Cytochrome c at charged interfaces. 1. Conformational and redox equilibria at the electrode/electrolyte interface probed by surface-enhanced resonance Raman spectroscopy

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Abstract
The structure and the electron-transfer properties of cytochrome c (cyt c) adsorbed on a silver electrode were analyzed by surface-enhanced resonance Raman spectroscopy. It was found that the adsorbed cyt c exists in various conformational states depending on the electrode potential. In state I the native structure of the heme protein is fully preserved and the redox potential (+0.02 V vs saturated calomel electrode) is close to the value for cyt c in solution. In state II the heme iron exists in a mixture of five-coordinated high-spin and six-coordinated low-spin configurations. It had been shown that these configurations form a thermal equilibrium [Hildebrandt, P., and Stockburger, M. (1986) J. Phys. Chem. 90, 6017]. It is demonstrated that these equilibria strongly depend on the charge distribution within the electrical double layer of the silver electrode/electrolyte interface, indicating that the changes in the coordination shell are induced by electrostatic interactions. The structural alterations in state II are apparently restricted to the heme crevice, which assumes an open conformation compared to the close structure in state I. This leads to a strong decrease of the redox potentials, which were determined to be -0.31 and -0.41 V for the five-coordinated high-spin and six-coordinated low-spin configurations, respectively. On the other hand, gross distortions of the protein structure can be excluded since the reversible proton-induced conformational change of cyt c as found in solution at low pH also takes place in state II of the adsorbed cyt c. The linkage of cyt c molecules to the surface is mediated by charged amino acid groups, and it depends on the potential which groups are thermodynamically favored to form such a molecular binding site. The conformational states I and II, which are in a potential-dependent equilibrium, therefore refer to two different molecular binding sites. At potentials below zero charge (< .apprx. -0.6 V) a rapid denaturation of the adsorbed cyt c is noted, which is reflected by drastic and irreversible changes in the surface-enhanced resonance Raman spectrum. Our results are discussed on the background of previous electrochemical studies of cyt c at electrodes.

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