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This manuscript describes the interaction of low-molecular-weight DNICs with short peptides designed to explore the stability and structure of DNIC-peptide/RRE-peptide constructs. Although characterization of protein-bound and low-molecular-weight DNICs is possible via EPR, XAS, and NRVS, this study demonstrates that the combination of aqueous IR ν(NO) and UV-vis spectra can serve as an efficient tool to characterize and discriminate peptide-bound DNICs and RREs. The de novo chelate-cysteine-containing peptides KC(A)(n)CK-bound (n = 1-4) dinitrosyliron complexes KC(A)(n)CK-DNIC (CnA-DNIC) and monodentate-cysteine-containing peptides KCAAK-/KCAAHK-bound Roussin's red esters (RREs) KCAAK-RRE/KCAAHK-RRE were synthesized and characterized by aqueous IR, UV-vis, EPR, CD, XAS, and ESI-MS. In contrast to the inertness of chelate-cysteine-containing peptide-bound DNICs toward KCAAK/KCAAHK, transformation of KCAAK-RRE/KCAAHK-RRE into CnA-DNIC triggered by CnA and reversible transformation between CnA-DNIC and CnA-RRE via {Fe(NO)(2)}(9)-{Fe(NO)(2)}(10) reduced-form peptide-bound RREs demonstrate that the {Fe(NO)(2)}(9) motif displays a preference for chelate-cysteine-containing peptides over monodentate-cysteine-containing peptides. Also, this study may signify that nitrosylation of [Fe-S] proteins generating protein-bound RREs, reduced protein-bound RREs, or protein-bound DNICs are modulated by both the oxidation state of iron and the chelating effect of the bound proteins of [Fe-S] clusters.

A combination of N/S/Fe K-edge X-ray absorption spectroscopy (XAS), X-ray diffraction data, and density functional theory (DFT) calculations provides an efficient way to unambiguously delineate the electronic structures and bonding characters of Fe-S, N-O, and Fe-N bonds among the direduced-form Roussin's red ester (RRE) [Fe2(μ-SPh)2(NO)4](2-)(1) with {Fe(NO)2}(10)-{Fe(NO)2}(10) core, the reduced-form RRE [Fe2(μ-SPh)2(NO)4](-)(3) with {Fe(NO)2}(9)-{Fe(NO)2}(10) core, and RRE [Fe2(μ-SPh)2(NO)4] (4) with {Fe(NO)2}(9)-{Fe(NO)2}(9) core. The major contributions of highest occupied molecular orbital (HOMO) 113α/β in complex 1 is related to the antibonding character between Fe(d) and Fe(d), Fe(d), and S atoms, and bonding character between Fe(d) and NO(π*). The effective nuclear charge (Zeff) of Fe site can be increased by removing electrons from HOMO to shorten the distances of Fe···Fe and Fe-S from 1 to 3 to 4 or, in contrast, to increase the Fe-N bond lengths from 1 to 3 to 4. The higher IR νNO stretching frequencies (1761, 1720 cm(-1) (4), 1680, 1665 cm(-1) (3), and 1646, 1611, 1603 cm(-1) (1)) associated with the higher transition energy of N1s →σ*(NO) (412.6 eV (4), 412.3 eV (3), and 412.2 eV (1)) and the higher Zeff of Fe derived from the transition energy of Fe1s → Fe3d (7113.8 eV (4), 7113.5 eV (3), and 7113.3 eV (1)) indicate that the N-O bond distances of these complexes are in the order of 1 > 3 > 4. The N/S/Fe K-edge XAS spectra as well as DFT computations reveal the reduction of complex 4 yielding complex 3 occurs at Fe, S, and NO; in contrast, reduction mainly occurs at Fe site from complex 3 to complex 1.

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.