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 protein's 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 electrolyte's (water-doped [ch][dhp]) slowest collective relaxation(s).

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-(CH(2))(n)-CH(3)] (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 degrees 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 A, 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.

Unusual mechanism for the short-range electron transfer within gold-alkanethiol-ionic-liquid films of subnanometer thickness.

Exploiting nanoscopically tunable composite gold-alkanethiol-ionic-liquid/ferrocene self-assembled systems with tunable electron transfer distance, we discovered in the case of thinner alkanethiol films a thermally activated electron transfer pattern totally controlled by the viscosity-related slow relaxation mode(s) of the ionic liquid acting as the reactant's fluctuating environment. This pattern manifested through the activation enthalpy and volume parameters that are identical to those for viscous flow was explained in terms of the extreme adiabatic mechanism with a vanishing Marcus barrier (via the exponential Franck-Condon-like term approaching unity).

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.

High-pressure testing of heterogeneous charge transfer in a room-temperature ionic liquid: evidence for solvent dynamic control.

We report the first application of a high-pressure electrochemical strategy to study heterogeneous charge transfer (CT) in a room-temperature ionic liquid, [BMIM][BTA]. High-pressure kinetic studies on electron exchange for two redox couples of different charge type, viz. [Fe(bipy)3]3+/2+ and [Fe(cp)2]+/0, at bare Au electrodes within the range of 0.1-150 MPa, revealed large positive volumes of activation that were found to be virtually the same for the two redox couples in terms of the CT rate constants and diffusion coefficients, despite the reactant's charge type. Independent viscosity (fluidity) studies at elevated pressure (up to 175 MPa), were also performed and revealed a pressure coefficient closely resembling the former ones. Complementary temperature-dependent kinetic studies within the range of 298-358 K also revealed the virtual similarity in activation enthalpies for the same kinetic and diffusion processes, as well as the viscosity of [BMIM][BTA]. A rigorous analysis of the complete variety of obtained results strongly indicates that dynamic (frictional) control of CT is operative by way of the full adiabatic mechanism. The contribution of the Franck-Condon term to the activation free energy of the kinetic process seems almost diminished because of the high value of electronic coupling and freezing out of the outer-sphere reorganization energy. Further analyses indicate that frictional control most probably takes place through slow translational modes (implying "minimal volume" cooperative dislocations) of constituent ions. This kind of motion seems further slowed down within the vicinity of the active site presumably located within the diffusive-like zone situated next to the compact (first) part of the metal/ionic liquid junction.

Impact of self-assembly composition on the alternate interfacial electron transfer for electrostatically immobilized cytochrome c.

We report on the effects of self-assembled monolayer (SAM) dilution and thickness on the electron transfer (ET) event for cytochrome c (CytC) electrostatically immobilized on carboxyl terminated groups. We observed biphasic kinetic behavior for a logarithmic dependence of the rate constant on the SAM carbon number (ET distance) within the series of mixed SAMs of C(5)COOH/C(2)OH, C(10)COOH/C(6)OH, and C(15)COOH/C(11)OH that is in overall similar to that found earlier for the undiluted SAM assemblies. However, in the case of C(15)COOH/C(11)OH and C(10)COOH/C(6)OH mixed SAMs a notable increase of the ET standard rate constant was observed, in comparison with the corresponding unicomponent (omega-COOH) SAMs. In the case of the C(5)COOH/C(2)OH composite SAM a decrease of the rate constant versus the unicomponent analogue was observed. The value of the reorganization free energy deduced through the Marcus-like data analysis did not change throughout the series; this fact along with the other observations indicates uncomplicated rate-determining unimolecular ET in all cases. Our results are consistent with a model that considers a changeover between the alternate, tunneling and adiabatic intrinsic ET mechanisms. The physical mechanism behind the observed fine kinetic effects in terms of the protein-rigidifying omega-COOH/CytC interactions arising in the case of mixed SAMs are also discussed.

Kinetic, thermodynamic, and mechanistic patterns for free (unbound) cytochrome c at Au/SAM junctions: impact of electronic coupling, hydrostatic pressure, and stabilizing/denaturing additives.

Combined kinetic (electrochemical) and thermodynamic (calorimetric) investigations were performed for an unbound (intact native-like) cytochrome c (CytC) freely diffusing to and from gold electrodes modified by hydroxyl-terminated self-assembled monolayer films (SAMs), under a unique broad range of experimental conditions. Our approach included: 1) fine-tuning of the charge-transfer (CT) distance by using the extended set of Au-deposited hydroxyl-terminated alkanethiol SAMs [-S-(CH(2))(n)-OH] of variable thickness (n=2, 3, 4, 6, 11); 2) application of a high-pressure (up to 150 MPa) kinetic strategy toward the representative Au/SAM/CytC assemblies (n=3, 4, 6); 3) complementary electrochemical and microcalorimetric studies on the impact of some stabilizing and denaturing additives. We report for the first time a mechanistic changeover detected for "free" CytC by three independent kinetic methods, manifested through 1) the abrupt change in the dependence of the shape of the electron exchange standard rate constant (k(o)) versus the SAM thickness (resulting in a variation of estimated actual CT range within ca. 15 to 25 A including ca. 11 A of an "effective" heme-to-omega-hydroxyl distance). The corresponding values of the electronic coupling matrix element vary within the range from ca. 3 to 0.02 cm(-1); 2) the change in activation volume from +6.7 (n=3), to approximately 0 (n=4), and -5.5 (n=6) cm(3) mol(-1) (disclosing at n=3 a direct pressure effect on the protein's internal viscosity); 3) a "full" Kramers-type viscosity dependence for k(o) at n=2 and 3 (demonstrating control of an intraglobular friction through the external dynamic properties), and its gradual transformation to the viscosity independent (nonadiabatic) regime at n=6 and 11. Multilateral cross-testing of "free" CytC in a native-like, glucose-stabilized and urea-destabilized (molten-globule-like) states revealed novel intrinsic links between local/global structural and functional characteristics. Importantly, our results on the high-pressure and solution-viscosity effects, together with matching literature data, strongly support the concept of "dynamic slaving", which implies that fluctuations involving "small" solution components control the proteins' intrinsic dynamics and function in a highly cooperative manner as far as CT processes under adiabatic conditions are concerned.

Local dense structural heterogeneities in liquid water from ambient to 300 MPa pressure: evidence for multiple liquid-liquid transitions.

Difference and double-difference near-infrared DO-D and HO-H stretching overtone (2nuOD and 2nuOH) spectroscopy and a rigorous (physically substantiated) band deconvolution technique were applied to reveal three different kinds of inherent (interstitial) structures of liquid water, which determine its high density (compared to ice lh under ambient conditions), its compressibility (under hydrostatic pressure, up to 300MPa), and its high fragility (manifested under temperature variation). Our data processing allowed the rigorous discrimination of up to six vibrational components. On the basis of an extensive comparative analysis combined with available structural data (X-ray and neutron scattering) and molecular dynamics (MD) simulations for liquid water, as well as with experimental and computed data for small non-tetrahedrally arranged water clusters, the major four components could be ascribed to: i) The basic lh icelike substructure; ii) the temperature-dependent remote interstitial "defects" due to tetrahedral displacements (primarily responsible for transport properties); iii) the interstitial "defects" most probably arranged in quasiplanar noncyclic tetramers (totally absent in the ice structure); and iv) the interstitial "defects" formed with increasing pressure, probably arranged in cubic water octamers and composed of two pairs of noncyclic and cyclic tetramer fragments. The latter structures include, essentially, bent hydrogen bonds stabilized by resonance effects.

Two-electron transfer for Tl(aq)(3+)/Tl(aq)(+) revisited. Common virtual [Tl(II)-Tl(II)](4+) intermediate for homogeneous (superexchange) and electrode (sequential) mechanisms.

Homogeneous and electrochemical two-electron transfers within the Tl(aq)(3+)/Tl(aq)(+) couple are considered on a common conceptual basis. For the 2 equiv electrochemical reduction of Tl(aq)(3+) to Tl(aq)(+), the intermediate state with a formal reduction potential, E(1) = 1.04 +/- 0.10 V vs the normal hydrogen electrode, was detected, different from the established value of 0.33 V for a Tl(3+)/Tl(2+) couple. Examination of obtained electrochemical (cyclic voltammetry (CV) and rotating disk electrode techniques, along with the CV-curve computer simulation procedure) and literature data indicate that the detected formal potential cannot be the property of electrode-adsorbed species, but rather of the covalently interacting dithallium intermediate [Tl(II)-Tl(II)](4+) located at the outer Helmholtz plane. The analysis of microscopic mechanisms, based on the recent hypothesis of H. Taube and the Marcus-Hush theory extended by Zusman and Beratan, and Koper and Schmickler, revealed that the homogeneous process most probably takes place through the superexchange inner-sphere two-electron-transfer mechanism, via an essentially virtual (undetectable) dithallium intermediate. In contrast, the electrochemical process occurs through a sequential mechanism, via the rate-determining step of Tl(aq)(2+) ion formation immediately followed by activationless formation of the metastable (CV-active) dithallium state. The second electrochemical electron-transfer step is fast, and shows up only in the peak height (but not in the shape) of the observed CV cathodic wave. The anodic wave for a microscopically reverse process of the oxidation of Tl(aq)(+) to Tl(aq)(3+) cannot be observed within the considered potential range due to the blocking of through-space electron transfer by the competitor process of ion transfer to the electrode.