Insights into the Dynamics and Dissociation Mechanism of a Protein Redox Complex Using Molecular Dynamics

<i>Leishmania major</i> peroxidase (LmP) is structurally and functionally similar to the well-studied yeast Cytochrome c peroxidase (CCP). A recent Brownian dynamics study showed that <i>L. major</i> Cytochrome c (LmCytc) associates with LmP by forming an initial complex with the N-terminal helix A of LmP, followed by a movement toward the electron transfer (ET) site observed in the LmP–LmCytc crystal structure. Critical to forming the active electron transfer complex is an intermolecular Arg-Asp ion pair at the center of the interface. If the dissociation reaction is effectively the reverse of the association reaction, then rupture of the Asp-Arg ion pair should be followed by movement of LmCytc back toward LmP helix A. To test this possibility, we have performed multiple molecular dynamics (MD) simulations of the LmP–LmCytc complex. In five separate simulations, LmCytc is observed to indeed move toward helix A, and in two of the simulations, the Asp–Arg ion pair breaks, which frees LmCytc to fully associate with the LmP helix A secondary binding site. These results support the “bind and crawl” or “velcro” mechanism of association, wherein LmCytc forms a nonspecific electrostatic complex with LmP helix A, followed by a “crawl” toward the ET-active site, where the Asp–Arg ion pair holds the LmCytc in position for rapid ET. These simulations also point to Tyr134_LmP as being important in the association/dissociation reactions. Experimentally mutating Tyr134 to Phe was found to decrease <i>K</i>_m by 3.6-fold, which is consistent with its predicted role in complex formation by MD simulations