Thermodynamics of binding of iron(III) by brasilibactin A

1
Thermodynamics of binding of iron(III) by
brasilibactin A
James Harrington, Heekwang Park, Yongcheng Ying,
Jiyong Hong, and Alvin L. Crumbliss, Department
of Chemistry, Duke University, Durham, NC,
27708-0346
Problem reversibility of protonation?
Comparison of stability constants
Introduction
Fe-BbtH spectrophotometric titration
Siderophore Log ß110 pFea
BbtH 26.961 22.73
Mycobactin S 26.62 N/A
Desferrioxamine B 30.63 26.6
Aerobactin 27.64 23.3
Exochelin MN 39.125 31.1
Rhodotorulic acid 62.2b 21.9

• Iron is necessary but problematic.

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
OH2
OH2
H2O
OH2
H2O
OH
Fe3
Fe3
H2O
apFe 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 ß230. Ref. 6.
OH2
H2O
OH2
Ksp 10-39
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.
OH2
OH2
H
H
H
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
Low pH spectrophotometric titration of the
Fe(III)-BbtH system
Microbial Iron Acquisition
Al
Fe
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.
Fe
Ca
Mg
SYNTHESIS
SOLUBILIZATION
Al
Microbial Cell
Na
Fe
Al
RELEASE
Fragment Potentiometric titration
Ca
Al
Fe
Environment
TRANSPORT
EXCHANGE AND UPTAKE
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.
Microbes produce small molecules called
siderophores, to solubilize iron, return it to
the cell, and facilitate transport into the cell.

• Bbt 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.

Fe(BbtH) stability constant
Conditions L 5.8 x 10-4 M, 25 C, µ 0.10 M
(NaClO4) Using 1 proton model, pKa1 9.05 .08

• Conclusions
• The Brasilibactin A analog hydrolyzes at basic
  pH.
• The presence of Fe stabilizes the Brasilibactin A
  analog through at least pH 8 (complex dissociates
  irreversibly ab).
• Molecule forms a stable complex with iron(III),
  but less stable than other hexadentate
  siderophores.
• BbtH exhibits a slower rate of complex formation
  with iron than AHA does.
• References
• 1 This work
• 2 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.

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.
Spectrophotometric titration of Fragment 1-2
pH 6.0-10.6
pH 2.7-6.0
EDTA D Fe(EDTA)
Objective
Competition of Fe(III)-BbtH complex with EDTA was
performed to determine the thermodynamic
stability constant of the Fe(III)-BbtH complex.
Characterize the pKas and thermodynamics of
interaction of iron(III) with brasilibactin A by
spectrophotometric/potentiometric titrations
Ligand Spectrophotometric titration
Conditions L 1.7 x 10-4 M, 25 C, µ 0.10 M
(NaClO4)
pKa1 10.090.03, pKa2 8.18 0.09
pKa1 4.8 0.2, pKa2 2.9 0.1
Conditions Fe3 2.5 x 10-4 M, BbtH 2.6 x
10-4 M, 25 C, µ 0.10 M (NaClO4).
Conditions L 1.4 x 10-4 M, 25 C, µ 0.10 M
(NaClO4)