Disabling the Entatic Control of Methionine Ligation through Additive Destabilization of Ferric Cytochrome <i>c</i>

The entatic state plays a key role in modulating the functional properties of metal sites, especially in proteins. In cytochrome (cyt) <i>c</i>, a hydrogen-bonding network contributes to stabilization of Met80 ligation to the heme iron, supporting the dual functions of this metalloprotein as an electron carrier in respiration and a peroxidase in apoptosis. We have prepared a cyt <i>c</i> variant in which both Thr49 and Thr78 within this network have been replaced with Val residues. Spectroscopic and electrochemical experiments suggest that the ferric form of the protein no longer has Met80 coordinating the heme iron, while the ferrous form preserves this interaction. Thermodynamic analyses demonstrate how perturbations at the periphery of the heme introduced by the mutations affect the stabilities of Met-, Lys-, and H₂O-ligated conformers. The foldon structure enables the propagation of destabilization effects to the region implicated in the entatic control of the Met80 ligation. The extent of destabilization is similar for the ferric and ferrous Met-ligated conformers, but the ligation outcome differs because the global stability of the protein and stabilities of its foldons depend on the redox state of the heme iron. The stability of low-energy foldons could be tuned in other metalloproteins to engineer redox-linked switchable functions.