Electrochemical characterization of an engineered red copper protein featuring an unprecedented entropic control of the reduction potential.

Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear Cu sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis.

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 .

Dramatic Electronic Perturbations of Cu Centers via Subtle Geometric Changes.

Cu is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, Cu is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima, σ* and π, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states.

Tuning of Enthalpic/Entropic Parameters of a Protein Redox Center through Manipulation of the Electronic Partition Function.

Manipulation of the partition function (Q) of the redox center Cu from cytochrome c oxidase is attained by tuning the accessibility of a low lying alternative electronic ground state and by perturbation of the electrostatic potential through point mutations, loop engineering and pH variation. We report clear correlations of the entropic and enthalpic contributions to redox potentials with Q and with the identity and hydrophobicity of the weak axial ligand, respectively.