## 4 Conclusions  
This work describes for the first time the potentiality of assembling fully functional S-layers from iron (II) salts (FeCl\({}_{2}\) and FeSO\({}_{4}\)). Such structures could only be achieved when exerting an external control over the naturally occurring oxidation of Fe\({}^{2+}\) to Fe\({}^{3+}\). In this regard, a combination of chemical (DTT, degassing) and electrical (fixed bulk potential) factors have been shown to most effectively act  
Figure 6: Influence of an underlying SCWP film on the SbpA film packing and ion permeability measured by Cyclic voltammetry measurements (\(-1\) to \(+1\) V) on Fe\({}^{2+}\) –containing S-layers (FeCl\({}_{2}\), dark to light blue solid lines versus FeSO\({}_{4}\), purple to pink open circles). Experiments were performed in 0.1 M HEPES and the SCWP-coated gold system (grey) was employed as signal reference. Inset at the AFM topography micrograph shows the resulting Fourier spectra from the SbpA recrystallized film in the presence of FeSO\({}_{4}\) salt. against such oxidizing trend. Formation of the respective crystalline films has been followed by means of both QCMD measurements, offering real time monitoring over the process of assembly and the ongoing structural transition and complementary Atomic Force Microscopy topography studies, which demonstrate the presence of squared lattice symmetry usually featuring SbpA films at the end of the process. Hence, FeCl\({}_{2}\) has been shown to induce the formation of a very compact and smooth protein layer which resembles that produced in the presence of CaCl\({}_{2}\). For FeSO\({}_{4}\) salts, contrarily, the packing of the resulting crystal layer is clearly compromised by its water content, because of the _kosmotropic_ counterion involved, which also impacts its final performance. Hence, cyclic voltammetry studies have proven these observations by testing the ion permeability of both types of S-layer coatings. The higher isolation shown by FeCl\({}_{2}\)-driven films in the -1 to +1 V range (up to a 50% more effective), could only be paired by sulphate-containing homologous samples when a supporting SCWP layer was placed underneath. This fact highlights the relevance of the homogeneous arrangement of the protein film on the final performance achieved. Then, new paths seem to open for the construction of crystalline S-layers in the presence of alternative divalent ions. Most important, it has to be remembered that S-layer morphogenesis might follow the theoretically simplest mechanism for a dynamic process of assembly of a closed container composed of monomolecular arrays of identical macromolecules. In addition, these results would confirm the paleo-geo-chemical hypothesis by which a simple protein membrane capable of dynamic growth in the presence of bivalent iron (Fe\({}^{2+}\)) would fulfil barrier and supporting functions in an early (anaerobic) stage of biological evolution. [PERSON] and [PERSON] conceived and designed the experiments; [PERSON] and [PERSON] performed the experiments; [PERSON] and [PERSON] analysed the data; [PERSON] contributed with image analysis tools; [PERSON] and [PERSON] wrote the paper; [PERSON], [PERSON] and [PERSON] proof-read the manuscript, contributed with the final discussions (data analysis) and complementary information. This research was funded by AFOSR Agreement Award, grant number Nr. FA9550-15-1-0459. The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript and in the decision to publish the results. Figure 14: Real-time monitoring of SbpA adsorption onto hydrophobic gold, under two different potential values (\(-1\) vs. \(+1\) V) being applied. The black arrow indicates the moment at which the oxidative value (\(+1\) V) the potential is changed to reducing values (\(-1\) V), which completely influences the protein-substrate interaction.