Using nanometer thick tunneling barriers with specifically attached cytochrome c, the electron-transfer rate constant was studied as a function of the SAM composition (alkane versus terthiophene), the omega-terminating group type (pyridine, imidazole, nitrile), and the solution viscosity. At large electrode-reactant separations, the pyridine terminated alkanethiols exhibit an exponential decline of the rate constant with increasing electron-transfer distance. At short separations, a plateau behavior, analogous to systems involving -COOH terminal groups to which cytochrome c can be attached electrostatically, is observed. The dependence of the rate constant in the plateau region on system properties is investigated. The rate constant is insensitive to the mode of attachment to the surface but displays a significant viscosity dependence, change with spacer composition (alkane versus terthiophene), and nature of the solvent (H(2)O versus D(2)O). Based on these findings and others, the conclusion is drawn that the charge-transfer rate constant at short distance is determined by polarization relaxation processes in the structure, rather than the electron tunneling probability or large-amplitude conformational rearrangement (gating). The transition in reaction mechanism with distance reflects a gradual transition between the tunneling and frictional mechanisms. This conclusion is consistent with data from a number of other sources as well.

Cytochrome c was coordinatively bound to self-assembled monolayers of pyridine-terminated alkanethiols on Au and Ag electrodes. The mechanism of heterogeneous electron transfer of the immobilized protein was investigated by cyclic voltammetry and time-resolved surface-enhanced resonance Raman spectroelectrochemistry. The temperature, distance, and overpotential dependencies of the electron transfer rates indicate a change of mechanism from a tunneling controlled reaction at long distances (thicker films) to a solvent/protein friction controlled reaction at smaller distances (thinner films).

Directly linking a gold electrode coated with pyridine‐terminated self‐assembled monolayers (SAMs) to the redox‐active heme unit of cytochrome c has enabled the electron‐transfer mechanism to be studied as a function of distance. Comparison of the kinetic data for this system (see graph) with earlier data on COOH‐terminated SAMs requires a change in the electron‐transfer mechanism with distance from the electrode surface that does not involve large‐amplitude conformational rearrangement.

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 ...