Lysine dissociation from ferric heme iron is affected by properties of the heme pocket

Amine is a common ligand to porphyrin metal centers. The number of heme proteins having this property keeps growing. In multiple biological systems, Lys dissociation serves to regulate enzymatic activity, and therefore it is important to understand mechanistic principles that govern this process. The redox-linked ligand switching in heme proteins having a thiolate axial ligand provides a convenient means to monitor the ligand dissociation kinetics, and we have previously applied this approach to characterize Lys80 dissociation in the M80K#/F82C (where M80K# denotes K72A/K73A/K79G/M80K/C102S) variant of yeast iso-1 cytochrome (cyt) c having Cys introduced at position 82. Herein, we present the T78C/M80K# variant, with Cys at the other side of Lys80 (position 78), which exhibits an order-of-magnitude faster Lys80 dissociation rate. Spectroscopic studies of protein unfolding suggest that the T78C/M80K# variant is more stable toward acid and thermal denaturation than the M80K#/F82C variant is, in both ferric and ferrous forms. Molecular dynamics simulations visualize differences in the heme pocket between the two variants and reveal distinct local protein dynamics. In T78C/M80K#, the dynamics of Ω loops C and D that surround the heme pocket are greater. The replacement of Thr82 with Cys in T78C/M80K# perturbs the hydrogen-bonding network that connects the two loops. The solvent (and proton) access to the heme pocket and to Lys80 is facilitated, resulting in faster dissociation rates. The studies highlight the intricate relationship between protein global stability and local dynamics.