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

摘要

Leishmania major peroxidase (LmP) is structurally and functionally similar to the well-studied yeast cytochrome c peroxidase (CCP). A recent Brownian dynamics study showed that L. major 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 carried out multiple molecular dynamics simulations of LmP-LmCytc complex. In 5 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 non-specific 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 Tyr134LmP as being important in the association/dissociation reactions. Experimentally mutating Tyr134 to Phe was found to decrease Km by 3.6 fold, consistent with its predicted role in complex formation by molecular dynamics simulations.