The coproporphyrin ferrochelatase of : mechanistic insights into a regulatory iron-binding site.

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from and are known to be inhibited by elevated iron concentrations. The work herein reports a (coproporphyrin III) for ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in is regulated by iron availability.