Ferric agrobactin

ACS Symposium Series American Chemical Society Washington, DC, 1980. 

Rhodotorulic acid, a hydroxamate type siderophore, binds 2/3 mole ferric ion to give an unchsurged, orange colored chelate which is fully formed at neutral pH (10). 

Titrations were performed as above except that 2 ymoles of ferric chloride and 3 Pmoles of rhodotorulic acid were mixed and neutralized prior to introduction of 2 ymoles of catechol type siderophore dissolved in ethanol. The eq.tiivalents of standard alkali required to neutralize the solution were then noted. 

From these data we conclude that agrobactin and parabactin form similar coordination compounds with ferric ion in which one of the atoms linked to the iron in the central bidentate portion of the complex is the tertiary N of the oxazoline ring. At neutral pH agrobactin A, in contrast to the tri-catechol enterobactin (l6), appeared to be not fully coordinated to the ferric ion. 

Similar experiments were performed by ligand exchange with the 1 1 ferric complex of nitrilotriacetate. The total proton yields per iron to the neutral pH zone were U.7 and for agrobactin and parabactin, respectively. Thus introduction of the iron (ill), either directly or by ligand exchange, gave complexes which for both agrobactin and parabactin should result in divalent anions at neutral pH. 

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Since the spectra of ferric agrobactin and ferric parabactin do show minor differences, a number of experiments were tried in which an attempt was made to mimic these differences via examination of the 1 3 ferric complexes of model salicyl and 2,3-dihy-droxyphenyloxazolines and the 1 1 1 ferric complexes of the model oxazolines with Tait s "Compound II", -bis-(2,3-dihyd oxyben-... 

Figure 5. Circular dichroism spectra of approximately 0.2mM solutions of A, ferric enterobactin B, ferric agrobactin C, blank D, ferric agrobactin A and E, ferric parabactin in O.IM phosphate pH 7.4...

Upon adding either agrobactin or parabactin to neutral solutions of ferric enterobactin there was little change in pH. However, mixing of enterobactin with either ferric agrobactin or ferric parabactin caused the pH to faiJ. to less than 6 and ca. 1 to 2 ymoles of alkali were required to neutralize the solutions. 

Exactly 0.5 ml of each of the two solutions was applied to individual 20 cm wide sheets of Whatman No. 1 paper and the separation performed in phosphate buffer pH 6.8 for 30 min. 30 ma and 2.5KV. Colored bands occurred at 9 and 13 cm from the origin, representing ferric agrobactin and ferric enterobactin, respectively, The zones were eluted into 5 ml of pH 6.8 phosphate and the spectra recorded from 6OO to UOO nm. 

In view of the several assumptions, such as the lack of a precise number for the extinction of ferric agrobactin, an exactly quantitative value cannot be placed on the affinity of agrobactin for ferric ion. However it is clear that the binding strength is comparable to that of enterobactin, which at pH 7. + is reported (I8) to have a formation constant 10 -larger than ferrichrome, a cyclohexapeptide trihydroxamate siderophore with a... 

Figure 4. Titration of agrobactin and parabactin in the presence of an equimolar quantity of ferric chloride. The siderophores differed significantly only at very high...

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... 

D. Electronic Absorption Spectra. The absorption spectra of the complexes formed by exchange from ferric nitrilotriacetate were determined in 0.1 M phosphate, pH 7- t, with a Beckman Model 25 recording spectrophotometer over the range kOO-700 nm. Agrobactin and parabactin gave wine colored iron complexes with a broad adsorption band centered at about 500 nm. At pH 7., the... 

The surprisingly large affinity of the spermidine siderophores for iron was confirmed by re-examination of the equilibria between enterobactin, agrobactin and ferric ion on a more quantitative basis. 

In an experiment in which equimolar amounts of enterobactin, agrobactin and ferric chloride were mixed and analyzed after titration to pH levels of 6, 7 and 8 the absorbancy ratios found, respectively, were 2.26, 1.36 and 1.55, all in favor of enterobactin, after electrophoretic sepaj ation at the same pH and elution of the bands into equivalent volumes of pH 6.8 phosphate buffer. 

Table II affords the evidence that parabactin and agrobactin, but not their open-form analogues, can counteract growth retardation of P. denitrificans caused by EDDA. The simplest interpretation of these data is that the synthetic chelator, like deferri-ferrichrome A, forms a non-transportable or otherwise unavailable anionic complex with ferric ion which effectively denies iron to the cell in the absence of a strongly competitive ligand which is, in fact, utilized ( ). Table II also records the activity of these compounds with Escherichia coli RW193, an organism defective in the synthesis of enterohactin. Thus it would appear that the A,els complexes are active in coli while those with an enantio-morphic configuration around the iron, A,cis, are utilized by P. denitrificans.

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