The role of molecular crowding in long-range metalloprotein electron transfer: Dissection into site- and scaffold-specific contributions

Mutational and structural analysis of an ancestral fungal dye‐decolorizing peroxidase

“…Last but not least, the mutants in which the arginine was replaced showed lower stability, which may reflect increased scaffold flexibility. Recent works have shown that the more flexible the scaffold, the higher the activation barrier of long-range electron transfer processes tends to be in proteins [48,49]. Such effect was described for several metalloenzymes, including hemecontaining cytochrome c [50,51].…”

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

“…This SAM composition has been shown to provide a suitable interface for adsorption and direct electrochemistry of cupredoxins in the nonadiabatic regime with retention of the active site structure. 29 , 82 Except for ; – Tt –Cu A and NiR– Tt –Cu A that gave no electrochemical signals, the voltammetries of the adsorbed chimeras yield quasi-reversible responses with charge transfer coefficients between 0.4 and 0.5, and peak currents that scale linearly with the scan rates, as expected for surface-confined redox active species (Fig. S13–S15 † ).…”

“…Before use Au electrodes were treated as described previously. 82 Briefly, after thorough chemical and electrochemical treatment, electrodes were incubated overnight in ethanolic solutions containing the desired alkanethiols to form self-assembled monolayer (SAM) coatings. After SAM-coating, electrodes were cycled repeatedly at 0.1 V s –1 within the potential windows appropriate for each protein in the measuring electrolyte solution (10 mM HEPES buffer, pH 7.0, containing 250 mM KNO 3 ).…”

Modulation of Functional Features in Electron Transferring Metalloproteins

“…A at the level of the three loops that surround the metal site (see Figure 6) As modification of the strong Cys and His ligands of the Cu A site are known to be disruptive, first coordination sphere modifications were restricted to the replacement of the weak axial ligand Met160 by a variety of amino acids. 3,[48][49][50][51][52][53][54] Second sphere modifications, on the other hand, were accomplished by replacement of either one or the three loops that surround the metal site (Figures 6 and Table 1) by the homologous sequences from Homo sapiens and Arabidopsis thaliana with preservation of the conserved ligand set. 50,54 Moreover, combined first and second coordination sphere modification was achieved by introducing the Met160His point mutation in the so-called Tt-3L-Hs chimera.…”

“…While this is certainly correct for most in vitro studies performed with diluted aqueous solutions (viscosity ≈ 1 cP), in vivo behavior can be drastically different since, for example, intracellular viscosities can be up to 500 times higher; therefore, long-range protein ET reactions are likely to be friction-controlled in spite of the weak coupling. 3,4 As mentioned above, the electronic coupling matrix element V is a measure of the strength of interactions between reactants and products at the nuclear configuration of the transition state. For a precise description of V in proteins (and macrobiomolecules in general) it is necessary to account for its dependence on the distance between redox centers, on the dielectric properties and atomistic description of the insulating (or conducting) material in between, and on fluctuations of electronic and structural parameters at different levels and time-scales of the multidimensional and hierarchical free energy landscapes that characterize macromolecules.…”