CuA-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(A)protein leads to a series of distorted T1-like sites that allow for independent modulation of reduction potentials (E degrees ') and electron transfer reorganization energies (lambda). On the one handE degrees ' values could be fine-tuned over 120 mV without affecting lambda. On the other,lambda values could be modulated by more than a factor of two while affectingE degrees ' 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.