In our lab, we use fluoride binding as a probe of the heme pocket structure of heme proteins. In our most recent study, we determined the reduction potentials (Em) of the heme-bound fluoride complex of adult hemoglobin (Hb-F) as a function of pH. The midpoint potentials of the reduction (Fe3+/Fe2+) of Hb-F were measured at room temperature by using UV/vis spectroelectrochemistry, from pH 4.5 to pH 7. Upon reduction, the spectra show that the reduced heme is a five-coordinate, ferrous heme (with no heme-bound fluoride). Our results show that the Em of the Hb-F complex is about 80 mV (vs SHE) between pH 5.5 and pH 7. However, below pH 5.5, the Em drops to a value of 53 mV (vs SHE) at pH 4.5. The drop in Em is indicative of the stabilization of the oxidized state (Hb-F) upon lowering the pH. To further understand the thermodynamics of the Hb-F redox reactions, we measured the enthalpy (△H) and entropy (△S) of the reduction (Fe3+/Fe2+) of Hb-F at pH 5 and pH 7. A detailed study of the thermodynamics revealed that the △S and △H at pH 5 were -57 J K-1 mol-1 and -24.0 kJ mol-1, respectively, and the △S and △H at pH 7 were -110 J K-1 mol-1 and -40.2 kJ mol-1, respectively. Using these values, the calculated Gibbs free energy (△G) at 25 oC for the reduction of Hb-F, is approximately 400 kJ more favorable at pH 7. Stated differently, the oxidized state (Hb-F) is favored at the lower pH. These measurements are in agreement with the Em measurements at room temperature, which indicates that the Hb-F is more stable at a lower pH. The pH effect on Em, △S, and △H can be attributed to the stabilization of the ferric Hb-F at low pH, due to the hydrogen bonding interactions between the heme-bound fluoride units and the protonated distal histidines of the α/β subunits of adult Hb. These hydrogen bonding interactions have been shown to regulate oxygen binding in adult Hb. This study demonstrates that the use of fluoride binding in Hb has the potential of helping us understand the molecular mechanism of oxygen transport in Hb.

Thermodynamics of the hemoglobin-fluoride complex redox reactions (Fe3+/Fe2+)

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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