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The actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae catalyzes the final reaction to generate heme b via the "coproporphyrin-dependent" heme biosynthesis pathway in the presence of hydrogen peroxide. The enzyme has a high reactivity toward H 2 O 2 used for the catalytic reaction and in the presence of an excess of H 2 O 2 new species are generated. Resonance Raman data, together with electronic absorption spectroscopy and mass spectrometry, indicate that an excess of hydrogen peroxide for both the substrate (coproheme) and product (heme b ) complexes of this enzyme causes a porphyrin hydroxylation of ring C or D, which is compatible with the formation of an iron chlorin-type heme d species. A similar effect has been previously observed for other heme-containing proteins, but this is the first time that a similar mechanism is reported for a coproheme enzyme. The hydroxylation determines a symmetry lowering of the porphyrin macrocycle, which causes the activation of A 2g modes upon Soret excitation with a significant change in their polarization ratios, the enhancement and splitting into two components of many E u bands, and an intensity decrease of the non-totally symmetric modes B 1g , which become polarized. This latter effect is clearly observed for the isolated ν 10 mode upon either Soret or Q-band excitations. The distal His118 is shown to be an absolute requirement for the conversion to heme d . This residue also plays an important role in the oxidative decarboxylation, because it acts as a base for deprotonation and subsequent heterolytic cleavage of hydrogen peroxide., (© 2022 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons Ltd.)

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 H 2 O 2 -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 90 o within the active site. Moreover, in the ferrous forms the frequency of the ν(Fe-N δ(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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)