Analysis of human ferrochelatase iron binding via amide hydrogen/deuterium exchange mass spectrometry

Human ferrochelatase (E.C. 4.99.1.1) is a membrane-associated enzyme that catalyzes the last step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Crystallographic structures have revealed that protoporphyrin binds in a cleft between two domains; however, the entry pathway and location of the iron binding site(s) is still contested. In an effort to address this issue, the structural elements involved in binding substrate iron were studied by amide hydrogen/deuterium exchange mass spectrometry. The deuterium incorporation rates into the backbone of apo- and iron-ferrochelatase in the absence of porphyrin substrate were measured. For the first time, it is demonstrated how the binding of ferrous iron specifically modulates ferrochelatase structure in solution. The distinct regions affected by the presence of iron provide insight into the mechanism by which iron is transported to the active site.