Bionanoconjugates were created with cytochrome c from horse heart (HCc) or yeast (YCc) and citrate-stabilized gold nanoparticles (AuNPs). Evidence for the formation of stable HCc−AuNP and YCc−AuNP bionanoconjugates came from a 5 nm red-shift of the surface plasmon resonance band of the AuNPs, increase of the ζ-potential, and direct visualization by atomic force microscopy. Langmuir isotherm fittings of ζ-potential data indicated that higher enthalpy changes are involved in the formation of the HCc−AuNP than in YCc−AuNP. UV−vis and circular dichroism studies of pH-induced aggregation of the bionanoconjugates revealed distinct protonation patterns with an aggregation pH of 8.8 and 6.2 for YCc−AuNP and HCc−AuNP, respectively. No appreciable changes were observed in the secondary structure of HCc in HCc−AuNP. In contrast, YCc in YCc−AuNP presented a decrease in α-helix content upon AuNP binding and an increase in β-sheet content upon pH-induced aggregation. Data discussion is based on the distinct binding modes of both proteins to the AuNPs via a covalent bond (Cys 102) for YCc and via electrostatic interaction for HCc.