Effect on intrinsic peroxidase activity of substituting coevolved residues from ω-loop C of human cytochrome c into yeast iso-1-cytochrome c

Naturally-occurring variants of human cytochrome c (Cytc) that induce thrombocytopenia IV occur within omega-loop C (residues 40-57). These variants enhance the peroxidase activity of human Cytc apparently by facilitating access to the heme by destabilizing omega-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 omega-loop C that coevolve and differ between yeast iso-1-Cytc and human Cytc. We prepared iso-1-Cytc variants with all possible combinations of the S40T, V57I and N63T substitutions to determine if these residues decrease the peroxidase activity of iso-1-Cytc to that of human Cytc 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 omega-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 Cytc, which is well-characterized by native-state hydrogen exchange methods, and K72A/K73A/K79A iso-1-Cytc show that local structural fluctuations and not subglobal cooperative unfolding of omega-loops C and D are sufficient to permit binding of a small molecule like peroxide to the heme. A 2.46 angstrom structure of N63T iso-1-Cytc identifies a change to a hydrogen bond network linking omega-loops C and D that could modulate the local fluctuations needed for the intrinsic peroxidase activity of Cytc.

Automated summary

Naturally-occurring variants of human cytochrome c with altered omega-loop C enhance peroxidase activity by destabilizing omega-loops C and D. We identified coevolving sites in omega-loop C between yeast iso-1-Cytc and human Cytc and found that these residues decrease the peroxidase activity of iso-1-Cytc. Structural fluctuations in the loop regions are important for the intrinsic peroxidase activity of Cytc.