The peroxidase activity of cytochrome c (Cytc), which leads to oxygenation of the mitochrondrial membrane lipid, cardiolipin, is an early step in the intrinsic pathway of apoptosis. Naturally occurring mutations to human Cytc, linked to thrombocytopenia, and known to enhance the peroxidase activity of human Cytc, all locate within Ω-loop C (residues 40 – 57). This observation has focused attention on the role of Ω-loop C in mediating the apoptotic peroxidase activity of Cytc. The intrinsic peroxidase activity of human Cytc is 20-fold lower than that of yeast iso-1-Cytc consistent with evolution of a tightly regulated off-state for peroxidase activity. The poor sequence conservation of Ω-loop C indicates that it may have played a role in this evolution. Furthermore, residues 40/57 and 57/63 show pairwise co-evolution and while sequentially distant are spatially proximal in the structure of Cytc. To test the hypothesis that these residues evolved to decrease the intrinsic peroxidase activity of Cytc, we prepared all possible single, double, and triple mutant variants of yeast iso-1-Cytc that replace the amino acids at positions 40, 57 and 63 with those of human Cytc. Global stability, local stability adjacent to Ω-loop C as measured by the alkaline conformational transition, and kcat and KM for peroxidase activity with guaiacol as substrate were measured for all variants. Overall, changes to these physical and functional properties are small with the final triple mutant variant S40T/V57I/N63T being most like WT iso-1-Cytc. This result indicates that co-evolution has acted as a buffer to maintain the overall properties of Ω-loop C and that evolution of an off-state for peroxidase activity is localized elsewhere in the protein. We discuss this result in the context of how the relative energies of the substructures of Cytc have changed from yeast to human.

Role of Ω-loop C in controlling the apoptotic peroxidase activity of cytochrome c

ACS Fall 2021

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