Raymond Group Research

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Coordination Chemistry of Biological Iron Transport Agents

All multi-cellular and essentially all single-celled organisms require iron for growth, even though the biological availability of iron is extremely limited by the insolubility of iron hydroxide. To overcome the poor availability and potential toxicity of iron and achieve effective homeostasis in aqueous aerobic conditions, organisms must tightly regulate their iron balance.

While mammals use the serum protein transferrin to transport iron through the blood to cells and the protein shell ferritin for its storage, micro-organisms synthesize low-molecular-weight chelating agents called siderophores which bind and solubilize iron. Characterized by their high specificity and affinity towards ferric ion, siderophores are exported from the cell in their apo form and can remove iron from minerals and host iron-binding proteins to form stable ferric complexes that are transported into bacterial cells through specific receptor proteins.

Removal of iron from environmental or host sources by siderophores and incorporation of the iron-siderophore complex into the cell are often the rate limiting factors in bacterial growth and pathogenesis. Understanding siderophore chemistry (including the coordination chemistry of the ferric-siderophore complex, kinetics of iron acquisition from a host, and bacterial uptake mechanisms) is therefore of direct medical relevance. One specific aim of our iron project is to characterize the mechanisms of siderophore-mediated iron uptake and to understand the role of siderophores in bacterial diseases. We are also applying our understanding of siderophore chemistry to the design of therapeutic synthetic iron chelators for the treatment of iron-overload diseases.

Siderophore Structure and Coordination Chemistry

The recent isolation of bacillibactin (BB) from many Bacilli species has provided a Gram-positive counterpart to the siderophore archetype produced by enteric bacteria, enterobactin (Ent). In the molecular structures of Ent and BB, three 2,3-catecholate moieties are linked to a trilactone backbone through amide bonds. While Ent is formed with a triserine backbone, BB incorporates glycine spacers between the catecholate groups and a threonine-based lactone. The apo forms of the siderophores exported from the bacterial cells are predisposed for iron binding; coordinated to ferric ion, both Ent and BB form very stable complexes (Kf = 1049 and 1047.6 respectively) with the respective characteristic Δ and Λ configurations at the metal center. Ent and BB have the highest known affinities for iron, which makes their structure, iron coordination and iron transport pathways interesting, complex and fascinating in the context of bacterial iron transport.

Trilactone-based natural siderophores

Modification of the seemingly perfect enterobactin structure invites many questions regarding the effect of the alterations on the uptake and stability of ferric bacillibactin as compared to ferric enterobactin. Several analogs of these trilactone-based siderophores have been synthesized to probe the effect of the spacer and trilactone structure on iron complex stability as well as on transport in Gram-negative and positive bacteria.

Current work focuses on structural characterization of the apo- and ferric-forms of these natural siderophores and their synthetic analogs. We are also investigating the effects of structure modifications on transport in Gram-negative and positive bacteria.

Abergel, R.J.; Zawadzka, A.M.; Hoette, T.M.; Raymond, K.N. “Enzymatic Hydrolysis of Trilactone Siderophores: Where Chiral Recognition Occurs in Enterobactin and Bacillibactin Iron Transport.”  J. Am Chem. Soc. 2009, 131, 12682–12692.

Abergel, R. J.; Zawadzka, A. M.; Raymond, K. N. "Petrobactin-Mediated Iron Transport in Pathogenic Bacteria: Coordination Chemistry of an Unusual 3,4-Catecholate/Citrate Siderophore." J. Am. Chem. Soc. 2008, 130, 2124-2125.

Abergel, R. J.; Warner, J. A.; Shuh, D. K.; Raymond, K. N. "Enterobactin Protonation and Iron Release: Structural Characterization of the Salicylate Coordination Shift in Ferric Enterobactin." J. Am. Chem. Soc. 2006, 128, 8920-8931.

Wilson, M. K.; Abergel, R. J.; Raymond, K. N.; Arceneaux, J. E. L.; Byers, B. R. "Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis." Biochem. Biophys. Res. Commun. 2006, 348, 320-325.

Siderophore Mediated Iron Transport

Most of our studies with synthetic analogs of natural siderophores have established important correlations between the coordination chemistry of the ferric complex and recognition at the outer membrane of Gram-negative (double cell membrane) organisms such as E. coli and A. hydrophila. Siderophore receptor proteins on the outer membrane bind specifically iron siderophore complexes and energy from the cytoplasm is shuttled to the outer membrane through intermediate proteins. We have also shown that metal exchange between two siderophores was essential for iron transport. The ligand exchange step occurs at the cell surface and involves the transfer of iron from a ferric-siderophore to an iron-free siderophore bound to its receptor. This shuttle mechanism implies an increase in the iron uptake rate with increasing concentrations of iron-free siderophore, a characteristic of ferric-siderophore poor environments in vivo.

  Proposed model of the siderophore shuttle iron exchange mechanism for iron transport in Gram-negative bacteria.  

While most studies about bacterial iron transport have been performed with Gram-negative organisms, very little is known about siderophore-mediated iron transport in Gram-positive organisms. Many human pathogens are Gram-positive organisms, having only one cell wall and potentially different iron transport machinery and pathways. What we understand thus far is that the iron transport system in Gram-positive bacteria consists of siderophore-binding proteins, permeases and ATPases, similar to the machinery seen in the periplasm and inner membrane of Gram-negative organisms.

We are now interested in characterizing the mechanisms of siderophore-mediated iron uptake in some representative Gram-positive bacteria such as B. subtilis and B. cereus.

Stintzi, A.; Barnes, C.; Xu, J.; Raymond, K. N. "Microbial Iron Transport via a Siderophore Shuttle: A Membrane Ion Transport Paradigm." Proc. Natl. Acad. Sci. USA 2000, 97, 10691-10696.

Dertz, E. A.; Stintzi, A.; Raymond, K. N. "Siderophore-mediated Iron Transport in B. subtilis and C. glutamicum." J. Biol. Inorg. Chem. 2006, 11, 1087-1097.

Pathogenic Bacterial Siderophores and “Stealth” Siderophores

Pathogenic bacteria such as Bacillus cereus and Bacillus anthracis are known to secrete stealth siderophores, or siderophores which are not recognized by the human immune system protein siderocalin (see Fig. “The arms race between the mammalian immune system and bacteria in the search for iron”). Stealth siderophores are necessarily secreted by some of the deadliest pathogens to fulfill their iron requirement and ensure full virulence. Recently, we have isolated, characterized and studied some of the first siderophore-binding proteins in Bacillus involved in siderophore-mediated iron uptake. Petrobactin is a stealth siderophore of B. anthracis and is also produced by B. cereus. We have crystallized a petrobactin-binding protein, YclQ, whose structure gives insight to the requirements and understanding of stealth siderophore uptake.

Current work focuses on identifying and characterizing the components of petrobactin-uptake systems in B. cereus and B. anthracis.

Ribbon drawing of YclQ, a substrate binding protein crystallized from Bacillus subtilis

Zawadzka, A.M.; Abergel, R.J.; Nichiporuk, R.; Andersen, U.N.; Raymond, K.N. “Siderophore-Mediated Iron Acquisition Systems in Bacillus cereus: Identification of Receptors for Anthrax Virulence-Associated Petrobactin.” Biochemistry 2009, 48, 3645-3657.


Zawadzka, A.M.; Kim, Y.; Maltseva, N.; Nichiporuk, R.; Fan, Y.; Joachimiak, A.; Raymond, K.N. “Characterization of a Bacillus subtilis Transporter for Petrobactin, an Anthrax Stealth Siderophore.” Proc. Natl. Acad. Sci. USA 2009, 106, 21854-21859.

Bacterial Siderophore-Human Protein Interaction

The binding of enterobactin by siderocalin (formally NGAL) is the first indication that human proteins may be produced to bind siderophores as an immunoresponse. Just as some siderophore receptors can bind a variety of siderophores, the generality of siderocalin-siderophore interaction is a major aspect of this project. 

The first human protein-
siderophore interaction

Determining which siderophores siderocalin binds can help elucidate the relevant molecular interactions and the function of the protein as an antagonist for siderophore-mediated iron transport. The specificity of siderocalin for a variety of siderophores and analogs is being probed in this collaboration with the research group of Dr. Roland Strong.

Siderocalin binds ferric enterobactin with an affinity close to that of the bacterial outer membrane receptor protein, FepA, however, the binding specificity of siderocalin differs notably from that of FepA. We have determined that the binding of ferric enterobactin by siderocalin is not significantly altered by changing the metal center, the siderophore scaffold or the chirality of the metal complex, but substitution of the 5' position on the catecholate rings can introduce enough steric hindrance to preclude protein binding.

Siderocalin can then act as an innate immune response to siderophore-mediated iron acquisition by sequestering siderophores such as enterobactin and bacillibactin, found in enteric bacteria and Bacilli species, respectively. Despite the great efficiency of these catecholate ligands at chelating iron, independent synthesis of additional stealth siderophores like aerobactin, salmochelin or petrobactin, which can evade siderocalin binding, is necessary to ensure full virulence of the lethal pathogens.

  The arms race between the mammalian immune system and bacteria in the search for iron: enterobactin removes iron from transferrin (a), siderocalin intercepts the ferric complex of enterobactin (b), bacteria produce alternative siderophores such as salmochelin S4 (c).  

Current work in our lab focuses on synthesizing siderophore analogs to further probe the specificity of the protein and to characterize spectroscopically the interactions between the protein and its substrates. We are also investigating the effect of siderocalin and similar proteins on the growth of different human pathogens.

Hoette, T.M.; Abergel, R.J.; Xu, J.; Strong, R.K.; Raymond, K.N. “The Role of Electrostatics in Siderophore Recognition by the Immunoprotein Siderocalin.” J. Am. Chem. Soc. 2008, 130, 17584-17592.

Goetz, D. H.; Holms, M. A.; Borregaard, N.; Bluhm, M. E.; Raymond, K. N.; Strong, R. K. "The Neutrophil Lipocalin NGAL is a Bacteriostatic Agent that Interferes with Siderophore-Mediated Iron Acquisition." Mol. Cell 2002, 10, 1033-1043. (Cover article.)

Flo, T. H.; Smith, K. D.; Sato, S.; Rodriguez, D. J.; Holmes, M. A.; Strong, R. K.; Akira, S.; Aderem, A. Nature 2004, 432, 917-921.

Abergel, R. J.; Moore, E. G.; Strong, R. K.; Raymond, K. N. "Microbial Evasion of the Immune System: Structural Modifications of Enterobactin Impair Siderocalin Recognition." J. Am. Chem. Soc. 2006, 128, 10998-10999.

Fischbach, M. A.; Lin, H.; Zhou, L.; Yu, Y.; Abergel, R. J.; Liu, D. R.; Raymond, K. N.; Wanner, B. L.; Strong, R. K.; Walsh, C. T.; Aderem, A.; Smith, K. D. "The Pathogen-associated IroA Gene Cluster Mediates Bacterial Evasion of Lipocalin 2." Proc. Nat. Acad. Sci. USA 2006, 103, 16502-16507.

Abergel, R. J.; Wilson, M. L.; Arceneaux, J. E. L.; Hoette, T. M.; Strong, R. K.; Byers, B. R.; Raymond, K. N. "The Anthrax Pathogen Evades the Mammalian Immune System Through Stealth Siderophore Production." Proc. Natl. Acad. Sci. USA 2006, 103, 18499-18503.

Mammalian Siderophores and Siderocalin

Aside from its bacteriostatic role, siderocalin is upregulated in other mammalian processes such as embryonic kidney development, apoptosis, and ischemia. From our studies of the interaction between bacterial siderophores and siderocalin, we have shown that siderocalin does not bind Fe3+ alone and requires an equivalent of an iron chelator or siderophore. In cases where non-bacterial stimuli induce the expression of siderocalin, we suspect that an equivalent of a mammalian siderophore exists in order to optimize the role that siderocalin plays in iron regulation. While mammals do not synthesize their own siderophores, bacterial and mammalian catecholate metabolites are often found in the body. These simple catechols are similar to the binding moieties of natural bacterial siderophores such as enterobactin and bacillibactin.

We have shown that simple catechols isolated from human urine exhibit proportional increases in concentration along with siderocalin expression in urine. Ongoing studies are dedicated to finding mammalian siderophores that bind iron and siderocalin, contributing to our understanding of the role of siderocalin in non-bacteriostatic mammalian processes.

Bao, G.; Clifton, M.; Hoette, T.M.; Mori, K.; Deng, S.; Qiu, A.; Viltard, M.; Williams, D.; Paragas, N.; Leete, T.; Kulkarni, R.; Li, X.; Lee, B.; Kalandadze, A.; Ratner, A.J.; Pizarro, J.C.; Schmidt-Ott, K.M.; Landry, D.W.; Raymond, K.N.; Strong, R.K.; Barasch, J, “Iron traffics in circulation bound to a siderocalin (Ngal)–catechol complex.” Nature Chemical Biology, 2010, 6, 602-609.

Therapeutic Iron Chelators

Treatments of blood-transfusion requiring diseases such as β-thalassemia, hemochromatosis or sickle cell anemia generate an iron overload condition in humans. Such serious clinical condition can be treated by iron chelation therapy effectively, but the goal of developing readily available, orally-active, effective and non-toxic sequestering agents has proven to be remarkably difficult to achieve. Desferrioxamine (Desferal©), a trihydroxamate ligand, remains the iron chelator of clinical use in the United States. Desferal© is expensive, has a short half-life in vivo, does not efficiently remove iron from transferrin, must be given on a regular, frequent basis by a subcutaneous or intravenous route, and its use can result in significant, irreversible toxicity. While other agents such as the tridentate chelator Deferasirox©, developed by Novartis, are being investigated as potential successors to Desferal©, orally active multidentate chelators that can bind iron more efficiently at lower doses are still desperately needed. The goal of this project is to develop new sequestering agents that would meet that need.

Iron chelators currently approved in the United States

The Raymond group has designed numbers of siderophore-like new iron chelators using hydroxypyridonates (HOPO) and 2,3-dihydroxyterephthalamide (TAM) binding units associated to polyamine backbones. Solution thermodynamics have shown that the affinities of our HOPO- and TAM-based ligands for ferric ion are among the highest for known iron chelators. Kinetics of iron removal from transferrin as well as lipophilicity measurements have also confirmed the high potential of these compounds as iron chelators. Our latest animal trials were designed to assess the therapeutic properties of these new ligands, with promising results since most of them proved more efficient than Desferal© at removing radiolabeled 59Fe in vivo.

Some of our HOPO- and TAM-based promising ligands for iron chelation therapy

We are currently interested in modifying our ligand design to achieve higher oral activities. Further animal trials will be designed to trace the path taken by the ligands and determine the origin of the removed iron. A collaboration with the Francis research group at UC Berkeley was recently started to investigate new ways of delivering our iron chelators in vivo.

Jurchen, K. M. C.; Raymond, K. N. "A bidentate terephthalamide ligand, TAMmeg, as an entry into terephthalamide-containing therapeutic iron chelating agents." Inorg. Chem. 2006, 45, 2438-2447.

Abergel, R. J.; Raymond, K. N. "Synthesis and thermodynamic evaluation of mixed hexadentate linear iron chelators containing hydroxypyridinone and terephthalamide units." Inorg. Chem. 2006, 45, 3622-3631.

Abergel, R. J.; Raymond, K. N. "Terephthalamide-Containing Ligands: Fast Removal of Iron from Transferrin." J. Biol. Inorg. Chem. 2008, 13, 229-240.


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