Distance and Orientation Dependence of Heterogeneous Electron Transfer:  A Surface-Enhanced Resonance Raman Scattering Study of Cytochrome c Bound to Carboxylic Acid Terminated Alkanethiols Adsorbed on Silver Electrodes

The distance and orientation dependence of the heterogeneous electron-transfer reaction between ferrocytochrome c (Fe²⁺Cc) and a silver film over nanosphere (AgFON) electrode is examined in detail using electrochemical surface-enhanced resonance Raman spectroscopy (SERRS) as a molecularly specific and structurally sensitive probe. The distance between the Fe²⁺ redox center and the electrode surface is controlled by varying the chain length x of an intervening carboxylic acid terminated alkanethiol, HS(CH₂)_xCOOH, self-assembled monolayer (SAM). The orientation of the heme in Fe²⁺Cc with respect to the AgFON/S(CH₂)_xCOOH electrode surface is controlled by its binding motif. Electrostatic binding of Fe²⁺Cc to AgFON/S(CH₂)_xCOOH yields a highly oriented redox system with the heme edge directed toward the electrode surface. The binding constants were determined to be K = 5.0 × 10⁶ M^-1 and 1.1 × 10⁶ M^-1, respectively, for the x = 5 and x = 10 SAMs. In contrast, covalent binding of Fe²⁺Cc yields a randomly oriented redox system with no preferred direction between the heme edge and the electrode surface. SERRS detected electrochemistry demonstrates that Fe²⁺Cc electrostatically bound to the x = 5 AgFON/S(CH₂)_xCOOH surface exhibits reversible oxidation to ferricytochrome c, whereas Fe²⁺Cc electrostatically bound to the x = 10 surface exhibits irreversible oxidation. In comparison, Fe²⁺Cc covalently bound to the x = 5 and x = 10 surfaces both exhibit oxidation with an intermediate degree of reversibility. In addition to these primary results, the work presented here shows that AgFON/S(CH₂)_xCOOH surfaces (1) are biocompatible − Fe²⁺Cc is observed in its native state and (2) are stable to supporting electrolyte changes spanning a wide range of ionic strength and pH thus enabling, for the first time, SERRS studies of these variables in a manner not accessible with either the widely used colloid or electrochemically roughened SERS-active surfaces.