# The Role of Metal Ions in the Electron Transport through Azurin-Based Junctions
## 3 Results and Discussion  
First, we inspect the influence of ion replacement on the protein structure. Henceforth, we refer to X-azurin (X = Ni, Cu, Co and Zn) as the azurin containing the X-ion. The four metals considered all belong to the first row of the \(d\) block of the periodic table. In Figure 1a-c, we represent the time averaged configuration obtained from a 0.5 \(\upmu\)s long MD simulation of the three different proteins, i.e., Cu-azurin (black), Co-azurin (red) and Ni-azurin (green). From the structure overlap between the different proteins and native azurin (i.e., Cu-azurin), it becomes apparent that the ion replacement has little influence on the protein conformation. This is further corroborated and quantified, by both small root-mean-square-deviation (RMSD) between average structures (shown in Figure 1a-c) as well as the preservation of the most prevalent secondary structure on the protein, i.e., \(\beta\)-sheets shown in Figure 1e. The \(\beta\)-sheets refer to a particular spacial organization of the protein's amino acids regulated/characterized by a specific hydrogen bond network among them. In the particular case of azurin, the most prevalent sort of secondary structure is the \(\beta\)-sheet, which are further arranged in a circular manner, thus forming a characteristic \(\beta\)-barrel. In Figure 1e, we quantify the content of this secondary-structure for the different variants to realize that the ion replacement does not alter/disrupt the H-bond network sustaining the characteristic shape and structure of the wild-type azurin. Moreover, from the RMSD, we observed that all proteins exhibit a structural deviation from the native protein smaller than 1.5 A. Such RMSD values are within the structural variations experienced by the protein resulting from thermal fluctuations. Moreover, the process of protein adsorption and subsequent tip indentation results in a structural deformation which is at least three times larger than the one observed from altering the protein ion (see Figure 2). All considered, one may state that, by altering the coordination ion, the structural modifications thus introduced are substantially smaller than the ones the protein experiences during the contact formation process. Note that the similarity of the parameters governing the bond motif of the different metal ions (e.g., charge and bond distances provided in Figures S2-S5) precluded the negligible structural modifications here observed. All considered, by the aforementioned reasons, the subsequent simulations were performed using a tip-protein contact equilibrated. Figure 2 shows the initial and final output geometries of the MD simulation performed on the Cu-azurin from which the structures used for the present work were obtained (see more details in [34]). In this simulation, the tip is brought close to the protein sideways and the contact is established via the \(\alpha\)-helix arm and hydrophobic patch. In the left column of Figure 3, we show the zero-bias transmission as a function of energy for the four proteins considered, at four different values of the tip-ion distance (d\({}_{t-X}\)). Such a distance was evaluated as the distance between the center of the lowest tip layer and the metal ion. In all curves, all resonances appear very sharp, indicating very low hybridization between the corresponding orbitals and the metallic leads. As expected, for most energy values the transmission decreases for increasing distance. Figure 1: Structural characterization of the X-azurin (X = Cu, Co and Ni) free solved in TIP3P water obtained via atomistic MD simulations. (**a–c**) Energetically equilibrated averaged configurations obtained over the last 200 ns of MD simulation for: (**a**) native azurin; (**b**) Co-azurin; and (**c**) Ni-azurin variants. The Zn-azurin is not included given the lack of reliable force-field parameters for this variant. Note that the Co-azurin (red) and Ni-azurin (green) configurations are aligned with the native one (black) and superposed to it.