Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome <i>c</i> Increases Its Peroxidatic Activity

Proteins performing multiple biochemical functions are called “moonlighting proteins” or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome <i>c</i> is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome <i>c</i> gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. The Y48H variant arises from one such mutation and is found in the 40–57 Ω-loop, the lowest-unfolding free energy substructure of the cytochrome <i>c</i> fold. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes compared to the wild-type structure, with the axial Met80 ligand coordinated to the heme iron. Despite this, the intrinsic peroxidase activity is enhanced, implying that a pentacoordinate heme state is more prevalent in the Y48H variant, corroborated through determination of a Met80 “off rate” of >125 s^–1 compared to a rate of ∼6 s^–1 for the wild-type protein. Heteronuclear nuclear magnetic resonance measurements with the oxidized Y48H variant reveal heightened dynamics in the 40–57 Ω-loop and the Met80-containing 71–85 Ω-loop relative to the wild-type protein, illustrating communication between these substructures. Placed into context with the G41S cytochrome <i>c</i> variant, also implicated in thrombocytopenia, a dynamic picture associated with this disease relative to cytochrome <i>c</i> is emerging whereby increasing dynamics in substructures of the cytochrome <i>c</i> fold serve to facilitate an increased population of the peroxidatic pentacoordinate heme state in the following order: wild type < G41S < Y48H