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Naturally-occurring variants of human cytochrome c (Cyt c) that induce thrombocytopenia IV occur within Ω-loop C (residues 40–57). These variants enhance the peroxidase activity of human Cyt c apparently by facilitating access to the heme by destabilizing Ω-loops C and D (residues 70–85). Given the importance of peroxidase activity in the early stages of apoptosis, we identified three sites with the EVmutation algorithm in or near Ω-loop C that coevolve and differ between yeast iso-1-Cyt c and human Cyt c. We prepared iso-1-Cyt c variants with all possible combinations of the S40T, V57I and N63T substitutions to determine if these residues decrease the peroxidase activity of iso-1-Cyt c to that of human Cyt c producing an effective off state for a peroxidase signaling switch. At pH 6 and above, all variants significantly decreased peroxidase activity. However, the correlation of peroxidase activity with local and global stability, expected if cooperative unfolding of Ω-loops C and D is required for peroxidase activity, was generally poor. The m -values derived from the guanidine hydrochloride dependence of the kinetics of imidazole binding to horse Cyt c , which is well-characterized by native-state hydrogen exchange methods, and K72A/K73A/K79A iso-1-Cyt c show that local structural fluctuations and not subglobal cooperative unfolding of Ω-loops C and D are sufficient to permit binding of a small molecule like peroxide to the heme. A 2.46 Å structure of N63T iso-1-Cyt c identifies a change to a hydrogen bond network linking Ω-loops C and D that could modulate the local fluctuations needed for the intrinsic peroxidase activity of Cyt c. Substitutions to yeast iso-1-cytochrome c at coevolved residues in Ω-loop C from human cytochrome c have compensatory effects on local and global stability but decrease intrinsic peroxidase activity. Imidazole binding to cytochrome c indicates that small dynamic fluctuations in Ω-loop C may control the on/off switch for apoptotic peroxidase signaling. [Display omitted] • Yeast iso-1-cytochrome c variants at three coevolved sites in Ω-loop C were made. • Effects on local and global stability are modest. • All variants reduce peroxidase activity at pH 6 and above. • Apoptotic peroxidase activity is not solely controlled by Ω-loop C/D stability. • Imidazole binding studies show that local dynamics may mediate peroxidase activity. [ABSTRACT FROM AUTHOR]