Figure 4.

Calorimetric titration of variant #1 (W93F/Y34W) with Fe²⁺ under anaerobic conditions. Plot of integrated heat versus Fe²⁺/variant #1 ratio. Inset: Raw data. The red line is the result of curve fitting to two independent classes of binding sites with n₁ = 12.0 ± 0.7, K₁ = (3.9 ± 2.2) × 10⁶ M^-1 and ΔH₁° = -0.45 ± 0.17 kJ/mol, corresponding to ΔG₁° = -37.6 ± 1.5 kJ/mol and ΔS₁° = 125 ± 5 J K^-1mol^-1, and n₂ = 6.8 ± 1.90, K₂= (1.5 ± 0.5) × 10⁵ M^-1 and ΔH₂° = 14.0 ± 7.3 kJ/mol, corresponding to ΔG₂°= -29.5 ± 0.8 kJ/mol and ΔS₂° = 146 ± 25 J/K-mol. Conditions: 3 μM protein in 0.1 M Mops and 2 mM Na₂S₂O₄, pH 7.05. The Fe²⁺ solution was prepared in anaerobic 0.1 M Mops, 2 mM Na₂S₂O₄, pH 7.05. Na₂S₂O₄ helps to maintain an oxygen-free solution during the titration. Its presence does not affect Fe²⁺ binding to the protein.²⁴