An active site at work – the role of key residues in C. diphteriae coproheme decarboxylase

Coproheme decarboxylases (ChdCs) are utilized by monoderm bacteria to produce heme b by a stepwise oxidative decarboxylation of the 2- and 4-propionate groups of iron coproporphyrin III (coproheme) to vinyl groups. This work compares the effect of hemin reconstitution versus the hydrogen peroxide-mediated conversion of coproheme to heme b in the actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) and selected variants. Both ferric and ferrous forms of wild-type (WT) CdChdC and its H118A, H118F, and A207E variants were characterized by resonance Raman and UV–vis spectroscopies. The heme b ligand assumes the same conformation in the WT active site for both the reconstituted and H2O2-mediated product, maintaining the same vinyl and propionate interactions with the protein. Nevertheless, it is important to note that the distal His118, which serves as a distal base, plays an important role in the stabilization of the cavity and for the heme b reconstitution. In fact, while the access of heme b is prevented by steric hindrance in the H118F variant, the substitution of His with the small apolar Ala residue favors the insertion of the heme b in the reversed conformation. The overall data strongly support that during decarboxylation, the intermediate product, a monovinyl-monopropionyl deuteroheme, rotates by 90o within the active site. Moreover, in the ferrous forms the frequency of the ν(Fe-Nδ(His)) stretching mode provides information on the strength of the proximal Fe-His bond and allows us to follow its variation during the two oxidative decarboxylation steps. Heme b in the reconstituted and H2O2-mediated product of wild-type maintains identical conformation and interactions. The proximal Fe-His bond strengthens during the decarboxylation. The distal His118 is important role for heme b reconstitution. The overall data strongly support that the intermediate product rotates by 90o within the active site. [Display omitted] •Reconstituted and H2O2-mediated product of wild-type show identical heme b interactions.•The access of heme b is prevented by steric hindrance in the H118F variant.•The H118A variant shows both reversed and canonical heme b insertion.•The monovinyl-monopropionyl deuteroheme rotates by 90o within the active site.•The proximal Fe-His bond strengthens during decarboxylation.