Coordination Chemistry of Biological Iron Transport
essentially all single-celled organisms require iron for growth, even
the biological availability of iron is extremely limited by the
iron hydroxide. To overcome the poor availability and potential
iron and achieve effective homeostasis in aqueous aerobic conditions,
must tightly regulate their iron balance.
mammals use the serum
protein transferrin to transport iron through the blood to cells and
protein shell ferritin for its storage, micro-organisms synthesize
low-molecular-weight chelating agents called siderophores which bind
solubilize iron. Characterized by their high specificity and affinity
ferric ion, siderophores are exported from the cell in their apo form
remove iron from minerals and host iron-binding proteins to form stable
complexes that are transported into bacterial cells through specific
of iron from
environmental or host sources by siderophores and incorporation of the
iron-siderophore complex into the cell are often the rate limiting
bacterial growth and pathogenesis. Understanding siderophore chemistry
(including the coordination chemistry of the ferric-siderophore
kinetics of iron acquisition from a host, and bacterial uptake
therefore of direct medical relevance. One specific aim of our iron
to characterize the mechanisms of siderophore-mediated iron uptake and
understand the role of siderophores in bacterial diseases. We are also
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
recent isolation of
bacillibactin (BB) from manyBacillispecies has provided a
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
bonds. While Ent is formed with a triserine backbone, BB incorporates
spacers between the catecholate groups and a threonine-based lactone.
forms of the siderophores exported from the bacterial cells are
iron binding; coordinated to ferric ion, both Ent and BB form very
1049and 1047.6respectively) with the
characteristic Δ and Λ configurations at the metal center. Ent and BB
highest known affinities for iron, which makes their structure, iron
coordination and iron transport pathways interesting, complex and
in the context of bacterial iron transport.
Trilactone-based natural siderophores
of the seemingly perfect
enterobactin structure invites many questions regarding the effect of
alterations on the uptake and stability of ferric bacillibactin as
ferric enterobactin. Several analogs of these trilactone-based
have been synthesized to probe the effect of the spacer and trilactone
structure on iron complex stability as well as on transport in
and positive bacteria.
work focuses on
structural characterization of the apo- and ferric-forms of these
siderophores and their synthetic analogs. We are also investigating the
of structure modifications on transport in Gram-negative and positive
Zawadzka, A.M.; Hoette, T.M.; Raymond, K.N. “Enzymatic Hydrolysis of
Siderophores: Where Chiral Recognition Occurs in Enterobactin and
Iron Transport.” J. Am Chem. Soc.2009, 131, 12682–12692.
Zawadzka, A. M.; Raymond, K. N. "Petrobactin-Mediated Iron Transport in
Pathogenic Bacteria: Coordination Chemistry of an Unusual
Chem. Soc.2008,130, 2124-2125.
Warner, J. A.; Shuh, D. K.; Raymond, K. N. "Enterobactin Protonation
Iron Release: Structural Characterization of the Salicylate
in Ferric Enterobactin."J.
Chem. Soc.2006,128, 8920-8931.
Abergel, R. J.; Raymond, K. N.; Arceneaux, J. E. L.; Byers, B. R.
Biophys. Res. Commun.2006,348, 320-325.
Siderophore Mediated Iron Transport
of our studies with
synthetic analogs of natural siderophores have established important
correlations between the coordination chemistry of the ferric complex
recognition at the outer membrane of Gram-negative (double cell
organisms such asE.
hydrophila. Siderophore receptor proteins on the outer membrane
specifically iron siderophore complexes and energy from the cytoplasm
shuttled to the outer membrane through intermediate proteins. We have
shown that metal exchange between two siderophores was essential for
transport. The ligand exchange step occurs at the cell surface and
transfer of iron from a ferric-siderophore to an iron-free siderophore
its receptor. This shuttle mechanism implies an increase in the iron
rate with increasing concentrations of iron-free siderophore, a
of ferric-siderophore poor environmentsin
Proposed model of the siderophore
shuttle iron exchange mechanism for iron transport in Gram-negative
most studies about
bacterial iron transport have been performed with Gram-negative
little is known about siderophore-mediated iron transport in Gram-positiveorganisms.
Many human pathogens are Gram-positive
organisms, having only one cell wall and potentially different iron
machinery and pathways.What
understand thus far is that the iron transport system in Gram-positive
consists of siderophore-binding proteins, permeases and ATPases,
similar to the
machinery seen in the periplasm and inner membrane of Gram-negative
are now interested in
characterizing the mechanisms of siderophore-mediated iron uptake in
representative Gram-positive bacteria such asB.
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. USA2000, 97,
Dertz, E. A.; Stintzi, A.; Raymond, K. N.
"Siderophore-mediated Iron Transport in B. subtilis and C.
glutamicum." J. Biol. Inorg. Chem.2006, 11,
and “Stealth” Siderophores
bacteria such as Bacillus cereus andBacillusanthracis are
known to secrete stealth siderophores, or siderophores which are not
by the human immune system protein siderocalin (see Fig. “The arms race
the mammalian immune system and bacteria in the search for iron”).
siderophores are necessarily secreted by some of the deadliest
fulfill their iron requirement and ensure full virulence. Recently,we have
characterized and studied some of the first siderophore-binding
proteins inBacillusinvolved insiderophore-mediated iron
is a stealth siderophore of B. anthracis
and is also produced by B. cereus. We
have crystallized a petrobactin-binding protein, YclQ, whose structure
insight to the requirements and understanding of stealth siderophore
work focuses on identifying and characterizing
the components of petrobactin-uptake systems in B. cereus and
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
Petrobactin.” Biochemistry2009, 48,
Zawadzka, A.M.; Kim, Y.; Maltseva, N.;
Nichiporuk, R.; Fan,
Y.; Joachimiak, A.; Raymond, K.N. “Characterization of a Bacillus
Transporter for Petrobactin, an Anthrax Stealth Siderophore.” Proc.
Acad. Sci. USA2009, 106, 21854-21859.
Bacterial Siderophore-Human Protein Interaction
binding of enterobactin by
siderocalin (formally NGAL) is the first indication that human proteins
produced to bind siderophores as an immunoresponse. Just as some
receptors can bind a variety of siderophores, the generality of
interaction is a major aspect of this project.
The first human protein-
siderocalin binds can help elucidate the relevant molecular
the function of the protein as an antagonist for siderophore-mediated
transport. The specificity of siderocalin for a variety of siderophores
analogs is being probed in this collaboration with the research group
enterobactin with an affinity close to that of the bacterial outer
receptor protein, FepA, however, the binding specificity of siderocalin
notably from that of FepA. We have determined that the binding of
enterobactin by siderocalin is not significantly altered by changing
center, the siderophore scaffold or the chirality of the metal complex,
substitution of the 5' position on the catecholate rings can introduce
steric hindrance to preclude protein binding.
can then act as an
innate immune response to siderophore-mediated iron acquisition by
siderophores such as enterobactin and bacillibactin, found in enteric
great efficiency of these catecholate ligands at chelating iron,
synthesis of additional stealth siderophores like aerobactin,
petrobactin, which can evade siderocalin binding, is necessary to
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).
work in our lab focuses
on synthesizing siderophore analogs to further probe the specificity of
protein and to characterize spectroscopically the interactions between
protein and its substrates. We are also investigating the effect of
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,
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. Cell2002,
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. Nature2004,
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. USA2006, 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. USA2006,
Siderophores and Siderocalin
from its bacteriostatic
role, siderocalin is upregulated in other mammalian processes such as
kidney development, apoptosis, and ischemia. From our studies of the
interaction between bacterial siderophores and siderocalin, we have
siderocalin does not bind Fe3+ alone and requires an
an iron chelator or siderophore. In cases where non-bacterial stimuli
the expression of siderocalin, we suspect that an equivalent of a
siderophore exists in order to optimize the role that siderocalin plays
regulation. While mammals do not synthesize their own siderophores,
and mammalian catecholate metabolites are often found in the body.
catechols are similar to the binding moieties of natural bacterial
such as enterobactin and bacillibactin.
have shown that simple
catechols isolated from human urine exhibit proportional increases in
concentration along with siderocalin expression in urine. Ongoing
dedicated to finding mammalian siderophores that bind iron and
contributing to our understanding of the role of siderocalin in
non-bacteriostatic mammalian processes.
Clifton, M.; Hoette, T.M.; Mori, K.; Deng, S.; Qiu, A.; Viltard, M.;
D.; Paragas, N.; Leete, T.; Kulkarni, R.; Li, X.; Lee, B.; Kalandadze,
Ratner, A.J.; Pizarro, J.C.; Schmidt-Ott, K.M.; Landry, D.W.; Raymond,
Strong, R.K.; Barasch, J, “Iron traffics in circulation bound to a
(Ngal)–catechol complex.” Nature Chemical Biology, 2010,
Some of our HOPO- and TAM-based promising
ligands for iron chelation therapy
are currently interested in
modifying our ligand design to achieve higher oral activities. Further
trials will be designed to trace the path taken by the ligands and
the origin of the removed iron. A collaboration with the Francis
at UC Berkeley was recently started to investigate new ways of
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.
Raymond, K. N. "Synthesis and thermodynamic evaluation of mixed
hexadentate linear iron chelators containing hydroxypyridinone and
Raymond, K. N. "Terephthalamide-Containing Ligands: Fast Removal of
Inorg. Chem.2008,13, 229-240.
Fred Hutchinson Cancer Research Center (FHCRC), Seattle, WA, USA
University of Mississippi Medical Center (UMMC), Jackson, MS, USA