Bacterial ferrochelatase turns human: Tyr13 determines the apparent metal specificity of Bacillus subtilis ferrochelatase

Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX. The enzymatic product heme (protoheme IX) is a well-known cofactor in a wide range of proteins. The insertion of metal ions other than Fe2+ occurs rarely in vivo, but all ferrochelatases that have been studied can insert Zn2+ at a good rate in vitro. Co2+, but not Cu2+, is known to be a good substrate of the mammalian and Saccharomyces cerevisiae ferrochelatases. In contrast, Cu2+, but not Co2+, has been found to be a good substrate of bacterial Bacillus subtilis ferrochelatase. It is not known how ferrochelatase discriminates between different metal ion substrates. Structural analysis of B. subtilis ferrochelatase has shown that Tyr13 is an indirect ligand of Fe2+ and a direct ligand of a copper mesoporphyrin product. A structure-based comparison revealed that Tyr13 aligns with a Met residue in the S. cerevisiae and human ferrochelatases. Tyr13 was changed to Met in the B. subtilis enzyme by site-directed mutagenesis. Enzymatic measurements showed that the modified enzyme inserted Co2+ at a higher rate than the wild-type B. subtilis ferrochelatase, but it had lost the ability to use Cu2+ as a substrate. Thus, the B. subtilis Tyr13Met ferrochelatase showed the same metal specificity as that of the ferrochelatases from S. cerevisiae and human.