The electron transfer (ET) scheme of cytochrome c (cyt. c) coupled to carboxylic acid-terminated alkanethiol self-assembled monolayers (SAMs) on well-defined gold (111) electrodes is a simplified model system to investigate both long range and intermolecular ET processes. The advantages of an electrochemical approach to investigate the ET mechanism are that one can both regulate the ET path length by using alkanethiol SAMs of varying chain lengths and deconvolute the intermolecular ET event at the interface from the intramolecular ET event. It has been shown that the interactions between cyt. c and the carboxylate termini are electrostatic in nature, analogous to those between cyt. c and negatively charged proteins such as cytochrome c peroxidase. In the present work, the effects of alkanethiol chain length, ionic strength, pH, and viscosity of supporting electrolyte on the ET kinetics were studied. The ET rates through long alkanethiol chains were observed to be slow because electron tunneling through the alkyl chain was the rate-limiting step in the process. On the other hand, the ET rate through shorter chain alkanethiols appeared to be independent of chain length, and the effect of ionic strength and pH on the observed ET rates was insignificant. It is proposed that the rate-limiting ET step through short alkyl chains results from a configurational rearrangement process preceding the ET event, and that its rate is 2.6 × 10 3 s -1 . This "gating" process arises from a rearrangement of the cyt. c from a stable binding form (binding complex) on the carboxylic acid terminus to a configuration (ET complex) which facilitates the most efficient ET pathway. The rate of the configurational rearrangement reaction that precedes the ET reaction was found to be markedly influenced by solution viscosity, but its equilibrium constant was independent of solution viscosity. The change in the configurational rearrangement reaction rate with solution viscosity follows a modified Kramers equation.

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The acid-base equilibrium of ω-functionalized alkanethiol monolayers on Au(111) has been studied using the change in double-layer capacitance accompanied by the protonation-deprotonation of ω-terminals. The pK of ω-carboxyl alkanethiols on Au(111) increases by four pH units. The shift becomes greater as the alkyl chain length increases. The same magnitude of the pK shift to the acidic side occurs in the monolayers of aminoethane thiol. The surface pK varies little with the electrode potential, whereas the increase in the supporting electrolyte concentration slightly diminishes the pK shift. The drawn-out shape of the titration curves is consistent with the mean-field model taking account of the repulsive interaction between adsorbed molecules, indicating the significance of the strong electrostatic repulsion between adsorbed thiol molecules in its charged states. The magnitude of the pK shift is, more than that predicted by the mean-field model, however, and suggests the considerable contribution from other factors that stabilize the uncharged state, for example, hydrogen bonding and low dielectric constant in the vicinity of the self-assembled monolayer. The possibility of counterion binding when the degree of deprotonation is large has been suggested.

Well-defined voltammetric responses of redox proteins with acidic-to-neutral pI values have been obtained on pure alkanethiol as well as on mixed self-assembled-monolayer (SAM) omega-derivatized alkanethiol/gold bead electrodes. Both azurin (P. aeruginosa) (pI = 5.6) and subunit II (Cu(A) domain) of ba(3)-type cytochrome c oxidase (T. thermophilus) (pI = 6.0) exhibit optimal voltammetric responses on 1:1 mixtures of [H(3)C(CH(2))(n)()SH + HO(CH(2))(n)()SH] SAMs. The electron transfer (ET) rate vs distance behavior of azurin and Cu(A) is independent of the omega-derivatized alkanethiol SAM headgroups. Strikingly, only wild-type azurin and mutants containing Trp48 give voltammetric responses: based on modeling, we suggest that electronic coupling with the SAM headgroup (H(3)C- and/or HO-) occurs at the Asn47 side chain carbonyl oxygen and that an Asn47-Cys112 hydrogen bond promotes intramolecular ET to the copper. Inspection of models also indicates that the Cu(A) domain of ba(3)-type cytochrome c oxidase is coupled to the SAM headgroup (H(3)C- and/or HO-) near the main chain carbonyl oxygen of Cys153 and that Phe88 (analogous to Trp143 in subunit II of cytochrome c oxidase from R. sphaeroides) is not involved in the dominant tunneling pathway. Our work suggests that hydrogen bonds from hydroxyl or other proton-donor groups to carbonyl oxygens potentially can facilitate intermolecular ET between physiological redox partners.

The reductive desorption process of self-assembled monolayers of 1-hexadecanethiol, 1-propanethiol, and 3-mercaptopropionic acid on Au(111) has been studied in 0.5 M KOH solution by in-situ scanning tunneling microscopy (STM) and cyclic voltammetry. In-situ STM images of the monolayers at the potentials between −0.2 V and the reduction potentials of each thiols show the pits that are commonly seen in STM images of thiol self-assembled monolayers. A drastic morphological change takes place in the STM image around the peak potential in a cyclic voltammogram for the reductive desorption of adsorbed thiols. The images indicate that 3-mercaptopropionic acid molecules diffuse away from the surface after the reduction because of its higher solubility, while 1-propanethiol and 1-hexadecanethiol molecules stay in the vicinity of the surface forming aggregates. The partial recovery of the 1-hexadecanethiol monolayer after the anodic scan, suggested by cyclic voltammograms, is confirmed by STM, whereas 1-propanethiol aggregates remain at the surface without being reoxidized. The difference in the reoxidation behavior reflects the different amphiphilic properties of the desorbed molecules and the resultant molecular organizations formed on the surface.

Electrochemistry of surface-modified cytochrome c (cyt c) bound electrostatically to carboxylate-terminated alkanethiol self-assembled monolayers (SAM) reveals highly anisotropic electronic coupling across the protein/ monolayer interface. Substitution of a lysine residue with alanine at position 13 in recombinant rat cyt c (RC9-K13A) lowers the interfacial electron transfer (ET) rate more than 5 orders of magnitude, whereas ET is only slightly affected by replacement of lysine-72 or lysine-79 with alanine. The results clearly show that lysine-13 is directly involved in coupling the protein to the SAM carboxylate terminus. Interfacial ET rates for both yeast iso-1 cyt c and the mutant RC9-K13R indicate that arginine-13 couples the protein to the carboxylate interface less well than lysine-13.

Replacement of self-assembled monolayers (SAM) of hexadecanethiol (HDT) on the Au(111) surface with 12-mercaptododecanoic acid (MDDA) in ethanol solution has been studied using voltammetry of the reductive desorption and scanning tunneling microscopy (STM). The exchange of adsorbed HDT molecules with MDDA dissolved in ethanol proceeds domainwise, that is, not in a random fashion. Two well-separated peaks, corresponding to the desorption of MDDA-rich domains and HDT-rich domains, appear in a voltammogram over the replacement process. The peak potential associated with the desorption of HDTrich domains remains unchanged in the course of the replacement, indicating that the solubility of MDDA in HDT-rich domains is very small. STM imaging of the substrate shows that domains whose sizes are greater than 15 nm 2 are predominant and occupy 85% of the area of HDT-rich domains. The entire exchange process is pseudo-first-order with the rate constant being 9.1 × 10 -3 h -1 in 1 mmol dm -3 MDDA in ethanol at 31 °C. The reverse process, i.e., the replacement of adsorbed MDDA with dissolved HDT in ethanol, is much slower, suggesting the stabilization of MDDA monolayers by lateral hydrogen bonding. A significant shift in the peak potential of MDDA-rich domains during the replacement indicates the considerable dissolution of HDT in MDDA domains.

This paper describes the results of an electrochemical and spectroscopic (infrared reflection and X-ray photoelectron spectroscopies) investigation of the modified gold electrode surfaces prepared from dilute ethanolic solutions of 4-mercaptopyridine (PySH) and 4,4‘-dipyridyl disulfide (PySS). Both precursors have been used extensively as facilitators for the electron transfer of redox proteins like cytochrome c (cyt c). During the course of an investigation of the interfacial architectures formed from the two different precursors, a previously unreported structural instability in the adlayers was discovered. This instability manifests itself as a decrease in the ability of the modified surfaces to facilitate the electron transfer of cyt c that correlates with an increase of the immersion time in the precursor solutions. Results are presented that delineate the decrease in facilitator performance and probe the structural changes resulting in the decrease in performance. Together, the electrochemical and surface spectroscopic findings reveal that the modified surfaces spontaneously decompose to yield an adlayer composed largely of adsorbed atomic and oligomeric sulfur, an adlayer that we found to be ineffective in the facilitation of the electron transfer reaction of cyt c. The implications of these findings on the use of this type of modifier to studies of electron transfer reactions of redox proteins and to issues of the general structural stability of organosulfur-based monolayers are briefly discussed.