactors are formed by the binding of discrete metal ion to sites composed from the side chains of histidine and/or the carboxylates aspartate and glutamate. These are principally employed to harness the oxidizing power of O2 for processes such as DNA synthesis and methane oxidation (2Gamba I. Codolà Z. Lloret-Fillol J. Costas M. Making and breaking of the O-O bond at iron complexes.Coord. Chem. Rev. 2017; 334: 2-2410.1016/j.ccr.2016.11.007Crossref Scopus (41) Google Scholar). Heme is formed by the incorporation of iron into the tetrapyrrole protoporphyrin IX. This chemically versatile cofactor is critical in many processes, including respiration, cycling of nitrogen, and sulfur and detoxification reactions in addition to also supporting electron transfer (3Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions.Biochim. Biophys. Acta. 1995; 1232 (8534676): 97-17310.1016/0005-2728(95)00092-5Crossref PubMed Scopus (468) Google Scholar, 4Kappler U. Maher M.J. The bacterial SoxAX cytochromes.Cell. Mol. Life Sci. 2013; 70 (22907414): 977-99210.1007/s00018-012-1098-yCrossref PubMed Google Scholar, 5Poulos T.L. Heme enzyme structure and function.Chem. Rev. 2014; 114 (24400737): 3919-396210.1021/cr400415kCrossref PubMed Scopus (610) Google Scholar). As a result of this versatility, the demand for iron is large in most organisms, including the majority of bacteria, with up to 25% of the proteome binding iron in some form (6Zhang Y.F. Sen S. Giedroc D.P. Iron acquisition by bacterial pathogens: beyond Tris-catecholate complexes.Chembiochem. 2020; 21 (32180318): 1955-196710.1002/cbic.201900778Crossref PubMed Scopus (2) Google Scholar). However, the same redox chemistry required for these roles (Reaction 1 and the Fenton reaction, Reaction 2) allows iron to catalyze the Haber–Weiss reaction (Reaction 3). Fe3++O2˙¯↔Fe2++O2Reaction 1 Fe2++H2O2↔Fe3++−OH+•OHReaction 2 O2˙¯+H2O2↔−OH+•OH+O2Reaction 3 Reactions 1–3 The resulting hydroxyl radicals (•OH) are highly reactive, causing damage to lipids, proteins, carbohydrates, and nucleic acids (7Touati D. Iron and oxidative stress in bacteria.Arch. Biochem. Biophys. 2000; 373 (10620317): 1-610.1006/abbi.1999.1518Crossref PubMed Scopus (621) Google Scholar). Superoxide (O2˙¯) and hydrogen peroxide (H2O2) are produced as by-products of aerobic respiration (8Imlay J.A. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium.Nat. Rev. Microbiol. 2013; 11 (23712352): 443-45410.1038/nrmicro3032Crossref PubMed Scopus (715) Google Scholar), and, therefore, any aerobically respiring organism faces the requirement not only to detoxify ROS but also to strictly regulate the concentration of iron in any form able to catalyze the Haber–Weiss reaction. This need is particularly acute in the case of bacteria because, in addition to endogenously produced ROS, they are often subjected to assault by ROS produced either by competitors in the environment or in phagocytes produced by the immune system of hosts during infection (9Reniere M.L. Reduce, induce, thrive: bacterial redox sensing during pathogenesis.J. Bacteriol. 2018; 200 (29891640): e00128-1810.1128/jb.00128-18Crossref PubMed Google Scholar). Nitric oxide is known to play an important role as a signaling molecule in biological systems but is also produced in elevated concentrations for defense or competition in a similar manner to ROS. Combination of nitric oxide with superoxide generates the peroxynitrite ion that is susceptible to further oxidation to either nitrogen dioxide or dinitrogen trioxide. Collectively, these RNS can cause damage to nucleic acids and modify the side chains of amino acids such that protein structure and function are impaired (9Reniere M.L. Reduce, induce, thrive: bacterial redox sensing during pathogenesis.J. Bacteriol. 2018; 200 (29891640): e00128-1810.1128/jb.00128-18Crossref PubMed Google Scholar). Furthermore, both ROS and RNS are known to lead to breakdown of iron-sulfur clusters, resulting in the displacement of iron from the cofactor. Thus, iron homeostasis and the generation of ROS and RNS are intimately connected, as are the regulatory networks for their management within bacterial cells. When considering the iron status of cells, it is important to distinguish between the quota, which is the total iron content of the cell, and that subset of the quota that is kinetically available for insertion into proteins and molecular cofactors, referred to as the “labile iron pool” (10Chandrangsu P. Rensing C. Helmann J.D. Metal homeostasis and resistance in bacteria.Nat. Rev. Microbiol. 2017; 15 (28344348): 338-35010.1038/nrmicro.2017.15Crossref PubMed Scopus (185) Google Scholar). The majority of the latter is likely in the Fe2+ oxidation state and coordinated by small molecules, such as low-molecular weight thiols (11Chandrangsu P. Loi V.V. Antelmann H. Helmann J.D. The role of bacillithiol in Gram-positive firmicutes.Antioxid. Redox Signal. 2018; 28 (28301954): 445-46210.1089/ars.2017.7057Crossref PubMed Scopus (48) Google Scholar, 12Hider R.C. Kong X.L. Glutathione: a key component of the cytoplasmic labile iron pool.Biometals. 2011; 24 (21769609): 1179-118710.1007/s10534-011-9476-8Crossref PubMed Scopus (129) Google Scholar). This represents the fraction of the quota available to fulfill metabolic requirement, but also that with the potential to catalyze unwanted ROS and RNS formation. Therefore, the first requirement of any regulatory system for iron homeostasis is the ability to sense the concentration of the labile iron pool across the physiologically relevant range, 1–10 μm according to most estimates (13Keyer K. Imlay J.A. Superoxide accelerates DNA damage by elevating free-iron levels.Proc. Natl. Acad. Sci. U. S. A. 1996; 93 (8942986): 13635-1364010.1073/pnas.93.24.13635Crossref PubMed Scopus (625) Google Scholar, 14Jacques J.F. Jang S. Prévost K. Desnoyers G. Desmarais M. Imlay J. Massé E. RyhB small RNA modulates the free intracellular iron pool and is essential for normal growth during iron limitation in Escherichia coli.Mol. Microbiol. 2006; 62 (17078818): 1181-119010.1111/j.1365-2958.2006.05439.xCrossref PubMed Scopus (92) Google Scholar, 15Beauchene N.A. Mettert E.L. Moore L.J. Keleş S. Willey E.R. Kiley P.J. O2 availability impacts iron homeostasis in Escherichia coli.Proc. Natl. Acad. Sci. U. S. A. 2017; 114 (29087312): 12261-1226610.1073/pnas.1707189114Crossref PubMed Scopus (21) Google Scholar). As one might expect, this is achieved by transcriptional regulators whose affinities for target DNA are modulated by either binding directly to iron or by the binding of iron-dependent prosthetic groups. Often these are global regulators, controlling the expression of a great many genes, including those involved in the biosynthesis and import of siderophores, import of ferrous iron, and the storage and/or efflux of iron present in excess of cellular requirements. This balancing of metal trafficking to fulfill nutritional requirements while suppressing potential toxicity, shown schematically in Fig. 1, is termed “nutritional passivation” and is a common strategy that extends beyond iron metabolism (16Koh E.I. Robinson A.E. Bandara N. Rogers B.E. Henderson J.P. Copper import in Escherichia coli by the yersiniabactin metallophore system.Nat. Chem. Biol. 2017; 13 (28759019): 1016-102110.1038/nchembio.2441Crossref PubMed Scopus (43) Google Scholar). Members of the Fur (ferric uptake regulator) superfamily are the most widespread transcriptional regulators controlling iron homeostasis in bacteria. The first member of the Fur family was identified in Escherichia coli some 35 years ago (17Schäffer S. Hantke K. Braun V. Nucleotide-sequence of the iron reglulatory gene fur.Mol. Gen. Genet. 1985; 200 (2993806): 110-11310.1007/BF00383321Crossref PubMed Google Scholar) and, as the name suggests, was reported to regulate the intake of Fe3+ into the cell. This is achieved by the binding of the protein to “Fur boxes,” AT-rich binding sites upstream of the regulated genes with the consensus sequence 5′-GATAATGATAATCATTATC-3′. It has been argued that the Fur box should be considered a 21-bp fragment containing two overlapping 7-1-7 inverted repeats that each bind a Fur dimer. These are positioned such that the two copies of Fur bind to opposite faces of the DNA helix (18Baichoo N. Helmann J.D. Recognition of DNA by Fur: a reinterpretation of the Fur box consensus sequence.J. Bacteriol. 2002; 184 (12374814): 5826-583210.1128/jb.184.21.5826-5832.2002Crossref PubMed Scopus (205) Google Scholar). Binding of Fur occludes access of RNA polymerase, thus repressing transcription of the responsive genes (19Escolar L. de Lorenzo V. Pérez-Martín J. Metalloregulation in vitro of the aerobactin promoter of Escherichia coli by the Fur (ferric uptake regulation) protein.Mol. Microbiol. 1997; 26 (9427409): 799-80810.1046/j.1365-2958.1997.6211987.xCrossref PubMed Google Scholar). However, despite the great deal of research effort directed at members of the Fur superfamily, an understanding of these processes at the molecular level has only recently been achieved. Despite reports of both monomeric (20Pecqueur L. D'Autreaux B. Dupuy J. Nicolet Y. Jacquamet L. Brutscher B. Michaud-Soret I. Bersch B. Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and x-ray crystallography.J. Biol. Chem. 2006; 281 (16690618): 21286-2129510.1074/jbc.M601278200Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and higher oligomeric (21Pérard J. Covès J. Castellan M. Solard C. Savard M. Miras R. Galop S. Signor L. Crouzy S. Michaud-Soret I. de Rosny E. Quaternary structure of Fur proteins, a new subfamily of tetrameric proteins.Biochemistry. 2016; 55 (26886069): 1503-151510.1021/acs.biochem.5b01061Crossref PubMed Scopus (14) Google Scholar) forms of Fur detected in solution, the physiologically relevant form of the protein is thought to be the homodimer. This is stabilized by a large buried interface between C-terminal dimerization domains (22Pohl E. Haller J.C. Mijovilovich A. Meyer-Klaucke W. Garman E. Vasil M.L. Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator.Mol. Microbiol. 2003; 47 (12581348): 903-91510.1046/j.1365-2958.2003.03337.xCrossref PubMed Scopus (245) Google Scholar) and, in most cases, the binding of a structural Zn2+ (23Fuangthong M. Helmann J.D. Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis.J. Bacteriol. 2003; 185 (14563870): 6348-635710.1128/jb.185.21.6348-6357.2003Crossref PubMed Scopus (96) Google Scholar) ion by four conserved Cys residues (24Sarvan S. Charih F. Askoura M. Butcher J. Brunzelle J.S. Stintzi A. Couture J.F. Functional insights into the interplay between DNA interaction and metal coordination in ferric uptake regulators.Sci. Rep. 2018; 8 (29739988)741010.1038/s41598-018-25157-6Crossref PubMed Scopus (7) Google Scholar). Occupancy of this structural site (S1) is required, but not sufficient, for DNA binding. The Fur family exhibits some structural variation, and in certain examples, the dimerization domain harbors a second structural site ligated by His and Glu residues (25Sarvan S. Butcher J. Stintzi A. Couture J.F. Variation on a theme: investigating the structural repertoires used by ferric uptake regulators to control gene expression.Biometals. 2018; 31 (30014354): 681-704410.1007/s10534-018-0120-8Crossref PubMed Scopus (11) Google Scholar). The dimerization domain is connected to the N-terminal DNA-binding domain via a flexible hinge region containing a regulatory site comprising His and Glu side chains that binds Fe2+ with a reported dissociation constant, Kd, of ∼1 μm when determined in vitro (26Mills S.A. Marletta M.A. Metal binding characteristics and role of iron oxidation in the ferric uptake regulator from Escherichia coli.Biochemistry. 2005; 44 (16216078): 13553-1355910.1021/bi0507579Crossref PubMed Scopus (82) Google Scholar). Whereas the regulatory site has been demonstrated to bind other di- and trivalent metals, it is thought that only Fe2+ is present at the concentration required to activate the protein in vivo. Occupancy of this site induces a rotation of the DNA-binding domain relative to the dimerization domain, creating an increased void area between the two DNA-binding domains such that they are able to accommodate dsDNA (25Sarvan S. Butcher J. Stintzi A. Couture J.F. Variation on a theme: investigating the structural repertoires used by ferric uptake regulators to control gene expression.Biometals. 2018; 31 (30014354): 681-704410.1007/s10534-018-0120-8Crossref PubMed Scopus (11) Google Scholar). It is thought that this conformational change forms the molecular basis of the increased affinity of Fur for DNA in vitro under elevated concentrations of the regulatory metal. In vitro studies utilizing gel-shift methods report Kd values of ∼10 nm for complex formation between activated Fur and target DNA sequences (23Fuangthong M. Helmann J.D. Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis.J. Bacteriol. 2003; 185 (14563870): 6348-635710.1128/jb.185.21.6348-6357.2003Crossref PubMed Scopus (96) Google Scholar). The recently reported crystal structure of Magnetospirillum gryphiswaldense Fur (27Deng Z.Q. Wang Q. Liu Z. Zhang M.F. Machado A.C.D. Chiu T.P. Feng C. Zhang Q. Yu L. Qi L. Zheng J.G. Wang X. Huo X.M. Qi X.X. Li X.R. et al.Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator.Nat. Commun. 2015; 6 (26134419)764210.1038/ncomms8642Crossref PubMed Scopus (55) Google Scholar) in complex with DNA has provided insight into the molecular basis for recognition of Fur-binding sites (Fig. 2). The AT-rich composition of the Fur box results in a narrowing of the minor groove and consequent increase in negative charge density from the phosphate backbone that persists upon repressor binding. This facilitates shape recognition by Fur via a favorable electrostatic interaction between a conserved lysine residue (Lys-15 in M. gryphiswaldense Fur numbering) and the minor groove. More specific interactions with bases in the major groove are facilitated by the rotation of the DNA-binding domains induced by metal binding at the regulatory site. This involves van der Waals interactions between Tyr-56 and consecutive thymine bases in the target sequences and hydrogen bonding between the guanidinium group of Arg-57 and the O6 and N7 atoms of a conserved guanine. A recent report suggests that Fur DNA binding can be tuned by protein-protein interactions (28Choi J. Ryu S. Regulation of iron uptake by fine-tuning the iron responsiveness of the iron sensor Fur.Appl. Environ. Microbiol. 2019; 85 (30824449): e03026-1810.1128/aem.03026-18Crossref PubMed Scopus (2) Google Scholar), in addition to the long-recognized effect of iron binding. EIIANtr, a component of the nitrogen metabolic phosphotransferase system, was shown to affect expression of Fur-regulated genes. In vitro gel shift measurements showed that this arises from formation of a protein-protein complex that lowers the affinity of holo-Fur for DNA. Consequently, repression of Fur-regulated genes requires a greater cytoplasmic Fe2+ concentration when EIIANtr is present. The Kd for the Fur-EIIANtr complex has not yet been determined; nor has the increase in Kd of the Fur-DNA complex in the presence of EIIANtr. In addition to the classic gene repression mechanism described above, Fur has been shown to act as an activator of gene expression, both directly (29Delany I. Rappuoli R. Scarlato V. Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis.Mol. Microbiol. 2004; 52 (15130126): 1081-109010.1111/j.1365-2958.2004.04030.xCrossref PubMed Scopus (0) Google Scholar, 30Seo S.W. Kim D. Latif H. O'Brien E.J. Szubin R. Palsson B.O. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli.Nat. Commun. 2014; 5 (25222563)491010.1038/ncomms5910Crossref PubMed Scopus (127) Google Scholar, 31Yu C.X. Genco C.A. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.J. 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The Fur-like protein Mur of Rhizobium leguminosarum is a Mn2+-responsive transcriptional regulator.Microbiology. 2004; 150 (15133106): 1447-145610.1099/mic.0.26961-0Crossref PubMed Scopus (0) Google Scholar), and Zur (43Fillat M.F. The Fur (ferric uptake regulator) superfamily: diversity and versatility of key transcriptional regulators.Arch. Biochem. Biophys. 2014; 546 (24513162): 41-5210.1016/j.abb.2014.01.029Crossref PubMed Scopus (156) Google Scholar), the zinc uptake regulator) and to peroxide-induced oxidative stress (Per) (23Fuangthong M. Helmann J.D. Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis.J. Bacteriol. 2003; 185 (14563870): 6348-635710.1128/jb.185.21.6348-6357.2003Crossref PubMed Scopus (96) Google Scholar). Genes identified as being regulated by Fur, such as that in E. coli, include those encoding iron-uptake systems, such as fhu, fec, and feo; the suf iron-sulfur cluster assembly system; iron-sulfur–containing proteins, such as fumA, acnA, acnB, and nuo; the iron-containing superoxide dismutase sodB; and the iron storage proteins bfr and ftnA (see below). Consistent with its role as a repressor of iron import systems, the transcriptional response of a Fur deletion mutant is similar to that evoked by iron limitation, even under iron-replete conditions. This inability to correctly sense the iron status of the cell has been demonstrated to result in an increase in ROS production (44Touati D. Jacques M. Tardat B. Bouchard L. Despied S. Lethal oxidative damage and mutagenesis are generated by iron in Δfur mutants of Escherichia coli—protective role of superoxide dismutate.J. 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