Electrochemistry in sensing of molecular interactions of proteins and their behavior in an electric field.

Electrochemical methods can be used not only for the sensitive analysis of proteins but also for deeper research into their structure, transport functions (transfer of electrons and protons), and sensing their interactions with soft and solid surfaces. Last but not least, electrochemical tools are useful for investigating the effect of an electric field on protein structure, the direct application of electrochemical methods for controlling protein function, or the micromanipulation of supramolecular protein structures. There are many experimental arrangements (modalities), from the classic configuration that works with an electrochemical cell to miniaturized electrochemical sensors and microchip platforms. The support of computational chemistry methods which appropriately complement the interpretation framework of experimental results is also important. This text describes recent directions in electrochemical methods for the determination of proteins and briefly summarizes available methodologies for the selective labeling of proteins using redox-active probes. Attention is also paid to the theoretical aspects of electron transport and the effect of an external electric field on the structure of selected proteins. Instead of providing a comprehensive overview, we aim to highlight areas of interest that have not been summarized recently, but, at the same time, represent current trends in the field.

Applicability of perturbed matrix method for charge transfer studies at bio/metallic interfaces: a case of azurin.

As the field of nanoelectronics based on biomolecules such as peptides and proteins rapidly grows, there is a need for robust computational methods able to reliably predict charge transfer properties at bio/metallic interfaces. Traditionally, hybrid quantum-mechanical/molecular-mechanical techniques are employed for systems where the electron hopping transfer mechanism is applicable to determine physical parameters controlling the thermodynamics and kinetics of charge transfer processes. However, these approaches are limited by a relatively high computational cost when extensive sampling of a configurational space is required, like in the case of soft biomatter. For these applications, semi-empirical approaches such as the perturbed matrix method (PMM) have been developed and successfully used to study charge-transfer processes in biomolecules. Here, we explore the performance of PMM on prototypical redox-active protein azurin in various environments, from solution to vacuum interfaces with gold surfaces and protein junction. We systematically benchmarked the robustness and convergence of the method with respect to the quantum-centre size, size of the Hamiltonian, number of samples, and level of theory. We show that PMM can adequately capture all the trends associated with the structural and electronic changes related to azurin oxidation at bio/metallic interfaces.

Tunneling-to-Hopping Transition in Multiheme Cytochrome Bioelectronic Junctions.

Multiheme cytochromes (MHCs) have attracted much interest for use in nanobioelectronic junctions due to their high electronic conductances. Recent measurements on dry MHC junctions suggested that a coherent tunneling mechanism is operative over surprisingly long long distances (>3 nm), which challenges our understanding of coherent transport phenomena. Here we show that this is due to (i) a low exponential distance decay constant for coherent conduction in MHCs (ß = 0.2 Å-1) and (ii) a large density of protein electronic states which prolongs the coherent tunneling regime to distances that exceed those in molecular wires made of small molecules. Incoherent hopping conduction is uncompetitive due to the large energy level offset at the protein-electrode interface. Removing this offset, e.g., by gating, we predict that the transport mechanism crosses over from coherent tunneling to incoherent hopping at a protein size of ∼7 nm, thus enabling transport on the micrometer scale with a shallow polynomial (∼1/r) distance decay.

Adsorption of Hexavalent Chromium Using Activated Carbon Produced from Sargassum ssp.: Comparison between Lab Experiments and Molecular Dynamics Simulations.

Adsorption is one of the most successful physicochemical approaches for removing heavy metal contaminants from polluted water. The use of residual biomass for the production of adsorbents has attracted a lot of attention due to its cheap price and environmentally friendly approach. The transformation of Sargassum-an invasive brown macroalga-into activated carbon (AC) via phosphoric acid thermochemical activation was explored in an effort to increase the value of Sargassum seaweed biomass. Several techniques (nitrogen adsorption, pHPZC, Boehm titration, FTIR and XPS) were used to characterize the physicochemical properties of the activated carbons. The SAC600 3/1 was predominantly microporous and mesoporous (39.6% and 60.4%, respectively) and revealed a high specific surface area (1695 m2·g-1). To serve as a comparison element, a commercial reference activated carbon with a large specific surface area (1900 m2·g-1) was also investigated. The influence of several parameters on the adsorption capacity of AC was studied: solution pH, solution temperature, contact time and Cr(VI) concentration. The best adsorption capacities were found at very acid (pH 2) solution pH and at lower temperatures. The adsorption kinetics of SAC600 3/1 fitted well a pseudo-second-order type 1 model and the adsorption isotherm was better described by a Jovanovic-Freundlich isotherm model. Molecular dynamics (MD) simulations confirmed the experimental results and determined that hydroxyl and carboxylate groups are the most influential functional groups in the adsorption process of chromium anions. MD simulations also showed that the addition of MgCl2 to the activated carbon surface before adsorption experiments, slightly increases the adsorption of HCrO4- and CrO42- anions. Finally, this theoretical study was experimentally validated obtaining an increase of 5.6% in chromium uptake.

Reorganization free energy of copper proteins in solution, in vacuum, and on metal surfaces.

Metalloproteins, known to efficiently transfer electronic charge in biological systems, recently found their utilization in nanobiotechnological devices where the protein is placed into direct contact with metal surfaces. The feasibility of oxidation/reduction of the protein redox sites is affected by the reorganization free energies, one of the key parameters determining the transfer rates. While their values have been measured and computed for proteins in their native environments, i.e., in aqueous solution, the reorganization free energies of dry proteins or proteins adsorbed to metal surfaces remain unknown. Here, we investigate the redox properties of blue copper protein azurin, a prototypical redox-active metalloprotein previously probed by various experimental techniques both in solution and on metal/vacuum interfaces. We used a hybrid quantum mechanical/molecular mechanical computational technique based on density functional theory to explore protein dynamics, flexibility, and corresponding reorganization free energies in aqueous solution, vacuum, and on vacuum gold interfaces. Surprisingly, the reorganization free energy only slightly decreases when azurin is dried because the loss of the hydration shell leads to larger flexibility of the protein near its redox site. At the vacuum gold surfaces, the energetics of the structure relaxation depends on the adsorption geometry; however, significant reduction of the reorganization free energy was not observed. These findings have important consequences for the charge transport mechanism in vacuum devices, showing that the free energy barriers for protein oxidation remain significant even under ultra-high vacuum conditions.

Effects of Externally Applied Electric Fields on the Manipulation of Solvated-Chignolin Folding: Static- versus Alternating-Field Dichotomy at Play.

The interaction between a protein and external electric field (EF) can alter its structure and dynamical behavior, which has a potential impact on the biological function of proteins and cause uncertain health consequences. Conversely, the application of EFs of judiciously selected intensity and frequency can help to treat disease, and optimization of this requires a greater understanding of EF-induced effects underpinning basic protein biophysics. In the present study, chignolin─an artificial protein sufficiently small to undergo fast-folding events and transitions─was selected as an ideal prototype to investigate how, and to what extent, externally applied electric fields may manipulate or influence protein-folding phenomena. Nonequilibrium molecular dynamics (NEMD) simulations have been performed of solvated chignolin to determine the distribution of folding states and their underlying transition dynamics, in the absence and presence of externally applied electric fields (both static and alternating); a key focus has been to ascertain how folding pathways are altered in an athermal sense by external fields. Compared to zero-field conditions, a dramatically different─indeed, bifurcated─behavior of chignolin-folding processes emerges between static- and alternating-field scenarios, especially vis-à-vis incipient stages of hydrophobic-core formation: in alternating fields, fold-state populations diversified, with an attendant acceleration of state-hopping folding kinetics, featuring the concomitant emergence of a new, quasi-stable structure compared to the native structure, in field-shifted energy landscapes.

HAB79: A new molecular dataset for benchmarking DFT and DFTB electronic couplings against high-level ab initio calculations.

A new molecular dataset called HAB79 is introduced to provide ab initio reference values for electronic couplings (transfer integrals) and to benchmark density functional theory (DFT) and density functional tight-binding (DFTB) calculations. The HAB79 dataset is composed of 79 planar heterocyclic polyaromatic hydrocarbon molecules frequently encountered in organic (opto)electronics, arranged to 921 structurally diverse dimer configurations. We show that CASSCF/NEVPT2 with a minimal active space provides a robust reference method that can be applied to the relatively large molecules of the dataset. Electronic couplings are largest for cofacial dimers, in particular, sulfur-containing polyaromatic hydrocarbons, with values in excess of 0.5 eV, followed by parallel displaced cofacial dimers. V-shaped dimer motifs, often encountered in the herringbone layers of organic crystals, exhibit medium-sized couplings, whereas T-shaped dimers have the lowest couplings. DFT values obtained from the projector operator-based diabatization (POD) method are initially benchmarked against the smaller databases HAB11 (HAB7-) and found to systematically improve when climbing Jacob's ladder, giving mean relative unsigned errors (MRUEs) of 27.7% (26.3%) for the generalized gradient approximation (GGA) functional BLYP, 20.7% (15.8%) for hybrid functional B3LYP, and 5.2% (7.5%) for the long-range corrected hybrid functional omega-B97X. Cost-effective POD in combination with a GGA functional and very efficient DFTB calculations on the dimers of the HAB79 database give a good linear correlation with the CASSCF/NEVPT2 reference data, which, after scaling with a multiplicative constant, gives reasonably small MRUEs of 17.9% and 40.1%, respectively, bearing in mind that couplings in HAB79 vary over 4 orders of magnitude. The ab initio reference data reported here are expected to be useful for benchmarking other DFT or semi-empirical approaches for electronic coupling calculations.

Dielectric properties of ice VII under the influence of time-alternating external electric fields.

The high-pressure solid phase of water known as ice VII has recently attracted a lot of attention when its presence was detected in large exoplanets, their icy satellites, and even in Earth's mantle. Moreover, a transition of ice VII to the superionic phase can be triggered by external electric fields. Here, we investigate the dielectric responses of ice VII to applied oscillating electric fields of various frequencies employing non-equilibrium ab initio molecular dynamics. We focus on the dynamical properties of a dipole-ordered ice VII structure, for which we explored external-field-induced electronic polarisation and the vibrational spectral density of states (VDOS). These analyses are important for the understanding of collective motions in the ice-VII lattice and the electronic properties of this exotic water phase.

Water Breakup at Fe2O3-Hematite/Water Interfaces: Influence of External Electric Fields from Nonequilibrium Ab Initio Molecular Dynamics.

The dynamical properties of physically and chemically adsorbed water molecules at pristine hematite-(001) surfaces have been studied by means of nonequilibrium ab initio molecular dynamics (NE-AIMD) in the NVT ensemble at room temperature, in the presence of externally applied, uniform static electric fields of increasing intensity. The dissociation of water molecules to form chemically adsorbed species was scrutinized, in addition to charge redistribution and Grotthus proton hopping between water molecules. Dynamical properties of the adsorbed water molecules and OH- and H3O+ ions were gauged, such as the hydrogen bonds between protons in water molecules and the bridging oxygen atoms at the hematite surface, as well as the interactions between oxygen atoms in adsorbed water molecules and iron atoms at the hematite surface. The development of Helmholtz charge layers via water breakup at Fe2O3-hematite/water interfaces is also an interesting feature, with the development of protonic conduction on the surface and more bulk-like water.

Dynamics of Meso-Chiral Interconversion in a Butterfly-Shape Overcrowded Alkene Rotor Tunable by Solvent Properties.

Elucidation of dynamics of molecular rotational motion is an essential part and challenging area of research. We demonstrate reversible diastereomeric interconversion of a molecular rotor composed of overcrowded butterfly-shape alkene (FDF). Its inherent dual rotatory motion (two rotors, one stator) with interconversion between two diastereomers, chiral trans-FDF and meso cis-FDF forms, has been examined in detail upon varying temperatures and solvents. The free energy profile of 180° revolution of one rotor part has a bimodal shape with unevenly positioned maxima (transition states). FDF in aromatic solvents adopts preferentially meso cis-conformation, while in non-aromatic solvents a chiral trans-conformation is more abundant owing to the solvent interactions with peripheral hexyl chains (solvophobic effect). Moderate correlations between the trans-FDF/cis-FDF ratio and solvent parameters, such as refractive index, polarizability, and viscosity were found.

Amino-acid interactions with the Au(111) surface: adsorption, band alignment, and interfacial electronic coupling.

The charge transport properties of biological molecules like peptides and proteins are intensively studied for the great flexibility, redox-state variability, long-range efficiency, and biocompatibility of potential bioelectronic applications. Yet, the electronic interactions of biomolecules with solid metal surfaces, determining the conductivities of the biomolecular junctions, are hard to predict and usually unavailable. Here, we present accurate adsorption structures and energies, electronic band alignment, and interfacial electronic coupling data for all 20 natural amino acids computed using the DFT+Σ scheme based on the vdW-DF and OT-RSH functionals. For comparison, data obtained using the popular PBE functional are provided as well. Tryptophan, compared to other amino acids, is shown to be distinctly exceptional in terms of the electronic properties related to charge transport. Its high adsorption energy, frontier-orbital levels aligned relatively close to the Fermi energy of gold and strong interfacial electronic coupling make it an ideal candidate for facilitating charge transfer on such heterogeneous interfaces. Although the amino acids in peptides and proteins are affected by the structural interactions hindering their contact with the surface, knowledge of the single-molecule surface interactions is necessary for a detailed understanding of such structural effects and tuning of potential applications.

Coherent Electron Transport across a 3 nm Bioelectronic Junction Made of Multi-Heme Proteins.

Multi-heme cytochromes (MHCs) are fascinating proteins used by bacterial organisms to shuttle electrons within, between, and out of their cells. When placed in solid-state electronic junctions, MHCs support temperature-independent currents over several nanometers that are 3 orders of magnitude higher compared to other redox proteins of similar size. To gain molecular-level insight into their astonishingly high conductivities, we combine experimental photoemission spectroscopy with DFT+Σ current-voltage calculations on a representative Gold-MHC-Gold junction. We find that conduction across the dry, 3 nm long protein occurs via off-resonant coherent tunneling, mediated by a large number of protein valence-band orbitals that are strongly delocalized over heme and protein residues. This picture is profoundly different from the electron hopping mechanism induced electrochemically or photochemically under aqueous conditions. Our results imply that the current output in solid-state junctions can be even further increased in resonance, for example, by applying a gate voltage, thus allowing a quantum jump for next-generation bionanoelectronic devices.

Possibility of realizing superionic ice VII in external electric fields of planetary bodies.

In a superionic (SI) ice phase, oxygen atoms remain crystallographically ordered while protons become fully diffusive as a result of intramolecular dissociation. Ice VII's importance as a potential candidate for a SI ice phase has been conjectured from anomalous proton diffusivity data. Theoretical studies indicate possible SI prevalence in large-planet mantles (e.g., Uranus and Neptune) and exoplanets. Here, we realize sustainable SI behavior in ice VII by means of externally applied electric fields, using state-of-the-art nonequilibrium ab initio molecular dynamics to witness at first hand the protons' fluid dance through a dipole-ordered ice VII lattice. We point out the possibility of SI ice VII on Venus, in its strong permanent electric field.

Ergodicity Breaking in Thermal Biological Electron Transfer? Cytochrome C Revisited II.

It was recently suggested that cytochrome c operates in an ergodicity-breaking regime characterized by unusually large energy gap thermal fluctuations and associated reorganization free energies for heme oxidation of up to 3.0 eV. The large fluctuations were reported to lower activation free energy for oxidation of the heme cofactor by almost a factor of 2 compared to the case where ergodicity is maintained. Our group has recently investigated this claim computationally at several levels of theory and found no evidence for such large energy gap fluctuations. Here we address the points of our earlier work that have raised criticism and we also extend our previous investigation by considering a simple linear polarizability model for cytochrome c oxidation. We find very consistent results among all our computational approaches, ranging from classical molecular dynamics, to the linear polarizability model to QM(PMM)/MM to full QM(DFT)/MM electrostatic emdedding. None of them support the notion of very large energy gap fluctuations or ergodicity breaking. The deviation between our simulations and the ones reported in [ J. Phys. Chem. B 2017, 121, 4958] is traced back to rather large electric fields at the Fe site of the heme c cofactor in that study, not seen in our simulations, neither with the AMBER nor with the CHARMM force field. While ergodicity breaking effects may well occur in other biological ET, our numerical evidence suggests that this is not the case for cytochrome c.

Ergodicity-Breaking in Thermal Biological Electron Transfer? Cytochrome C Revisited.

It was recently suggested that certain redox proteins operate in an ergodicity-breaking regime to facilitate biological electron transfer (ET). A signature for this is a large variance reorganization free energy (several electronvolts) but a significantly smaller Stokes reorganization free energy due to incomplete protein relaxation on the time scale of the ET event. Here we investigate whether this picture holds for oxidation of cytochrome c in aqueous solution, at various levels of theory including classical molecular dynamics with two additive and one electronically polarizable force field, and QM/MM calculations with the QM region treated by full electrostatic DFT embedding and by the perturbed matrix method. Sampling the protein and energy gap dynamics over more than 250 ns, we find no evidence for ergodicity-breaking effects. In particular, the inclusion of electronic polarizability of the heme group at QM/MM levels did not induce nonergodic effects, contrary to previous reports by Matyushov et al. The well-known problem of overestimation of reorganization free energies with additive force fields is cured when the protein and solvent are treated as electronically polarizable. Ergodicity-breaking effects may occur in other redox proteins, and our results suggest that long simulations, ideally on the ET time scale, with electronically polarizable force fields are required to obtain strong numerical evidence for them.

Multimodal switching of a redox-active macrocycle.

Molecules that can exist in multiple states with the possibility of toggling between those states based on different stimuli have potential for use in molecular switching or sensing applications. Multimodal chemical or photochemical oxidative switching of an antioxidant-substituted resorcinarene macrocycle is reported. Intramolecular charge-transfer states, involving hemiquinhydrones are probed and these interactions are used to construct an oxidation-state-coupled molecular switching manifold that reports its switch-state conformation via striking variation in its electronic absorption spectra. The coupling of two different oxidation states with two different charge-transfer states within one macrocyclic scaffold delivers up to five different optical outputs. This molecular switching manifold exploits intramolecular coupling of multiple redox active substituents within a single molecule.

Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF.

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.

Adsorption of Amino Acids on Gold: Assessing the Accuracy of the GolP-CHARMM Force Field and Parametrization of Au-S Bonds.

The interaction of amino acids with metal electrodes plays a crucial role in bioelectrochemistry and the emerging field of bionanoelectronics. Here we present benchmark calculations of the adsorption structure and energy of all natural amino acids on Au(111) in vacuum using a van-der-Waals density functional (revPBE-vdW) that showed good performance on the S22 set of weakly bound dimers (mean relative unsigned error (MRUE) wrt CCSD(T)/CBS = 13.3%) and adsorption energies of small organic molecules on Au(111) (MRUE wrt experiment = 11.2%). The vdW-DF results are then used to assess the accuracy of a popular force field for Au-amino acid interactions, GolP-CHARMM, which explicitly describes image charge interactions via rigid-rod dipoles. We find that while the force field underestimates adsorption distances, it does reproduce the binding energy rather well (MRUE wrt revPBE-vdW = 11.3%) with the MRUE decreasing in the order Cys, Met > amines > aliphatic > carboxylic > aromatic. We also present a parametrization of the bonding interaction between sulfur-containing molecules and the Au(111) surface and report force field parameters that are compatible with GolP-CHARMM. We believe the vdW-DF calculations presented herein will provide useful reference data for further force field development, and that the new Au-S bonding parameters will enable improved simulations of proteins immobilized on Au-electrodes via S-linkages.

Pressure dependence of structural properties of ice VII: An ab initio molecular-dynamics study.

The observed anomalous self-diffusivity of ice VII in the region of 10 GPa at ∼400 K has been suggested to arise from a change in proton-hopping mechanism involving a transition from ionic-defect-driven diffusivity to that dominated by diffusion of rotational defects. Here, we report ab initio molecular dynamics to study the structural, hydrogen bonding, electronic, vibrational, and Raman properties of ice VII at this temperature and between 5 and 20 GPa to elucidate any possible hints of intramolecular strain that may serve as precursor events for proton hopping to unfold. We determine such equilibrium properties to be in reasonable agreement with experimental Raman spectra, although we do not detect any water-dissociation and proton-hopping events per se, owing to still-large water-dissociation free-energy barriers.