Structural and dynamic properties of hemeproteins that control electroreactivity

Abstract

The main objective of this project was to gain a better understanding of the structural and dynamic contributors to hemeprotein electroreactivity. To this end we employed cytochrome b5 as a model system. We have been successful in structurally characterizing cytochrome b5 in both the A and the B conformations which differ in the orientation of the bound heme. This was the first complete structural characterization of such a heterogeneous system. Differences in the structures have revealed the possible origins of differences in the reduction potential of the two conformers. Also, the dynamic behavior of cytochrome b5 was analyzed in the oxidized and reduced state. The results of this analysis have shown that changes in backbone dynamics of cytochrome b5 on oxidation contribute significantly to the electromotive driving force and that the dramatic difference in the dynamics of the two oxidation states are probably a result of redox linked hydrogen bond strength modulation. The observations have opened new avenues in terms of strategies that can be used for the development of therapeutic agents for the treatment of methemoglobinemia. A generalized hydrophobic binding domain on cytochrome b5 has been identified using an experimental NMR based strategy and binding to this domain could be further optimized to give a potential drug. Preliminary evidence based on the temperature dependence of the reduction potential suggests that the entropic changes associated with oxidation of myoglobin and hemoglobin are diametrically opposite to the behavior observed for cytochrome b5. An analysis of the backbone dynamics of myoglobin would give us insight into whether the backbone dynamics of a protein determine the electrochemical properties essential to the physiological role of a hemeprotein. In order to test this hypothesis, we have developed a high yield overexpression system for sperm whale myoglobin so as to obtain large quantities of the protein for NMR experiments. Further, a high yield expression system for the beta-globin subunit of hemoglobin has also been developed, the motivation being the study of the cytochrome b5-hemoglobin complex and the development of suitable remedial agents for sickle cell anemia.