Figure 3.

Anaerobic titration of 5.0 µM wild-type BxmR monomer (2.5 µM dimer) (μ), α3NΔ (λ) and α5Δ (υ) BxmRs with Zn^II as monitored by change in the Tyr fluorescence intensity, F_i/F_o. The solid lines through the data correspond to a nonlinear least squares fit to model that invokes sequential binding of Zn^II to a single high affinity site (for α3NΔ and α5Δ BxmRs) or two high affinity sites (for wild-type BxmR) each with a characteristic F_i/F_o signature. After filling these high affinity zinc binding sites (one α5 site with an F_i/F_o>1; one α3N site with an F_i/F_o <1), Zn(II) binds to each of the remaining low affinity α5′ and α′ sites on the dimer. Fitted parameters are as follows, with K_Zn1>10¹⁰ M⁻¹ (see Fig. 2A; Table 1): α3NΔ BxmR: F_i/F_o values, 1.045 (±0.001), 1.03 (fixed) for the high affinity and low affinity α5 Zn(II) sites, respectively; α5Δ BxmR: F_i/F_o values, 0.753 (±0.002), 0.915 (±0.011) for the high affinity and low affinity α3N Zn(II) sites, respectively; wild-type BxmR, F_i/F_o constrained to the above values for first three binding steps, with F_i/F_o for filling the final binding site, 0.80 (±0.03).