The [2Fe2S] cluster and active-site architecture of Corynebacterium diphtheriae coproporphyrin ferrochelatase: Structure, stability, and catalysis

Iron ferrochelatases (fCs) catalyse the insertion of ferrous iron into porphyrins. In the coproporphyrin-dependent heme biosynthesis pathway, utilized by Gram-positive bacteria, ferrochelatases are responsible for the penultimate step, yielding ferric iron coproporphyrin III (coproheme) starting from coproporphyrin III. Some representative fCs carry a [2Fe2S] cluster, like the coproporphyrin ferrochelatase (CpfC) from the actinobacterium Corynebacterium diphtheriae (Cd). The general role of the iron‑sulfur cluster, whether catalytic or else, is still unclear. Thus, we have studied (i) the structure of the protein and the [2Fe2S] cluster coordination in detail by UV–vis electronic absorption and resonance Raman spectroscopies as well as by solving the X-ray crystal structure of apoprotein (apo) – WT CdCpfC; (ii) active site variants to elucidate the role of a conserved distal histidine and glutamate pair for iron insertion and stabilization of the heme cavity.Our results show that the iron‑sulfur cluster is stably coordinated and that the type of the cluster, the nature of the ligands and the coordination motif are similar to those of human fC. The investigation of active site variants allows for a comprehensive comparison of the roles of conserved distal residues with those in previously studied coproporphyrin ferrochelatases from Firmicutes.