Naturally Occurring Disease-Related Mutations in the 40–57 Ω‑Loop of Human Cytochrome <i>c</i> Control Triggering of the Alkaline Isomerization

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome <i>c</i> are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome <i>c</i> peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71–85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p<i>K</i>_trigger). A second deprotonation stabilizes a pentacoordinate heme form (p<i>K</i>_a2). In this study, site-directed variants at the 41 and 48 positions in the 40–57 Ω-loop and at the 81 and 83 positions in the 71–85 Ω-loop reveal that conformational transitions in the 71–85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40–57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the p<i>K</i>_trigger and increase p<i>K</i>_a2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80–Fe­(III) bond strength