Time-lapse anomalous X-ray diffraction shows how Fe2+substrate ions move through ferritin protein nanocages to oxidoreductase sites

Abstract

Ferritin superfamily protein cages reversibly synthesize internal biominerals, Fe2O3·H2O. Fe2+and O2(or H2O2) substrates bind at oxidoreductase sites in the cage, initiating biomineral synthesis to concentrate iron and prevent potentially toxic reactions products from Fe2+and O2or H2O2chemistry. By freezing ferritin crystals ofRana catesbeianaferritin M (RcMf) at different time intervals after exposure to a ferrous salt, a series of high-resolution anomalous X-ray diffraction data sets were obtained that led to crystal structures that allowed the direct observation of ferrous ions entering, moving along and binding at enzyme sites in the protein cages. The ensemble of crystal structures from both aerobic and anaerobic conditions provides snapshots of the iron substrate bound at different cage locations that vary with time. The observed differential occupation of the two iron sites in the enzyme oxidoreductase centre (with Glu23 and Glu58, and with Glu58, His61 and Glu103 as ligands, respectively) and other iron-binding sites (with Glu53, His54, Glu57, Glu136 and Asp140 as ligands) reflects the approach of the Fe2+substrate and its progression before the enzymatic cycle 2Fe2++ O2→ Fe3+—O—O—Fe3+→ Fe3+—O(H)—Fe3+and turnover. The crystal structures also revealed different Fe2+coordination compounds bound to the ion channels located at the threefold and fourfold symmetry axes of the cage.