Cyclic voltammetry was employed to determine the formal reduction potential and heterogeneous electrontransfer rate constant of cytochrome c immobilized on three different metal substrates chemically modified with 11- mercaptoundoecanoic acid. The metal substrates include smooth gold and silver electrodes as well as nanoscopically rough silver electrodes obtained via an oxidation-reduction cycle. Electrode roughening followed a protocol typically employed to prepare surface-enhanced Raman active surfaces such that the electrochemical results can be compared with those determined by surface-enhanced resonance Raman spectroscopy of cytochrome c. The roughness of the surfaces was estimated by means of atomic force microscopy. For all systems midpoint potentials were found to be -0.068 V (vs SCE), although for rough silver electrode the midpoint potential slightly shifted in time from -0.051 V to -0.068 V within 24 h. The heterogeneous electron-transfer rate constants differ for the various metal substrates and were found to be smaller by a factor of 2.5 for the rough and smooth Ag substrates compared to Au electrodes. These findings imply that it is primarily the kind of metal rather than its surface morphology that controls the thermodynamics and kinetics of interfacial redox processes of immobilized cytochrome c. The present paper reconciles the partly conflicting results obtained by electrochemical methods, usually done on Au, and surface-enhanced resonance Raman spectroscopic techniques which are usually performed on Ag electrodes. © 2009 American Chemical Society.