The Metal Core Structures in the Recombinant Escherichia coli Transcriptional Factor SoxR

X‐ray absorption, circular dichroism, and EPR spectroscopy were employed to investigate the metal‐core structures in the Escherichia coli transcriptional factor SoxR under reduced, oxidized, and nitrosylated conditions. The spectroscopic data revealed that the coordination environments of the metal active centers varied only very slightly between the reduced and oxidized states, similar to most other proteins containing iron–sulfur clusters. Upon nitrosylation of oxidized SoxR, however, we observed a low‐temperature EPR spectrum characteristic of a protein dinitrosyl iron complex (DNIC), with an intensity corresponding to about two DNICs per iron sulfur cluster in the protein, according to spin quantification relative to a low‐molecular‐weight DNIC standard. In addition, there was no evidence for dichroic spectral features in the responsive region of the nitrosyl iron complexes, as well as for FeFe back‐scattering in the fitting of the Fe extended X‐ray absorption fine structure (EXAFS) spectrum. Instead the Fe EXAFS spectrum of the nitrosylated SoxR core exhibited the same first‐ and second‐shell coordination environments characteristic of modeled small molecular DNICs, indicating that each of the [2 Fe2 S] cores in the homodimeric SoxR was dissociated into two individual DNICs. Similar nitrosylation of the reduced mixed‐valence SoxR for 1 min led to degradation of the iron–sulfur clusters to give several iron species, including one with EPR signals characteristic of a reduced Roussin’s red ester (rRRE), a diamagnetic species, presumably Roussin’s red ester (RRE), and a small amount of DNIC. We also undertook in vivo time‐course studies of E. coli cells containing recombinant SoxR after rapid purging of the cells with exogenous NO gas. Rapid freeze‐quenched EPR experiments demonstrated rapid formation of the SoxR rRRE species, followed by fast breakup of this precursor intermediate to form the stable protein‐bound DNIC species. Accordingly, under nitrosative stress, we believe that the response of SoxR to NO could depend on the intracellular redox state of E. coli, the central modulator of which could be exploited to deduce the appropriate mechanism to sense the presence of NO for physiological regulation. NO sense! Spectroscopic studies of the reduced and oxidized forms of the E. coli transcriptional factor SoxR, followed by mechanistic analyses of the nitrosylation process of these species, allowed the elucidation of the redox chemistry associated wit