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 ...

Charge transfer studies have been performed for self-assembled monolayers of single-stranded and double-stranded peptide nucleic acids (PNAs) having a C-terminus cysteine and an N-terminus ferrocene group as a redox reporter. The decay of the charge transfer rate with distance was strong for short single-stranded PNA molecules and weak for long single-stranded and double-stranded PNAs. Possible mechanisms for this "softening" of the distance dependence are discussed. The nature of the mechanism change can be explained by a transition of the charge transport mechanism from superexchange-mediated tunneling for short PNAs to a "hopping" mechanism for long PNAs.

This study examines quantitative correlations between molecular conductances and standard electrochemical rate constants for alkanes and peptide nucleic acid (PNA) oligomers as a function of the length, structure, and charge transport mechanism. The experimental data show a power-law relationship between conductances and charge transfer rates within a given class of molecules with the same bridge chemistry, and a lack of correlation when a more diverse group of molecules is compared, in contrast with some theoretical predictions. Surprisingly, the PNA duplexes exhibit the lowest charge-transfer rates and the highest molecular conductances. The nonlinear rate-conductance relationships for structures with the same bridging chemistries are attributed to differences in the charge-mediation characteristics of the molecular bridge, energy barrier shifts and electronic dephasing, in the two different experimental settings.

The rotational motion of cytochrome c has been restricted by cross-linking it to mixed self-assembled monolayers (SAMs) with the compositions S-(CH 2 ) m COOH/S-(CH 2 ) n OH on gold electrodes via the formation of amide bonds between lysine residues on the protein and terminal carboxylate groups of the SAM. The effect of SAM thickness on the electron-transfer rate has been studied, and two main observations are drawn. First, the electron-transfer rate displays the same qualitative dependence on SAM thickness that was previously reported for electrostatically adsorbed and pyridine-ligated assemblies, suggesting a tunneling mechanism at long distance and some other rate-limiting process at short distance. Second, a significant effect on the rate is observed for mixed SAMs having a hydroxyl-terminated alkanethiol diluent when the diluent is more than one methylene group shorter than the carboxylic acid alkanethiol. These conclusions suggest that largeamplitude protein motion (i.e., gating) is not rate-limiting at short distance, though smaller-amplitude motions cannot be ruled out.