Role of Peripheral Contacts in Structure and Function of c-Type Cytochromes

Structural perturbations influence many properties of proteins, but sequence variations are frequently observed in nature without perturbing the overall stability, fold, and function. In this thesis work, heme proteins cytochrome c and c2 have been used to provide insight into the relationship between peripheral contacts and its function. Recent studies with pathogenic cytochrome c mutations G41S and Y48H, and growth-inhibiting mutation K72A, have highlighted the importance of contacts between loops C and D in function of the protein. Characterization of these variants shows that loop D local stability and dynamics are altered upon loop C perturbation, influencing electron transfer, alkaline transition, and peroxidase activity. Effects of these structural perturbations are different for human and horse heart proteins. Computational models suggest these differences may depend on whether the native peripheral contacts involving loops C and D are preserved. These contacts are also important for the cooperative acid unfolding transition. Loop D packs closely to the 60’s helix, forming additional intermolecular contacts for the protein to fold into a compact state with a Met80-ligated 6-coordinate heme iron. Y67R mutation perturbs these inner peripheral contacts and triggers a conformational rearrangement to induce Lys-ligation. Conformational rearrangements are important in cytochrome c. Interaction between cardiolipin and cytochrome c breaks up native peripheral contacts, which triggers a conformational rearrangement that enhances peroxidase activity so the protein functions as a peroxidase instead of an electron carrier. The heme ligation state of the species responsible for the increased peroxidase activity has been under debate. Characterization of Y67H, a variant that readily forms bisHis-ligation upon structural perturbation, suggests that bisHis-ligated species is not the species responsible for the enhanced peroxidase activity. Peripheral contacts are key participants in the proton-coupled electron transfer mechanism of cytochrome c2, an electron carrier in the bacterial photosynthetic electron transfer pathway. A calorimetric characterization of Rhodospirillum rubrum cytochrome c2 suggests that His42, a residue hydrogen-bonded to heme propionate 7, participates in proton transfer and is responsible for the pH-dependent reduction potential. Preliminary studies designed to probe the relationship between peripheral contacts and the heterogeneity of unfolded states are also discussed