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-trans fer reaction between ferrocytochrome c (Fe(2+)Cc) and a silver film over na nosphere (AgFON) electrode is examined in detail using electrochemical surf ace-enhanced resonance Raman spectroscopy (SERRS) as a molecularly specific and structurally sensitive probe. The distance between the Fe2+ redox cent er and the electrode surface is controlled by varying the chain length x of an intervening carboxylic acid terminated alkanethiol, HS(CH2)(x)COOH, sel f-assembled monolayer (SAM). The orientation of the heme in Fe(2+)Cc with r espect to the AgFON/S(CK2)(x) COOH electrode surface is controlled by its b inding motif. Electrostatic binding of Fe(2+)Cc to AgFON/S(CH2)(x)COOH yiel ds a highly oriented redox system with the heme edge directed toward the el ectrode surface. The binding constants were determined to be K = 5.0 x 10(6 ) M-1 and 1.1 x 10(6) M-1, respectively, for the x = 5 and s = 10 SAMs. In contrast, covalent binding of Fe(2+)Cc yields a randomly oriented redox sys tem with no preferred direction between the heme edge and the electrode sur face. SERRS detected electrochemistry demonstrates that Fe(2+)Cc electrosta tically bound to the x = 5 AgFON/S(CH2)(x)COOH surface exhibits reversible oxidation to ferricytochrome c, whereas Fe2+Ce electrostatically bound to t he x = 10 surface exhibits irreversible oxidation. In comparison, Fe(2+)Cc covalently bound to the x = 5 and x = 10 surfaces both exhibit oxidation wi th an intermediate degree of reversibility. in addition to these primary re sults, the work presented here shows that AgFON/S(CH2)(x)COOH surfaces (1) are biocompatible - Fe(2+)Cc is observed in its native state and (2) are st able to supporting electrolyte changes spanning a wide range of ionic stren gth and pH thus enabling, for the first time, SERRS studies of these variab les in a manner not accessible with either the widely used colloid or elect rochemically roughened SERS-active surfaces.