Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion

Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe(2+)-pyoverdine than for Fe(3+)-pyoverdine. At pH 7.4, about 90% of Fe(3+) was bound by pyoverdine Pa-C after 24 h whereas Fe(2+) was bound by the pyoverdine completely in only 5 min. The possibility that Fe(2+) underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe(2+) concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe(2+) with pyoverdine in the presence of 8-hydroxyquinoline, an Fe(3+)-specific chelating agent, resulted in the formation of a Fe(3+)-hydroxyquinoline complex, suggesting that the iron in the Fe(2+)-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.