Sortase-mediated ligation of cytochrome c peroxidase and cytochrome c highlights the roles of dynamics and conformational specificity for interprotein electron transfer.

The complex between cytochrome c peroxidase (CcP) and cytochrome c (Cc) is an important model system for studying interprotein electron transfer (ET). Low ionic strength conditions stabilize the CcP:Cc complex, but promote unfavorable second-site binding of Cc. Conversely, high ionic strengths favor the 1:1 complex but promote its dissociation. We sought to stabilize the complex and minimize second-site binding by linking the two proteins together via sortase-mediated transpeptidation. Ligation efficiency of the two proteins depends on the length of the flexible linker and the conditions of the ligation reaction. Structural comparisons and AI-based predictions indicate that the conformations assumed by the fusion proteins depend substantially on the linker length. A short linker allows the association mode found in the 1:1 non-covalent complex but favors more extended states. Longer linkers are more conducive to productive complex formation but still sample other conformational states that disfavor interprotein ET. The degree of predicted conformational variation in the fusion proteins agrees well with their ET reactivity and structural analyses by crystallography and small-angle x-ray scattering. Our findings underscore that specific interaction modes between redox partners influence their electronic communication and reveal that interdomain linkers have the potential to control intermolecular reactions and alter the sampling of productive interfaces.