Journal
BIOMOLECULES
Volume 13, Issue 6, Pages -Publisher
MDPI
DOI: 10.3390/biom13060946
Keywords
coproheme decarboxylase; stopped-flow spectroscopy; HPLC; mass spectrometry
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Coproheme decarboxylases (ChdCs) are enzymes involved in the biosynthesis of heme. This study focuses on the second part of the decarboxylation reaction catalyzed by ChdCs, which has not been previously studied. The researchers optimized the production and purification of a intermediate compound called monovinyl, monopropionate deuteroheme (MMD), and used it to study the reaction mechanism. The results indicate that the second part of the reaction is similar to the first part, with slight differences in the active site architecture and H-bonding network.
Coproheme decarboxylases (ChdCs) are terminal enzymes of the coproporphyrin-dependent heme biosynthetic pathway. In this reaction, two propionate groups are cleaved from the redox-active iron-containing substrate, coproheme, to form vinyl groups of the heme b product. The two decarboxylation reactions proceed sequentially, and a redox-active three-propionate porphyrin, called monovinyl, monopropionate deuteroheme (MMD), is transiently formed as an intermediate. While the reaction mechanism for the first part of the redox reaction, which is initiated by hydrogen peroxide, has been elucidated in some detail, the second part of this reaction, starting from MMD, has not been studied. Here, we report the optimization of enzymatic MMD production by ChdC and purification by reversed-phase chromatography. With the obtained MMD, we were able to study the second part of heme b formation by actinobacterial ChdC from Corynebacterium diphtheriae, starting with Compound I formation upon the addition of hydrogen peroxide. The results indicate that the second part of the decarboxylation reaction is analogous to the first part, although somewhat slower, which is explained by differences in the active site architecture and its H-bonding network. The results are discussed in terms of known kinetic and structural data and help to fill some mechanistic gaps in the overall reaction catalyzed by ChdCs.
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