With this set of structures at hand, a series of modifications were introduced to each of them, to facilitate a faithful determination of the electrostatic patterns around the respective active sites. In first instance, to enable proper detachment of the catalytic cluster from the protein matrix without inducing artificial distortions of the external electric field, the cysteine residue, coordinating the catalytic unit through a (deprotonated) thiol, was replaced by an alanine residue. Subsequently, Propka 33 was used to assign physically relevant protonation states across the protein matrix. Next, the histidine residues adjacent to the catalytic unit, for which the protonation state depends on the parametrization of the cofactor, were corrected so that their states match those in respectively Head-Gordon's, 2 as well as Bjornsson's 16 and Siegbahn's, 17 computational work. In the following step, all 'non-residue' substances (e.g., waters, substrates, counterions, etc.) were removed so that only the actual protein matrix remained. The positions of the hydrogens on the residues were subsequently refined through a minimization with 250 cycles of steepest descent, followed by 750 cycles of conjugate gradients, while keeping the positions of all the heavy atoms fixed. Minimizations were run using sander module of AMBER suite. Next, the electrostatic environment, induced by the protein matrix, was extracted as point charges localized at the atomic position of the minimized structure, with partial charges assigned based on the ff14SB protein force field. To this end, electric field strengths were computed on spherical grids around the positions of the Fe atoms of the FeMoco/FeVco/FeFeco cluster with the help of the TITAN code, 36 making use of Coulomb's law,