Prediction of Reduction Potentials of Copper Proteins with Continuum Electrostatics and Density Functional Theory

Abstract

Blue copper proteins, such as azurin, show dramatic changes in Cu2+/Cu+ reduction potential upon mutation over the full physiological range. Hence, they have important functions in electron transfer and oxidation chemistry and have applications in industrial biotechnology. The details of what determines these reduction potential changes upon mutation are still unclear. Moreover, it has been difficult to model and predict the reduction potential of azurin mutants and currently no unique procedure or workflow pattern exists. Furthermore, high‐level computational methods can be accurate but are too time consuming for practical use. In this work, a novel approach for calculating reduction potentials of azurin mutants is shown, based on a combination of continuum electrostatics, density functional theory and empirical hydrophobicity factors. Our method accurately reproduces experimental reduction potential changes of 30 mutants with respect to wildtype within experimental error and highlights the factors contributing to the reduction potential change. Finally, reduction potentials are predicted for a series of 124 new mutants that have not yet been investigated experimentally. Several mutants are identified that are located well over 10 Å from the copper center that change the reduction potential by more than 85 mV. The work shows that secondary coordination sphere mutations mostly lead to long‐range electrostatic changes and hence can be modeled accurately with continuum electrostatics.

A Comparative Review on the Catalytic Mechanism of Nonheme Iron Hydroxylases and Halogenases

“…This enables studies on full proteins that keep the orientation and constraints of the enzyme in the model. These studies are reasonably accurate, and recent benchmark and calibration studies that compare a range of density functional theory methods versus experimental rate constants, kinetic isotope effects, and spectroscopic parameters are in good agreement [187][188][189][190][191][192][193].…”

Ab initio electronic structure calculations of entire blue copper azurins

“…A. Fereiro et al 38 However, a close-up inspection reveals slight differences between the mutants, as expected from the observed variations of the redox potentials. 23,25 See Fig. S15 of the ESI, † where some graphical pairwise comparisons have been carried out.…”

“…For this purpose, several theoretical approaches are currently available to estimate the redox potential of different mutants. [24][25][26] However, this complex behavior, which determines the changes in the reduction potential upon mutation, is still unclear and a deep understanding of what governs them is to date still missing.…”

Does Substrate Positioning Affect the Selectivity and Reactivity in the Hectochlorin Biosynthesis Halogenase?

“…A detailed benchmark study comparing computational free energies of activation with experimental data from Eyring plots gave good agreement between experiment and theory for the methods described here (Cantú Reinhard et al, 2016a ). Furthermore, these methods were also seen to excellently reproduce reduction potentials of copper proteins, (Fowler et al, 2017 ) and regio- and chemoselectivities of reaction mechanisms (Jastrzebski et al, 2014 ; Barman et al, 2016b ; Yang et al, 2016 ).…”