Molecular basis of HO/O/OH discrimination during electrochemical activation of DyP peroxidases: The critical role of the distal residues.

Here, we show that the replacement of the distal residues Asp and/or Arg of the DyP peroxidases from Bacillus subtilis and Pseudomonas putida results in functional enzymes, albeit with spectroscopically perturbed active sites. All the enzymes can be activated either by the addition of exogenous HO or by in situ electrochemical generation of the reactive oxygen species (ROS) OH, O and HO. The latter method leads to broader and upshifted pH-activity profiles.

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.

Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial.

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 .

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.

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.