## 3 Results and Discussion
At this point, it is important to bring up the fact that the signal peptides of Cyt \(c_{6}\) and Cyt \(c_{6}\)-like proteins from the cyanobacterium _Anabaena_ sp. PCC 7119, having a processing site (AXA) downstream from the N-terminal end, are recognized by _E. coli_ which results in transport of the proteins to periplasmic space, a subcellular compartment equivalent to thylakoid lumen, where the signal peptide is correctly processed [21; 22; 23]. However, the hydrophobic tail of the _Synechocystis_ Cyt \(c_{\text{M}}\) is not recognized by _E. coli_ as a signal peptide. For this reason, it has only been possible to express the soluble region of _Synechocystis_ Cyt \(c_{\text{M}}\) in _E. coli_ by substituting its hydrophobic tail for the signal peptide of _Anabaena_ Cyt \(c_{6}\)[6]. The template protein used by Phyre2 for calculating those 15 structures happened to be the \(c\) subunit of nitric oxide reductase (NorC) from _Pseudomonas aeruginosa_. Thus, the similarity between the returned models and the original template were compared. Surprisingly, the overlap between both proteins was clear and almost total (Figure 3A). To confirm this, a second, independent prediction for the same 15 sequences was performed using I-TASSER, a different structure prediction software that incorporates the ab initio approach in its algorithm. In this new test, the structures obtained were, once again, almost identical to that of NorC (Figure 3B). We obtained similar results once more using AlphaFold, a novel deep learning algorithm that incorporates physical and biological knowledge about protein structure and that leverages multi-sequence alignments [18]. Crucially, NorC is a subunit of nitric oxide reductase that has never been found in cyanobacteria, while the core subunit NorB is present in several cyanobacterial species [24] and its expression decreases in \(\Delta\)cytM mutants [7]. Furthermore, Cyt \(c_{\text{M}}\) sharply increases its expression in oxidative stress conditions [9], a condition in which nitric oxide reductase is highly active [25]. In order to determine similarities in charges between Cyt \(c_{\text{M}}\) and NorC, we subsequently evaluated their surface electrostatic potential (Figure 4). Also in this aspect, we found similarities between Cyt \(c_{\text{M}}\) and NorC that might be indicative of a similar function, with Cyt \(c_{\text{M}}\) acting as a functional part of a nitric oxide reductase and therefore being involved in the oxidative stress response. These included a positively charged tip at the end of the tail, a negatively charged region at the body closest to the tail, another positively charged region on the opposite side of the body and yet one more negatively charged pocket on the bottom. On the other hand, there are some clear differences too, such as a more extensive positive zone in the case of NorC, but the resemblances between both structures here are again undeniable. Moreover, the midpoint redox potential of Cyt \(c_{\text{M}}\) and NorC, a fundamental parameter to establish the function of redox proteins, are relatively close with +150 mV and +183 mV, respectively [6; 26]. This is interestingly opposed to the major divergences found previously between the surface  
Figure 3: Comparative of the predicted structures of Cyt \(c_{\text{M}}\) and NorC. (**A**) shows the predicted structure for Cyt \(c_{\text{M}}\) by Phyre2 (blue) vs. NorC (red) while (**B**) shows the same prediction (blue) vs. the one by I-TASSER (yellow). In both panels, the structure predicted by Phyre2 is the one for Cyt \(c_{\text{M}}\) of _Nostoc_ sp. PCC 7120, as in Figure 2.  
electrostatic potential and midpoint redox potential of Cyt \(c_{\text{M}}\) and the ones of Cyt \(c_{6}\) and Pc [6].