Coordination Chemistry of Microbial Iron Transport

Abstract

This Account focuses on the coordination chemistry of the microbial iron chelators called siderophores. The initial research (early 1970s) focused on simple analogs of siderophores, which included hydroxamate, catecholate, or hydroxycarboxylate ligands. The subsequent work increasingly focused on the transport of siderophores and their microbial iron transport. Since these are pseudo-octahedral complexes often composed of bidentate ligands, there is chirality at the metal center that in principle is independent of the ligand chirality. It has been shown in many cases that chiral recognition of the complex occurs. Many techniques have been used to elucidate the iron uptake processes in both Gram-positive (single membrane) and Gram-negative (double membrane) bacteria. These have included the use of radioactive labels (of ligand, metal, or both), kinetically inert metal complexes, and Mössbauer spectroscopy. In general, siderophore recognition and transport involves receptors that recognize the metal chelate portion of the iron-siderophore complex. A second, to date less commonly found, mechanism called the siderophore shuttle involves the receptor binding an apo-siderophore. Since one of the primary ways that microbes compete with each other for iron stores is the strength of their competing siderophore complexes, it became important early on to characterize the solution thermodynamics of these species. Since the acidity of siderophores varies significantly, just the stability constant does not give a direct measure of the relative competitive strength of binding. For this reason, the pM value is compared. The pM, like pH, is a measure of the negative log of the free metal ion concentration, typically calculated at pH 7.4, and standard total concentrations of metal and ligand. The characterization of the electronic structure of ferric siderophores has done much to help explain the high stability of these complexes. A new chapter in siderophore science has emerged with the characterization of what are now called siderocalins. Initially found as a protein of the human innate immune system, these proteins bind both ferric and apo-siderophores to inactivate the siderophore transport system and hence deny iron to an invading pathogenic microbe. Siderocalins also can play a role in iron transport of the host, particularly in the early stages of fetal development. Finally, it is speculated that the molecular targets of siderocalins in different species differ based on the siderophore structures of the most important bacterial pathogens of those species.

Figure 1

Chemical structure of siderophores with various iron-binding moieties; including catecholates (enterobactin, bacillibactin, petrobactin), hydroxamates (rhodotorulic acid, aerobactin, alcaligin), and carboxylates (petrobactin, aerobactin).

Figure 2

Chiralty of metal–siderophore complex. (A) Rhodotorulic acid forms a complex with two metals and three siderophores. (B) The E. coli ferrichrome receptor FhuE transports native rhodotorulic acid (Δ configuration) less than the unnatural enantiomer (Λ configuration). (C) Propeller chirality of Λ (left) and Δ (right) configurations.

Figure 3

Active site of the enterobactin hydrolase Fes. The crystal structure of Fes from Shigella flexneri (PDB entry 2B20) shows an active site buried deep within the enzyme scaffold (gray ribbon). The active site is composed of a putative oxyanion hole (yellow sticks) and catalytic (orange sticks) residues.

Figure 4

Siderophore uptake systems in E. coli. Siderophore uptake is both receptor and energy dependent. The outer membrane receptors are the most selective component of the systems. They have significantly different affinities or uptake rates for siderophores within the same class, for example, enterobactin and the enterobactin hydrolysis product, 2,3-dihydroxybenzoylserine (DBS).

Figure 5

Proposed model of the siderophore shuttle iron exchange mechanism for iron transport in Gram-negative bacteria. (A) In vivo, apo-siderophore (red) may often be in excess of the ferric siderophore (blue), and thus the cognate receptor is predominantly loaded with the apo-siderophore. (B) A ferric siderophore approaches the receptor-bound apo-siderophore and transfers a ferric ion in a mechanism likely facilitated by the receptor. (C) Iron-binding by the siderophore inside the receptor barrel induces a conformational change that signals the iron-loaded status. Energized TonB then triggers translocation of the ferric siderophore to the periplasm. (D) Finally, the receptor returns to its initial conformation bound to an apo-siderophore. Reproduced with permission from ref (31). Copyright 2000 National Academy of Sciences, U.S.A.

Figure 6

Models of the Gram-positive siderophore-shuttle mechanism and displacement mechanism of YxeB. YxeB is initially bound to an apo-siderophore. (1) An Fe–siderophore approaches YxeB and rests near the binding pocket occupied by the apo-siderophore. At this step, two pathways are possible. Steps 2–4 are the shuttle pathway. (2) Iron exchanges from the Fe–siderophore to the apo-siderophore in the binding pocket. The protein facilitates this step by increasing the local concentration of the entering ligand and the ferric complex. (3) The new Fe–siderophore (B) is transported and the created iron-released ligand (A) may remain bound by the YxeB protein. (4) The receptor is bound to an apo-siderophore. Steps 5–7 are the displacement pathway. (5) The Fe–siderophore displaces the apo-siderophore and occupies the binding pocket. (6) The original Fe–siderophore (A) is transported. (7) The SBP is bound to an apo-siderophore. In the Gram-positive siderophore-shuttle, both pathways operate but the shuttle pathway is preferred. Reproduced with permission from ref (32). Copyright 2014 American Chemical Society.

Figure 7

Catecholate (left) and salicylate (right) iron binding modes.

Figure 8

Siderocalin, the first human protein found to specifically bind siderophores. Reproduced with permission from the cover of Molecular Cell, vol 10, iss 5. Copyright 2002 Elsevier.

Figure 9

Siderocalin binding of 2,3-catechol amides versus 3,4-catechol amides. Shown at upper left is the protein calyx and its interaction with the iron catechol complex, with the detail shown at upper right. Below are shown the metal complexes and resultant binding by the protein. The intense red is due to the bound ferric complex, which is absent at lower right. Reproduced with permission from ref (49). Copyright 2006 National Academy of Sciences, U.S.A.

Figure 10

Bacillus cereus uses ferric citrate as one vehicle for iron delivery. FctC is a ferric-citrate siderophore binding protein of B. cereus; it binds Fe₃Cit₃ (K_d = 0.27 nM), although this is a trace species in solution. The structural model proposed has all carboxylate oxygens coordinated to the Fe(III) center (red is O, blue is Fe). The remaining ligands are from the protein receptor. Adapted with permission from ref (57). Copyright 2012 National Academy of Sciences, U.S.A.