The electrochemical behavior of three proteins fragments from the respiratory chain of the extremophilic bacterium Therm us thermophilus, namely, cytochrome c(1) (Cyt-c(1)), cytochrome c(552) (Cyt-c(552)), and Cu-A, immobilized on three-dimensional gold nanoparticles electrodes was investigated by cyclic voltammetry. The gold nanoparticles were modified by either dithiobissuccinimidyl propionate (DTSP) or a mixed self-assembled monolayer of 6-mercaptohexan-1-ol and hexanethiol, depending on the surface of the protein. High surface coverages with enzymes and good electron transfer rates were achieved in the case of Cyt-c(1) immobilized on DTSP-modified gold nanoparticles and Cyt-c(552) or Cu-A immobilized on mixed SAMs-modified gold nanoparticles. Interestingly, high surface coverages with Cu-A were also observed on DTSP-modified gold nanoparticles, but a slower electron transfer rate was determined in this case. The gold nanoparticle/protein assemblies were characterized by surface-enhanced IR spectroscopy and transmission electron microscopy.