Fine Tuning of Functional Features of the Cu Site by Loop-Directed Mutagenesis.

Here we report the spectroscopic and electrochemical characterization of three novel chimeric Cu proteins in which either one or the three loops surrounding the metal ions in the Thermus thermophilus protein have been replaced by homologous human and plant sequences while preserving the set of coordinating amino acids. These conservative modifications mimic basic differences between Cu sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of Cu. This results in a systematic modulation of thermodynamic and kinetic electron transfer (ET) parameters and in the selection of one of two possible redox-active molecular orbitals, which differ in the ET reorganization energy by a factor of 2. Moreover, the ET mechanism is found to be frictionally controlled, and the modifications introduced into the different chimeras do not affect the frictional activation parameter.