Furthermore, this study aimed to identify the physical and electrochemical effects that immobilising Cyt c to AuNPs has upon the redox ability of the protein by varying the conjugation strategies, self-assembled monolayer (SAM) ligands, AuNP size, and presence of other adsorbate molecules. Using circular dichroism (CD) spectroscopy, we observed changes in the secondary and tertiary structures of Cyt c after its conjugation to AuNPs . This technique can help monitor any modifications in a protein's structure, such as folding or unfolding . Also, timeof-flight secondary ion mass spectrometry (ToF-SIMS) was used to discern the molecular composition of the surface, thereby helping us understand the orientation of Cyt c . By employing these techniques in conjunction, we linked any observed changes in structural orientation to potential shifts in the redox capacity of Cyt c . The evaluation of electrochemical properties was conducted using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The heterogeneous rate constant (k 0 ) for Cyt c or Zn Porph was calculated using Cyt c.