Spectroscopic description of the two nitrosyl-iron complexes responsible for fur inhibition by nitric oxide.

Ferric uptake regulation protein (Fur) is a global regulator, ubiquitous in Gram negative bacteria, that acts as a transcriptional repressor when it binds ferrous ion. Fur is involved in responses to several types of stress related to iron metabolism, such as stress induced by nitric oxide (NO) generated by macrophages against bacterial invasion. NO was recently shown to react with Fe(2+) ions in FeFur (iron substituted Fur protein) leading to an Fur bound iron-nitrosyl complex, unable to bind DNA, and characterized by a g = 2.03 EPR signal, associated with an S = (1)/(2) ground state. This electronic configuration could arise from either a mononitrosyl-iron [Fe(NO)](7) or a dinitrosyl-iron [Fe(NO)(2)](9) complex. The use of several spectroscopic tools such as EPR, ENDOR, FTIR, Mössbauer, and UV-visible spectroscopies as well as mass spectrometry analysis was necessary to characterize the iron-nitrosyl species in Fur. Furthermore, changes of C132 and C137 into glycines by site directed mutagenesis reveal that neither of the two cysteines is required for the formation of the g = 2.03 signal. Altogether, we found that two species are responsible for Fur inhibition in NO stress conditions: the major species, S(1/2), is an [Fe(NO)(2)](9) (S = (1)/(2)) complex without bound thiolate and the minor species is probably a diamagnetic [Fe(NO)(2)](8) (S = 0) complex. This is the first characterization of these physiologically relevant species potentially linking iron metabolism and the response to NO toxicity in bacteria.