Enterobactins iron complexes

Once the siderophore-iron complexes are inside the bacteria, the iron is released and utilized for vital cell functions. The iron-free hydroxamate siderophores are commonly re-excreted to bring in an additional iron load (Enterobactin is at least partially degraded by a cytoplasmic esterase This cycle is repeated until specific intracellular ferric uptake regulation proteins (Fur proteins) bind iron, and signal that the intracellular iron level is satisfactory, at -which point ne-w siderophore and siderophore-receptor biosynthesis are halted and the iron-uptake process stops. This intricate feedback mechanism allows a meticulous control over iron(III) uptake and accumulation against an unfavorable concentration gradient so as to maintain the intracellular iron(III) level within the required narrow window. Several excellent reviews concerning siderophore-iron transport mechanisms have been recently published i.3,i6, is,40,45,60-62 ... 

The best known example is enterobactin (otherwise called enterochelin), which is produced apparently by all enteric bacteria. It has three 2,3-dihydroxybenzoyl groups attached to a macrocyc-lic lactone derived from three residues of L-serine condensed head-to-tail. The structures of enterobactin and its iron complex are shown in Figure 45, which shows that the iron is bound by six phenolate oxygen atoms in an octahedral environment. Enterobactin has the highest known affinity for Fem, with log K = 52 at pH 7.4.1182 The iron(III) complex can exist as isomeric forms, which may be associated with selectivity in binding to the receptor site. 

It can be seen from molecular models that two diastereoisomers are possible for the ferric enterobactin complex, A-cis and A-cis. These are not mirror images because of the optical activity of the ligand. The similarity of the roles played by the ferrichromes and enterobactin lent additional speculative interest to the preferred absolute configuration of the iron complex (20). The structural studies of the tris catechol complexes (vide infra) and the spectroscopic properties of the chromic... 

For the ferric siderophore complexes, comparison of the CD spectra of the chromium complexes of ferrichrome and enterobactin with the CD spectra of their iron complexes [and the separation of optical isomers of even ferric(benzhydroxamate)3 complexes in nonaqueous solution 192)] have shown that the same rule applied to the CD spectra for chromium complexes can be used for iron siderophore complexes as well iron(III) complexes will have a predominant A configuration in solution if the CD band in... 

Thus, we can have an isotropic g = 4.3 even for near axial symmetry (A- -0) as long as fi =3/4. Over the years, g =4.3 has come to be known as the signature for extreme Rhombic iron complexes, but now it is clear that this is not necessarily so. Several experimental examples such as enterobactin and mycobactin-P have shown this to be true. However, the pure axial case with E=0 will still give the electronic states as described in Fig. 6a. 

N, 6-N-di(2,3-dihydroxybenzoyl)-L-lysine (58) is a siderophore produced by Azotobacter vinelandii which has only two catechol groups. However, of the catecholate siderophores by far the best studied is enterobactin. A major difference between hydroxamate and catecholate siderophores occurs in their utilization as transport agents. For the former, the iron complex is taken up by the bacterial cell, the iron released, and the hydroxamate siderophore re-secreted for additional iron chelation. In contrast, enterobactin is destroyed by enzymatic hydrolysis within the cell and therefore the ligand is not recycled. This hydrolysis of the amide linkages of the iron(III) enterobactin lowers the redox potential of the chelate complex sufficiently to allow iron reduction — and thus uptake of iron into the cell metabolism (59, 60). 

In addition to being an indirect measurement of the formation constant (Ki) of the iron complex, the reduction potential of the ferric siderophore complex is an important factor in developing the iron-release mechanism for siderophore-mediated iron transport. Under standard conditions, the reduction potentials for most known siderophores (ferric enterobactin —750 mV NHE V" ferriferrioxamine B 450mV NHE ) seem to preclude the use of biological reduc-tants (NAD(P)H/NAD(P)+ —324mV NHE ) to reduce the ferric ion to the ferrous ion and therefore prompt release of the ion from the siderophore. However, this potential is highly sensitive to the ratio of [Fe +]/[Fe +], as predicted by the Nernst equation. 

C. Net Electrical Charge. The iron complexes of agrobactin, agrobactin A, parabactin and enterobactin were prepared by neutralization of the ligands in the presence of ferric chloride and their electrophoretic mobilities compared with that of ferrichrome... 

Althou we have characterized the siderophore of P. denitri-ficans as parabactin (Figure 2, R=H) and not parabactin A (Figure 1, R=H), there is some question as to which form was isolated from the same organism by Tait ( 5 ) The relative stability of the oxazoline to acid hydrolysis, the spectral shifts observed by Tait in acidic media and not found in our parabactin A ( 8), and the properties he ascribes to the iron complex can only be reconciled with the structure in Figure 2, R H. Although crude preparations of ferric enterobactin contain a number of colored species, the reddish form is the tris-catecholate bluish tints are associated with oxidized/polymerized or otherwise coordinated forms of iron ( 23). Parabactin A yields a relatively inferior complex with ferric ion which fails to develop a red color even at quite alkaline pH. 

The potentiometric titration curve of ferric enterobactin, shown in Figure 3, has a sharp inflection after the addition of six equivalents of base. Such a break indicates that the six phenolic oxygens from the three dihydroxybenzoyl groups are displaced by ferric ion in the ferric enterobactin complex. This interpretation is further supported by the absorbance maximum at 490 nm (e 5600), which is very similar to simple tris(catecho-lato)iron(III) complexes (, T). The very low pH at which com-plexatlon of enterobactin occurs, with virtually complete complex formation by pH 6, is a strong indication of a very stable complex. However, the titration is prematurely terminated at pH 3.8 by the precipitation of a purple neutral iron complex (whose composition and structure will be discussed later) which makes it impossible to determine the stability consteint of ferric enterobactin from potentiometric data alone. 

Enterobactin (ent), the cycHc triester of 2,3-dihydroxy-A/-benzoyl-l-serine, uses three catecholate dianions to coordinate iron. The iron(III)-enterobactin complex [62280-34-6] has extraordinary thermodynamic stabiUty. For Fe " +ent , the estimated formal stabiUty constant is 10 and the reduction potential is approximately —750 mV at pH 7 (23). Several catecholate-containing synthetic analogues of enterobactin have been investigated and found to have lesser, but still impressively large, formation constants. 

The effect of the amino acid spacer on iron(III) affinity was investigated using a series of enterobactin-mimic TRENCAM-based siderophores (82). While TRENCAM (17) has structural similarities to enterobactin, in that it is a tripodal tris-catechol iron-binding molecule, the addition of amino acid spacers to the TRENCAM frame (Fig. 10) increases the stability of the iron(III) complexes of the analogs in the order ofbAla (19)complex stability is attributed to the intramolecular interactions of the additional amino acid side chains that stabilize the iron-siderophore complex slightly. 

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