Cu-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential.

Attaining rational modulation of thermodynamic and kinetic redox parameters of metalloproteins is a key milestone towards the (re)design of proteins with new or improved redox functions. Here we report that implantation of ligand loops from natural T1 proteins into the scaffold of a Cu protein leads to a series of distorted T1-like sites that allow for independent modulation of reduction potentials (°') and electron transfer reorganization energies (). On the one hand °' values could be fine-tuned over 120 mV without affecting . On the other, values could be modulated by more than a factor of two while affecting °' only by a few millivolts. These results are in sharp contrast to previous studies that used T1 cupredoxin folds, thus highlighting the importance of the protein scaffold in determining such parameters.