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

Federico Sebastiani,Riccardo Risorti,Chiara Niccoli,Hanna Michlits,Maurizio Becucci,Stefan Hofbauer,Giulietta Smulevich

doi:10.1016/j.jinorgbio.2022.111718

Abstract

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.

Highlights

Coproheme decarboxylases (ChdCs) catalyze the last step of heme b synthesis in monoderm bacteria by a two-step oxidative decarboxylation of the two propionate groups of iron coproporphyrin III [1,2,3,4,5]
We have shown that both coproheme decarboxylase from Corynebacterium diphtheriae (CdChdC) WT and H118A convert coproheme mainly to heme b upon titration with H2O2 with small re sidual amounts of mon ovinyl monopropionyl deuteroheme (MMD), while the variant A207E accumulates MMD along with a minor amount of heme b and H118F generates only MMD [11,12]
The complex is characterized by a maximum of the Soret band at around 401–402 nm and a band at 586 nm, while the heme b-CdChdC complex of wild-type and the H118A variant is a mixture of 5- and 6-coordinated (6c) high spin (HS) species (Fig. S1 right) [11]

Summary

Introduction

Coproheme decarboxylases (ChdCs) catalyze the last step of heme b synthesis in monoderm bacteria by a two-step oxidative decarboxylation of the two propionate groups of iron coproporphyrin III (coproheme) [1,2,3,4,5]. Upon titration with H2O2, this variant accumulates exclusively MMD, where only the propionate at position 2 was Abbreviations: ChdC, coproheme decarboxylase; CdChdC, coproheme decarboxylase from Corynebacterium diphtheriae; LmChdC, coproheme decarboxylase from Listeria monocytogenes; SaChdC, coproheme decarboxylase from Staphylococcus aureus; DyPs, dye-decolorizing peroxidases; Clds, chlorite dismutases; LS, low-spin; HS, high-spin; QS, quantum mixed-spin; 6c, six coordinated; 5c, five coordinated; RR, resonance Raman; MMD, monovinyl mono-propionate deuteroheme; WT, wildtype. In addition to the catalytic Y135, the distal H118 is important in actinobacterial ChdCs for deprotonation of hydrogen peroxide and, Compound I formation [12] This histidine residue is lacking in firmicute ChdCs and is located on a loop that connects the two ferredoxin-like folds, which is decisive for the respective activities of the entire structural superfamily [13]. In the present work we study the wild-type and the H118A and A207E variants (Fig. 1) reconstituted with heme b in order to understand whether heme b maintains the same geometry of

Methods

Results

Conclusion