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 CuA 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. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the CuA scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.

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

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

Here we report the spectroscopic and electrochemical characterization of three novel chimeric CuA 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 CuA sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of CuA.. 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.

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 CuA 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.