Figure 8.

Stopped-flow fluorescence quenching by O₂ oxidation of Fe²⁺ prebound to variants #1 (curve a) and #2 (curve b) containing 48 Fe²⁺/shell. Fitted curves are shown in red. The rate constants and intrinsic fluorescence constants for variant #1 from curve-fitting (curve a) are: k₁ = 19.0 ± 3.1 s^-1 and k₂ = 1.86 ± 0.04 s^-1, I_A = 1.01 ± 0.02 μM^-1 on a per iron dimer basis, I_B = 0.655 ± 0.021 μM^-1 dimer, and I_C = 0.0425 + 0.0012 μM^-1 dimer (curve a); and for variant #2 (curve b) are: k₁ = 16.6 ± 2.3 s^-1 and k₂ = 2.38 ± 0.15 s^-1, I_A = 0.711 ± 0.011 μM^-1 dimer, I_B = 0.320 ± 0.030 μM^-1 dimer, and I_C = 0.0158 ± 0.0013 μM^-1 dimer (curve b) from eq 5 with the equations for the concentrations of species given elsewhere.³¹ The residual is the difference between the fitted and experimental curves. The stated rate constants and associated errors are averages and standard deviations, respectively, from curve fits to three separate kinetic runs. The observed quenching kinetics is from the oxidation of the prebound Fe²⁺ and not from the binding of Fe²⁺ itself. Final conditions: anaerobic 1.5 μM apo-variants containing 48 Fe²⁺/shell in 50 mM Mops pH 7.15 rapidly mixed with H₂O saturated with 100% O₂, 25 °C.