Heme proteins carry out a wide range of biological functions, such as oxygen storage and transport, electron transfer, peroxidase, and gas sensing. Our research focuses on understanding how heme-protein interactions can specifically define the biological role of a heme protein. In our studies, we use fluoride binding as a heme pocket probe for Hb, Mb, horseradish peroxidase (HRP), and clam hemoglobins. Using this approach, we have performed pH-dependence studies of fluoride binding in these systems. Fluoride binding affinity in heme proteins that have a distal histidine, as in Mb and Hb, is affected by the protonation state of the histidine with a measured pK<sub>a</sub> of 5.7. HRP and hemoglobins II and III from the clam <i>Lucina pectinata</i> which have instead distal arginine and tyrosine, respectively, bind fluoride ion with an affinity that is 10 times stronger than that of Mb and Hb. These results indicate how the pH profile of fluoride binding is affected by the identity of the distal amino acid. Additionally, to achieve a greater scope in understanding the role of the protein structure, we have performed temperature-controlled experiments to study the thermodynamic properties of fluoride binding. We measured the enthalpies and entropies of fluoride binding in Hb, Mb, and HRP. We have observed that all three proteins show a strong correlation between the entropy and enthalpy of fluoride binding (see Figure). We have also noticed that despite their similarities in the heme pocket structure, Mb and Hb show differences in their thermodynamics of fluoride binding. Unlike Mb, Hb is able to utilize entropy for favorable fluoride binding. These thermodynamic differences between Mb and Hb could be a reflection of the difference in the protein structures, mainly the quaternary structure of Hb which is lacking in Mb.

Molecular mechanisms in heme protein function: A thermodynamic perspective from fluoride-binding studies

254th National Meeting (2017)

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