Many heme proteins undergo switching of axial ligands at the heme iron, often as a mechanism to modulate electron transfer (ET) and catalytic activity. In cytochrome c, the axial ligands are His18 and Met80, but the Met ligand is a poor match for the ferric heme iron and is readily displaced. Networks of adjoining hydrogen bonds formed through the second coordination sphere create the protein scaffold necessary for maintaining Met ligation to the ferric heme iron. A key component of these interactions are the propionate moieties on the periphery of the heme, which have been shown to affect the dynamics of ligand replacement, the reduction potential at the heme iron, as well as the pK_a of the ligand switch reaction from Met to Lys known as the alkaline transition. Heme propionate 6 (HP6) makes connections to two low stability foldons in the protein, Ω-loops C and D, through hydrogen bonding interactions with the highly conserved residues Thr49 and Thr78. Recent work has shown that perturbing each of these connections resulted in shifting the pK_a of the Met-to-Lys transition to near-neutral pH. We have created and characterized the variant T49V/T78V/K79G in which both these HP6 connections are broken. Electronic absorption and NMR studies suggest Met/His ligation in the ferrous protein throughout a wide pH range, but a complete absence of Met ligation in the ferric protein. Analysis of the pH titration spectral data for the ferric protein supports a transition from a high-spin H₂O-ligated species to a low-spin Lys-ligated species with a pK_a value of 6.4 ± 0.1. Thermal and chemical denaturation experiments suggest that the integrity of the secondary structure is largely maintained, and the thermodynamic parameters yielded from such experiments indicate that the global stability of the T49V/T78V/K79G variant is comparable to those of T49V/K79G and T78V/K79G with consideration of the error bars. In contrast, the vicinity of Met80 and dynamics of surrounding Ω-loops C and D are perturbed more. These results reveal a cooperative nature of propionate interactions in controlling ligation in heme proteins and a role of perturbations of these contacts in switching ET properties of these proteins.

Yunling Deng

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