Structural Changes in Adsorbed Cytochrome c upon Applied Potential Characterized by Neutron Reflectometry

The structural behavior of an electron-transfer protein, cytochrome c, at the 316L stainless steel electrode/aqueous interface was investigated over a range of applied potentials using neutron reflectometry supported by solution depletion isotherms, X-ray reflectometry, and quartz crystal microbalance measurements. A custom-made electrochemical cell allowed in situ observation of the adsorbed protein across a range of applied potentials; models fitted to the NR data showed a compact inner protein layer at the metal/electrolyte interface and a further thicker but highly diffuse layer that could be removed by rinsing. The overall amount adsorbed was found to be strongly dependent on the applied potential and buffer pH. Subtle but significant changes in the structure of the adsorbed protein layer were seen as the potential was swept between ±0.40 V, reflecting changing attractive/repulsive interactions between the protein's charged side groups and the surface. At greater applied potentials, irreversible changes in the stainless steel film structure were also observed and attributed to deuterium absorption into the metal.