Hydrogen peroxide‐mediated conversion of coproheme to heme b by HemQ—lessons from the first crystal structure and kinetic studies

Heme biosynthesis in Gram‐positive bacteria follows a recently described coproporphyrin‐dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme‐HemQ from Listeria monocytogenes) at 1.69 Å resol...

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Bibliographic Details
Published in:The FEBS journal 2016-12, Vol.283 (23), p.4386-4401
Main Authors: Hofbauer, Stefan, Mlynek, Georg, Milazzo, Lisa, Pühringer, Dominic, Maresch, Daniel, Schaffner, Irene, Furtmüller, Paul G., Smulevich, Giulietta, Djinović‐Carugo, Kristina, Obinger, Christian
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Catalytic Domain
coproheme
Crystal structure
Crystallography, X-Ray
Enzymes
Firmicutes
Gram-Positive Bacteria - enzymology
Gram-Positive Bacteria - genetics
Gram-Positive Bacteria - metabolism
Gram‐positive pathogens
Heme - chemistry
Heme - metabolism
heme b
heme biosynthesis
Hemeproteins - chemistry
Hemeproteins - genetics
Hemeproteins - metabolism
Hemin - analogs & derivatives
Hemin - chemistry
Hemin - metabolism
HemQ
Hydrogen Bonding
Hydrogen peroxide
Hydrogen Peroxide - chemistry
Hydrogen Peroxide - metabolism
Iron
Kinetics
Listeria monocytogenes
Listeria monocytogenes - enzymology
Listeria monocytogenes - genetics
Listeria monocytogenes - metabolism
Mass Spectrometry
Models, Molecular
Original
Propionates - chemistry
Propionates - metabolism
Protein Binding
Protein Domains
Sequence Homology, Amino Acid
Spectrophotometry
Spectrum Analysis, Raman
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Summary:Heme biosynthesis in Gram‐positive bacteria follows a recently described coproporphyrin‐dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme‐HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV‐vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five‐coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H‐bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H2O2 molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (kcat/KM = 1.8 × 102 m−1·s−1 at pH 7.0) and occurs in a stepwise manner with a three‐propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two‐step reaction mechanism. Furthermore, the structure of coproheme‐HemQ is compared to that of the phylogenetically related heme b‐containing chlorite dismutases. Database Structural data are available in the PDB under the accession number 5LOQ. HemQ is an enzyme involved in the late stages of heme biosynthesis in monoderm bacteria. Here we present the first coproheme‐bound structure of HemQ from Listeria monocytogenes, which delivers valuable insight into the architecture of the active site. Furthermore, we report that two moles of hydrogen peroxide are required to transform one coproheme to heme b.
ISSN:1742-464X
1742-4658
DOI:10.1111/febs.13930