thermodynamics of binding of iron(iii) by brasilibactin a james harrington, heekwang park, yongcheng...
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Thermodynamics of binding of iron(III) by brasilibactin AJames Harrington, Heekwang Park, Yongcheng Ying, Jiyong Hong, and Alvin L. Crumbliss, Department of Chemistry, Duke University, Durham, NC, 27708-0346
Iron is necessary but problematic.
Fe3
+
OH2
OH2
OH2
OH2
H2
O
H2
O
Fe3
+OHOH2
OH2
OH2
H2
OH2
OH+ H+ H+
Ksp = 10-39
However, iron(III) easily hydrolyzes and forms insoluble hydroxide and oxide salts, resulting in low aqueous concentrations at physiological conditions. Iron can also take part in redox reactions that produce reactive oxygen species and can harm organisms
Microbial Iron Acquisition
Fe
Environment
Na
Ca
SYNTHESIS SOLUBILIZATION
TRANSPORT
EXCHANGE AND UPTAKE
RELEASE
Mg
Al
Al
Al
Ca
Microbial Cell
Fe
Fe
Fe
FeAl
Microbes produce small molecules called siderophores, to solubilize iron, return it to the cell, and facilitate transport into the cell.
Fe
Brasilibactin A is a membrane-bound siderophore produced by Nocardia brasiliensis, which has been found to be cytotoxic at low concentrations (~ 50 nM). It is hypothesized that this is due to iron binding, as iron(III) inhibits caspase 3, an enzyme in the apoptosis pathway.
N
O
O
HN
OHN
OHO
NH
O
O
RO
NCHOHO
Brasilibactin A, R = C15H31
ObjectiveCharacterize the pKa’s and thermodynamics of interaction of iron(III) with brasilibactin A by spectrophotometric/potentiometric titrations
Ligand Spectrophotometric titration
Conditions: [L] = 1.4 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4)
Problem: reversibility of protonation?
N
O
O
HN
OHN
OHO
NH
O
O
O
NCHOHO
OH-
The irreversible spectral transition suggests chemical reaction, possibly hydrolysis. Ester moiety may be susceptible to hydrolysis at high pH. Similar behavior has been observed in other siderophores, such as enterobactin, fusarinines, and fusigens.
Fragment Potentiometric titration
Conditions: [L] = 5.8 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4) Using 1 proton model, pKa1 = 9.05 ± .08
NOHO
NH
O
OH
Fragment 3&4
Spectrophotometric titration of Fragment 1-2
pKa1 = 10.09±0.03, pKa2 = 8.18 ±0.09
pKa1 = 4.8 ±0.2, pKa2 = 2.9 ±0.1
pH 6.0-10.6 pH 2.7-6.0
N
O
O
HN
OH
HO
O
NCHOHO
Fragment 1&2
H
N
O
O
HN
OHN
OHO
NH
O
O
O
NCHOHO
BbtH
Fe-BbtH spectrophotometric titration
Conditions: [Fe3+] = 2.3 x 10-4 M, [BbtH] = 2.4 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4)The transition at high pH is not reversible. Likely dissociation of the complex, then hydrolysis of the ligand.Low pH spectrophotometric
titration of the Fe(III)-BbtH system
Conditions: [Fe3+] = 2.1 x 10-4 M, [BbtH] = 2.1 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4)At low pH, the spectrum slowly decreases to baseline. This shift is reversible by returning the pH to its original value. Indicates reversible dissociation of the Fe(III)-BbtH complex.
Competition of Fe(III)-BbtH complex with EDTA was performed to determine the thermodynamic stability constant of the Fe(III)-BbtH complex.
[Fe(BbtH)] stability constant
NO
O
NH
O
NO
O
HN
O
OO
N
C
O
O
H
Fe3+
N
O
O
HN
OHN
OHO
NH
O
O
RO
NCHOHO
+ EDTA Fe(EDTA) +
Conditions: [Fe3+] = 2.5 x 10-4 M, [BbtH] = 2.6 x 10-
4 M, 25 °C, μ = 0.10 M (NaClO4).
N
O
O
HN
OHN
OHO
NH
O
O
O
NHO
O 12
Mycobactin S
Siderophore Log β110 pFea
BbtH 26.961 22.73Mycobactin S 26.62 N/A
Desferrioxamine B
30.63 26.6
Aerobactin 27.64 23.3Exochelin MN 39.125 31.1
Rhodotorulic acid 62.2b 21.9apFe is the concentration of free aqueous iron(III) in solution at set conditions of [M] = 10-6 M, [L] = 10-5 M, and pH = 7.4.bThis stability constant is a log β230. Ref. 6.
ConclusionsoThe Brasilibactin A analog hydrolyzes at basic pH.oThe presence of Fe stabilizes the Brasilibactin A analog through at least pH 8 (complex dissociates irreversibly ab). oMolecule forms a stable complex with iron(III), but less stable than other hexadentate siderophores.oBbtH exhibits a slower rate of complex formation with iron than AHA does.
References:1 – This work2 – MacCordick, Schleiffer, and Duplatre, Radiochim. Acta 1985, 38, 43.3 – Schwarzenbach and Schwartzenbach, Helv. Chim. Acta 1963, 46, 1390.4 – Kupper, Carrano, Kuhn, and Butler, Inorg. Chem. 2006, 45, 6028.5 – Dhungana, Miller, Dong, Ratledge, Crumbliss, J. Am. Chem. Soc. 2003, 125, 7654.6 – Spasojevic, Armstrong, Brickman, Crumbliss, Inorg. Chem. 1999, 38, 449.
Acknowledgements: We thank Duke University, the Center for Biomolecular and Tissue Engineering, the NIH, NSF Grants CHE 0418006 and CHE 0809466, and the rest of the Crumbliss and Hong labs.
O
NH
N
OOH
NH3
HN
O
NH2+
N
OH
O
HN
O
HN
O
NH+HN
HO
+H3N
Exochelin MN
N
O OHNH
O
O
NHO
NH
O
O
NHO
+H3N
Desferrioxamine B
HN
NHO
O
NN
OHOOHO
Rhodotorulic acid
O
N
HN
HO
HO
O O
HO
COOH
NH
O
N OHO
OH
OAerobactin
OH
N
OHN
O
NHO
O
O
O
NH
N
O
OH
H
pKa = 10.09 pKa = 2.9
pKa = 8.18
pKa = 9.05
O
H
Iron is necessary for a variety of cellular processes, i.e. small molecule transport, electron transport. Microbes require an effective concentration of at least 10-5 M for survival
Introduction
Conditions: [L] = 1.7 x 10-4 M, 25 °C, μ = 0.10 M (NaClO4)
Comparison of stability constants
oBbt complex exhibits slow formation kinetics (relative to AHA). Addition of iron(III) to solution of BbtH at low pH (~2) resulted in complex formation over 3 times as long as complex formation was observed with AHA as evidenced by change in solution color.