| Abstract
| - Cytochrome c (Cyt-c) is immobilized on Ag and Au electrodes coated with self-assembled monolayers (SAM),comprised of pyridine-terminated alkanethiols and a shorter chain diluent thiol. Surface-enhanced resonanceRaman (SERR) spectroscopy of coated Ag electrodes reveals that the adsorbed Cyt-c forms a potential-dependent coordination equilibrium with a predominant five-coordinated high-spin (5cHS) state in the reducedform and six-coordinated low-spin (6cLS) state prevailing in the oxidized form. In the oxidized species, thenative Met-80 ligand of the heme is replaced by a pyridinyl residue of the bifunctional thiols that accordingto earlier scanning tunneling microscopy form islands in the hydrophobic monolayer. The redox potentialsderived from the SERR band intensities are estimated to be −0.24 and −0.18 V (vs AgCl) for the 6cLS and5cHS states, respectively, and lie in the range of the midpoint potential determined for Cyt-c on coated Auelectrodes by cyclic voltammetry (CV). Whereas in the latter case, a nearly ideal Nernstian behavior for aone-electron couple was observed, the SERR spectroscopic analysis yields about 0.4 for the number oftransferred electrons for each spin state. This discrepancy is mainly attributed to a distribution of substates ofthe immobilized protein in both the 6cLS and 5cHS forms, as indicated by substantial band broadening in theSERR spectra. These substates may arise from different orientations and heme pocket structures and exhibitdifferent redox properties. Whereas SERR spectroscopy probes all adsorbed Cyt-c species including thosethat are largely redox inactive, CV measurements reflect only the substates that are electrochemically active.
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