Dimitri E. Khoshtariya

Fundamental signatures of short- and long-range electron transfer for the blue copper protein azurin at Au/SAM junctions

The blue copper protein from Pseudomonas aeruginosa, azurin, immobilized at gold electrodes through hydrophobic interaction with alkanethiol self-assembled monolayers (SAMs) of the general type [-S - (CH2)n - CH3] (n = 4, 10, and 15) was employed to gain detailed insight into the physical mechanisms of short- and long-range biomolecular electron transfer (ET). Fast scan cyclic voltammetry and a Marcus equation analysis were used to determine unimolecular standard rate constants and reorganization free energies for variable n, temperature (2–55 °C), and pressure (5–150 MPa) conditions. A novel global fitting procedure was found to account for the reduced ET rate constant over almost five orders of magnitude (covering different n, temperature, and pressure) and revealed that electron exchange is a direct ET process and not conformationally gated. All the ET data, addressing SAMs with thickness variable over ca. 12 Å, could be described by using a single reorganization energy (0.3 eV), however, the values for the enthalpies and volumes of activation were found to vary with n. These data and their comparison with theory show how to discriminate between the fundamental signatures of short- and long-range biomolecular ET that are theoretically anticipated for the adiabatic and nonadiabatic ET mechanisms, respectively.

Multiple Mechanisms for Electron Transfer at Metal/Self-Assembled Monolayer/Room-Temperature Ionic Liquid Junctions: Dynamical Arrest versus Frictional Control and Non-Adiabaticity

Electrochemical devices consisting of gold electrodes coated by electronically well-behaved self-assembled alkanethiol monolayers of variable thickness, a ferrocene/ferrocenium redox probe and a typical room-temperature ionic liquid (RTIL) [bmim][NTf(2)] (1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide) as a unique reaction medium with an exceptionally broad spectrum of relaxational modes (probed under variable temperature and pressure conditions), have been used to vary the intrinsic electron-transfer (ET) rate constant over eight orders of magnitude (from 0.1 to 3x10(7) s(-1)) by further tuning of the overvoltage. A remarkable interplay of ET mechanisms was observed, which was accompanied by the stepwise drop in the reorganisation free energy of the medium from 1.0 to 0.1 eV. The first mechanistic changeover to the dynamically arrested regime, with a locking ultra-slow relaxation time of approximately 50 micros, occurred at donor-acceptor separations below 20 A. Another mechanistic changeover to the full solvent friction regime, controlled by a medium relaxation process of approximately 100 ns, emerged for ET distances smaller than 8 A.

Heterogeneous electron transfer at Au/SAM junctions in a room-temperature ionic liquid under pressure.

Proven electrochemical approaches were applied to study heterogeneous electron transfer (ET) between selected redox couples and gold electrodes modified with alkanethiol self-assembled monolayers (SAMs), using the room-temperature ionic liquid (RTIL) [bmim][NTf2] as reaction medium; ferrocene as freely diffusing redox probe in the RTIL was tested for ET through both thin (butanethiol) and thick (dodecanethiol) assemblages at pressures up to 150 MPa; well behaved kinetic patterns and reproducibility of data were demonstrated for ET within the unique Au/SAM/RTIL arrays.

Simplicity within the complexity: bilateral impact of DMSO on the functional and unfolding patterns of α-chymotrypsin.

New understanding of the fundamental links between protein stability, conformational flexibility and function, can be gained through synergic studies on their catalytic and folding/unfolding properties under the influence of stabilizing/destabilizing additives. We explored an impact of dimethyl sulfoxide (DMSO), the moderate effector of multilateral action, on the kinetic (functional) and thermodynamic (thermal unfolding) patterns of a hydrolytic enzyme, α-chymotrypsin (α-CT), over a wide range of additive concentrations, 0-70% (v/v). Both the calorimetric and kinetic data exhibited rich behavior pointing to the complex interplay of global/local stability (and flexibility) patterns. The complex action of DMSO is explained through the negative and positive preferential solvation motifs that prevail for the extreme opposite, native-like and unfolded states, respectively, implying essential stabilization of compact domains by enhancement of interfacial water networks and destabilization of a flexible active site by direct binding of DMSO to the unoccupied specific positions intended for elongated polypeptide substrates.

The solvent friction mechanism for outer-sphere electron exchange at bare metal electrodes. The case of Au/Ru(NH3)63+/2+ redox system

The heterogeneous rate constant for the Ru(NH3)63+/2+ electron exchange at the bare gold electrode displays a power-law dependence on the solution viscosity (varied by addition of glucose, 0–600 g l−1) with a negative power index of δ=1, indicative of the “full” solvent friction (adiabatic) mechanism for the intrinsic charge-transfer step. Comparison with related processes suggests that this mechanism mainly occurs at bare metal electrodes irrespective of the reactants’ charged state and the method of viscosity variation. The adiabatic regime operates notwithstanding the presence of at least one layer of solvating water, and/or specifically adsorbed ions, contributing to the charge-transfer distance.

Temperature, pressure and solvent isotope effects on the precursor formation constant and reorganization dynamics in outer-sphere charge transfer between free mobile [Fe(CN)6]3– and [Fe(CN)6]4– ions

Temperature (298–348 K), high-pressure (0.1–150 MPa) and solvent isotope (H2O/D2O) effects upon the maximum energy and absorption intensity of the outer-sphere metal–metal charge-transfer (MMCT) absorption band in encounter complexes of free mobile [Fe(CN)6]3– and [Fe(CN)6]4– ions have been investigated. ΔHA=(– 1.82 ± 0.05) kJ mol–1, ΔSA=(– 30.7 ± 1.4) J mol–1, K–1, and ΔVA=(– 1.0 ± 0.2) cm3 mol–1 have been obtained for the precursor formation of the absorbing species at 298 K and 0.1 MPa. The absorption maximum shifts by 120 cm–1 to higher energy upon increasing the temperature from 298 to 348 K while a decrease by 200 cm–1 has been observed in the pressure range 0.1–150 MPa. Replacement of H2O by D2O as the solvent results in a blue shift of the MMCT absorption maximum by 460 cm–1 without change in the intensity. Water molecules of the first solvation sphere, hydrogen bonded to the reactants, contribute considerably to the reorganization Gibbs energy of the optical transition.

Electron transfer with azurin at Au–SAM junctions in contact with a protic ionic melt: impact of glassy dynamics

Gold electrodes were coated with alkanethiol SAM-azurin (Az, blue cupredoxin) assemblies and placed in contact with a water-doped and buffered protic ionic melt as the electrolyte, choline dihydrogen phosphate ([ch][dhp]). Fast-scan protein-film voltammetry was applied to explore interfacial biological electron transfer (ET) under conditions approaching the glass-transition border. The ET rate was studied as a function of the water amount, temperature (273-353 K), and pressure (0.1-150 MPa). Exposure of the Az films to the semi-solid electrolyte greatly affected the proteins conformational dynamics, hence the ET rate, via the mechanism occurring in the extra complicated dynamically-controlled regime, is compared to the earlier studies on the reference system with a conventional electrolyte (D. E. Khoshtariya et al., Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 2757-2762), allowing for the disclosure of even more uncommon mechanistic motifs. For samples with low water content (ca. 3 or less waters per [ch][dhp]), at moderately low temperatures (below ca. 298 K) and/or high pressure (150 MPa), the voltammetric profiles systematically deviated from the standard Marcus current-overvoltage pattern, deemed as attributable to a breakdown of the linear response approximation through the essential steepening of the Gibbs energy wells near the glass-forming threshold. Electrolytes with a higher water content (6 to 15 waters per [ch][dhp]) display anomalous temperature and pressure performances, suggesting that the system crosses a broad nonergodic zone which arises from the interplay of ET-coupled large-scale conformational (highly cooperative) modes of the Az protein, inherently linked to the electrolytes (water-doped [ch][dhp]) slowest collective relaxation(s).

Charge-Transfer Patterns for [Ru(NH3)6]3+/2+ at SAM Modified GoldElectrodes: Impact of the Permeability of a Redox Probe

Electrochemical performance of a (Ru(NH3)6) 3+/2+ redox couple at gold electrodes modified by alkanethiol self assembled monolayer (SAM) films of the type (-SH -(CH2)n - CH3) with different number of methylene units (n = 2 to 10) in the presence and absence of glucose additives has been studied using fast scan cyclic and steady-state voltammetry. Specific scatter of measured rate constants caused by enhanced sensitivity of this probe to minor defects of SAMs has been observed in a general agreement with the published data for thicker SAMs (n = 9 to 18). In addition, we have disclo- sed the anomalous viscosity-imposed drop of the heterogeneous rate constant for the case of Au electrodes modified by thinner n-alkanethiol SAMs (n = 2, 4). Taking into the account the fact of (Ru(NH3)6) 3+/2+ couples capability to penetrate into the SAM interior, we ascribe the obtained results to the manifestation of the solvent-friction mechanism under the condition where the redox species presumably together with a few of solvating water molecules reside in a SAMs peri- pheral interior marked by much higher local viscosity (slower dielectric relaxation) compared to the electrolyte solution.

Compact high pressure unit for ultraviolet-visible-near-infrared spectroscopic measurements at pressures up to 400 MPa

The construction of a compact high pressure unit for UV-VIS-NIR spectroscopic measurements at pressures up to 400 MPa is described. The pressure generating system can be operated with different pressure liquids, depending on the spectral characteristics required for specific applications. The system can be used for thermodynamic as well as a variety of kinetic measurements.

Fundamental Studies of Long- and Short-Range Electron Exchange Mechanisms between Electrodes and Proteins

Electron transfer1-7 by thermally activated hopping through localized centers is a common motif that occurs over a broad spectrum of matter such as non-conducting crystalline solids, amorphous glasses, fluidic/viscous liquids, and biomolecules. This basic kinetic motif is an essential element for a broad variety of vital biological and technological processes.8-10 New directions in electrochemistry, a discipline which has contributed greatly to developing the theoretical underpinnings of charge-transfer phenomena,1-7,11-13 promise to reveal important features of electron transfer (ET) in biomolecular redox chains. This new frontier in electrochemistry, which has been developing over the past two decades,14-53 uses chemically tunable, nanoscopic electrode structures for addressing fundamental questions in charge exchange between electrodes and biomolecules.14-21,54-88