Chemical Identification of [Fe(III)(OH)(H2O)]2+ Complex Ion at the Ferroxidase Active Site in Cryo-EM Maps.

Cryo-EM generates images of the distribution of the electrostatic potential (ESP) in objects. The contributions to ESP maps made by atoms that are charged extend over distances far greater than their own diameters and thus make a disproportionately large contribution to the low-frequency terms in the Fourier transforms of these maps. For this reason, analysis of the frequency-dependence of the information in cryo-EM maps can distinguish hydroxyl anions from water molecules or ferric ions from ferrous ions. Here, this approach is used to show that the metal ion bound in the ferroxidase active site in a recently published cryo-EM map (emd-35984) of recombinant murine heavy-chain ferritin is Fe³⁺. It is coordinated by E27, E62, and H65 in a distorted square pyramidal geometry with its other two ligands being a hydroxyl anion and a water molecule. That metal ion was originally identified as a sodium ion. Additionally, it is found that there is a Cl^- anion bound to the 4-fold axial gate formed by partially protonated H173 residues.