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-BbtHspectrophotometric titration 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 Iron is necessary but problematic. 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 β230. 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 TRANSPORT Environment 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. Acta1985, 38, 43. • 3 – Schwarzenbach and Schwartzenbach, Helv. Chim. Acta1963, 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 pKa’s 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 = 10.09±0.03, 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)