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 − ðCH 2 Þ n − CH 3 ] (n ¼ 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. electron transfer mechanism | pressure | protein friction | reorganization | temperature T he intrinsic electron transfer (ET) mechanisms of even small and otherwise well-characterized proteins such as cytochrome c or azurin (Az) are difficult to identify conclusively because of the proteins' complexity, i.e., inhomogeneous structural and dynamic properties (1-14). The use of bioelectrochemical tunneling junctions, such as self-assembled monolayer (SAM) films of variable composition and thickness on metal electrodes, with redox proteins immobilized at the solution interface (or freely diffusing to the SAM terminal groups) have been shown to provide an assembly with well-defined and variable control parameters. As such, these assemblies are well suited for fundamental studies (15-32) and offer promise for versatile nanotechnology applications (32,33). On the basis of earlier fundamental efforts, this work studies ET between a Au electrode that is coated with a SAM alkanethiol film of variable thickness and a "model" biomolecular target, the blue copper protein, Az, from Pseudomonas aeruginosa that is immobilized through hydrophobic interactions onto the SAM. As a decisive development of the preceding work (19,20,25,26), we offer unique kinetic data obtained through temperature-and pressure-variation and the mechanistic analysis through a unique global fitting procedure accounting for ET at different SAM thickness, temperature, and pressure conditions that provided the variation of the reduced ET rate constant over almost five orders of magnitude. Importantly, our previous work demonstrated that Au-deposited SAMs can withstand pressure-related stress within 5 to 150 MPa (27, 28, 34).The rate constant, k ...

By variation of the electronic coupling strength, the transition between the solvent-controlled regime (in which the electron-transfer rate constant depends on the solvent friction) and the nonadiabatic electron-transfer limit was observed for the Au/Fe(CN) 6 3-/4redox system. The solvent friction regime was demonstrated for a bare Au electrode by showing that the apparent standard rate constant was inversely proportional to the viscosity in water/glucose solutions containing 1 M KCl. The magnitude of the electronic coupling between the Au and the redox species was reduced by preparing n-alkanethiol-coated gold electrodes (Au-S-(CH 2 ) n-1 -CH 3 with n ) 2, 4, 6, 8) of different thicknesses. For the case of a Au electrode coated by an ethanethiol monolayer (n ) 2) the rate constant exhibited a fractional viscosity dependence, whereas the electrodes with n ) 4, 6, and 8 methylenes in the film showed no viscosity dependence. This trend is indicative of an overall gradual turnover between the two regimes. In the nonadiabatic regime the distance dependence of the electronic coupling decay is 1.04 Å -1 , and its extrapolated value at the closest electrode-reactant distance is 3.5 kcal mol -1 . Analysis of the kinetic data, together with some results available in the literature, determines the intrinsic parameters of the charge-transfer step in both regimes. Corrections for the significant variation in the reactive site potential near the electrode (at the outer Helmholtz plane, OHP) and the reorganization free energy with the charge-transfer distance are taken into account. Evidence for a freezing out of the Marcus barrier (lowering by a factor of 2) was found for the process at the bare Au electrode, in accordance with theoretical prediction (Zusman, L. D. Chem. Phys. 1983, 80, 29).

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.