Insight into the function of active site residues in the catalytic mechanism of human ferrochelatase.

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

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© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.