Iron(III) Uptake and Release by Chrysobactin, a Siderophore of the Phytophatogenic Bacterium Erwinia chrysanthemi Vladislav Tomis ˇic ´, † Sylvie Blanc, † Mourad Elhabiri, † Dominique Expert,* ,‡ and Anne-Marie Albrecht-Gary* ,† Laboratoire de Physico-Chimie Bioinorganique, ULP-CNRS (UMR 7177), Institut de Chimie, ECPM, 25 rue Becquerel, 67200 Strasbourg, France, and Laboratoire Interactions Plantes-Pathoge `nes, INRA/INA-PG/UPMC (UMR 217), 16 rue Claude Bernard, 75231 Paris Cedex 05, France Received June 20, 2008 The plant pathogenic enterobacterium Erwinia chrysanthemi causes important soft-rot disease on a wide range of plants including vegetables and ornamentals of economic importance. It produces a major mono(catecholate) siderophore, chrysobactin (R-N-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine). To unravel the role of chrysobactin in the virulence of E. chrysanthemi, its iron(III) coordination properties were thus investigated in aqueous solutions using electrospray ionization mass spectrometric, potentiometric, and spectrophotometric methods. Moreover, kinetic experiments allowed us to determine the uptake and release mechanisms. The formation mechanism of the 1:1 complex reveals a key role of the terminal carboxylic group of chrysobactin in the binding of either FeOH 2+ or Fe 2 (OH) 2 4+ . The proton-driven dissociation of the ferric tris-, bis-, and mono(chrysobactin) complexes was also studied. For these three ferric complexes, a single protonation triggers the release of the bound chrysobactin molecule. Interestingly, the dissociation of the last ligand proceeded via the formation of an intermediate for which a salicylate-type mode of bonding was proposed. Introduction Under iron-deficient conditions, almost all microorganisms excrete highly efficient and specific ferric ion chelators, termed siderophores. 1-3 These molecules typically possess hydroxamate, catecholate, and/or R-hydroxycarboxylate li- gating groups. Although many siderophores are hexadentate ligands, several natural iron sequestering agents of lower denticity have been described, 4 and their iron(III) complex- ation properties were thoroughly studied. 5,6 One of them is chrysobactin (noted Cb), R-N-(2,3-dihydroxybenzoyl)-D- lysyl-L-serine (Figure 1), a mono(catecholate) siderophore first isolated by Persmark et al. 7 from the plant pathogenic enterobacterium Erwinia chrysanthemi, which causes soft- rot disease on a wide range of plants including vegetables and ornamentals of economic importance. More recently, chrysobactin was shown to be produced by several strains of E. carotoVora subsp. carotoVora deleterious to potato plants. 8 * To whom correspondence should be addressed. E-mail: amalbre@ chimie.u-strasbg.fr (A.-M.A.-G.), Dominique.Expert@inapg.inra.fr (D.E.). † ULP-CNRS (UMR 7177), Institut de Chimie. ‡ INRA/INA-PG/UPMC (UMR 217). (1) Neilands, J. B. Struct. Bonding (Berlin) 1984, 58, 1–24. (2) Raymond, K. N.; Mu ¨ ller, G.; Matzanke, B. F. Top. Curr. Chem. 1984, 123, 49–102. (3) Winkelmann, G. CRC Handbook of Microbial Iron Chelates; CRC Press: Boca Raton, FL, 1991; pp 15-64. (4) (a) Albrecht-Gary, A. M.; Crumbliss, A. L. In Metal Ions in Biological Systems; Sigel A., Siegel H. Eds.; Marcel Dekker, Inc.: New York, 1998; pp 239-327, and references cited therein. (b) Albrecht-Gary, A. M., Crumbliss, A. L. Scientific Bridges for 2000 and Beyond, TEC & DOC Editions, Acade ´mie des Sciences; Institut de France: Paris, 1999; pp 73-89. (5) Telford, J. R.; Raymond, K. N. Inorg. Chem. 1998, 37, 4578–4583. (6) Boukhalfa, H.; Brickman, T. J.; Armstrong, S. K.; Crumbliss, A. L. Inorg. Chem. 2000, 39, 5591–5602. (7) Persmark, M.; Expert, D.; Neilands, J. B. J. Biol. Chem. 1989, 264, 3187–3193. (8) Barnes, H. H.; Ishimaru, C. A. Biometals 1999, 12, 83–87. Figure 1. Structure of fully deprotonated chrysobactin and attribution of the protonation sites. Inorg. Chem. 2008, 47, 9419-9430 10.1021/ic801143e CCC: $40.75  2008 American Chemical Society Inorganic Chemistry, Vol. 47, No. 20, 2008 9419 Published on Web 09/20/2008