Figure 2. Redox potential of the RsrA^red.Zn²⁺–σ^R complex.

(a) RsrA^red.Zn²⁺ binding to σ^R monitored by intrinsic tryptophan emission fluorescence spectroscopy. Figure shows emission spectra (λ_ex 295 nm) of σ^R at different RsrA^red.Zn²⁺ concentrations collected at 25 °C in 50 mM Tris (pH 7.5) buffer, 100 mM NaCl and 2 mM DTT. For clarity, traces are coloured with incrementally darker tone of grey for increasing RsrA concentration. Inset figure shows fraction of σ^R in complex as measured by the change in fluorescence intensity at 343 nm with increasing RsrA^red.Zn²⁺ concentration. Data show stoichiometric binding of RsrA^red.Zn²⁺ to σ^R. (b) Redox potential of RsrA^red.Zn²⁺ in complex with σ^R complex relative to a redox couple of reduced/oxidized glutathione. The degree of oxidation was measured by the change in tryptophan emission fluorescence of σ^R as the complex dissociates (open triangles) and by the release of Zn²⁺ using the PAR assay (filled triangles). Both sets of data were fitted to the Nernst equation to determine the redox potential of the RsrA^red-Zn²⁺–σ^R complex as −193.04±2.01 mV. Experiments were conducted in 50 mM Tris (pH 7.5) buffer containing 100 mM NaCl and at 25 °C. (c) Redox ruler showing the redox potentials of selected proteins and small molecules. Figure adapted from ref. 27. The estimated redox potential of the bacterial cytoplasm is indicated in grey.