Mutation of Pro76 affects the dynamics of Ω-loop D of yeast Iso-1-cytochrome c: Application of gated electron transfer

Cytochrome c (Cytc) contains two Ω-loops (C, residues 40–57 and D, residues 70–85) that control access of Cytc to alternate conformers needed to convert it from an electron transfer (ET) protein to an enzyme with peroxidase activity. As a peroxidase, Cytc oxygenates the mitochondrial lipid cardiolipin, which is an early signal in apoptosis. We probe the role of Pro76, a highly conserved residue in Ω-loop D, in controlling the access to alternate conformers of Cytc. We introduce histidines into Ω-loop D as specific probes of the alkaline conformational transition. We replaced Pro76 with His in the presence of a K72A mutation (A72H76 variant). Variants with K73H and P76G mutations (A72H73G76) and a K79A mutation added (A72H73G76A79) were also prepared. pH titrations monitored in the visible region show that, unlike the previously studied K73H variant, the native state (Met80 ligation) of Cytc is not populated between pH 2 and 11 for the A72H73G76 and A72H73G76A79 variants, indicating that the P76G mutation strongly destabilizes the native conformer. The A72H76 variant fully populates the native conformer near pH 5. GdnHCl unfolding studies and characterization of the alkaline transition by pH jump and gated ET kinetic studies were used to construct an energy landscape for the A72H76 variant. Comparison to other Ω-loop D histidine variants shows that the P76H mutation strongly destabilizes the native state and that Pro76 contributes to a native-like transition state. Both observations indicate that Pro76 is critical for controlling access to alternate conformers needed for peroxidase activity.