Figure 4. Zinc release is the rate-limiting step in RsrA oxidation.

Experiments were conducted at 25 °C in 50 mM Tris (pH 7.5) buffer containing 100 mM NaCl. (a) Oxidation of the RsrA–σ^R complex on treatment with diamide under second-order conditions (25 μM). The fraction of reduced RsrA was determined by the change in diamide absorbance at 320 nm (Supplementary Fig. 2a) in the presence and absence of bound zinc. Zinc release was monitored at 500 nm using the PAR assay. The two methods showed good agreement for the bimolecular rate constant for diamide-induced oxidation of RsrA^red.Zn²⁺ (183±6 and 195±11 M⁻¹ s⁻¹, respectively). (b) Zinc release from the RsrA^red.Zn²⁺–σ^R complex (2 μM) on treatment with increasing concentrations of diamide under pseudo-first-order conditions monitored by the PAR assay; 25 μM (filled diamonds), 50, 100, 150 and 200 μM (open diamonds). Inset shows variation of k_obs with diamide concentration (with associated error bars). Data were fitted to the Michealis–Menten equation, with fitted parameters of K₁=0.7 mM and k₂=0.15 s⁻¹ (Supplementary Fig. 2b). The corresponding bimolecular rate constant (k₂/K₁=214 M⁻¹ s⁻¹) is in reasonable agreement with values obtained in a. (c) Oxidation-induced dissociation of the RsrA^red.Zn²⁺–σ^R complex (2 μM) monitored by tryptophan fluorescence on treatment with H₂O₂ under pseudo-first-order conditions. Three H₂O₂ concentrations are shown as follows: 1 mM (triangles), 2 mM (open circles) and 6 mM (closed circles). Inset, pseudo-first-order plot (with associated error bars) from which the bimolecular rate constant for H₂O₂-induced dissociation of the complex was obtained (0.39±0.08 M⁻¹ s⁻¹). (d) Zinc release from the RsrA^red.Zn²⁺–σ^R complex (2 μM) on treatment with increasing concentrations of H₂O₂ (2–10 mM) under pseudo-first-order conditions. Inset: pseudo-first-order plot (with associated error bars) from which the bimolecular rate constant for the H₂O₂-induced zinc dissociation was obtained (0.32±0.06 M⁻¹ s⁻¹).