Redox Cofactor Rotates during Its Stepwise Decarboxylation: Molecular Mechanism of Conversion of Coproheme to Heme b

Abstract

Coproheme decarboxylase (ChdC) catalyzes the last step in the heme biosynthesis pathway of monoderm bacteria with coproheme acting both as redox cofactor and substrate. Hydrogen peroxide mediates the stepwise decarboxylation of propionates 2 and 4 of coproheme. Here we present the crystal structures of coproheme-loaded ChdC from Listeria monocytogenes (LmChdC) and the three-propionate intermediate, for which the propionate at position 2 (p2) has been converted to a vinyl group and is rotated by 90° compared to the coproheme complex structure. Single, double, and triple mutants of LmChdC, in which H-bonding interactions to propionates 2, 4, 6, and 7 were eliminated, allowed us to obtain the assignment of the coproheme propionates by resonance Raman spectroscopy and to follow the H 2 O 2 -mediated conversion of coproheme to heme b . Substitution of H 2 O 2 by chlorite allowed us to monitor compound I formation in the inactive Y147H variant which lacks the catalytically essential Y147. This residue was demonstrated to be oxidized during turnover by using the spin-trap 2-methyl-2-nitrosopropane. Based on these findings and the data derived from molecular dynamics simulations of cofactor structures in distinct poses, we propose a reaction mechanism for the stepwise decarboxylation of coproheme that includes a 90° rotation of the intermediate three-propionate redox cofactor.

Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase

“…In SaChdC, the melting temperature rises by 14°C (59-73°C) and in LmChdC by 20°C (35-55°C) compared to the respective apo-proteins, whereas heme b bound ChdCs show the same unfolding behavior as the apoprotein [19]. Therefore, the stabilization is only due to the additional interaction of p2 and p4, and in LmChdC, a pronounced H-bonding network between coproheme and the respective amino acid side chains is established, spanning from p2 to p4 [20,21].…”

“…The inability to form Compound I upon addition of hydrogen peroxide, due to the structural restraints of iron ligation discussed above, is the reason that no catalase and no ChdC activity is detected in LmCpfC. Interestingly, p2 in coproheme-LmCpfC is in close proximity to a tyrosine residue (Y124), similar to Firmicutes ChdCs, where p2 is interacting with a catalytically relevant essential tyrosine (radical site), which triggers decarboxylation of p2 and later p4 [3,21,27,28]. The inactivity toward peroxides is a necessity, since the only task of a ferrochelatase is to insert ferrous iron into a porphyrin.…”

“…UV-vis spectra were recorded using a Cary 60 (Agilent, Santa Clara, CA, USA) scanning spectrophotometer. LmChdC was expressed and purified as described previously [21]. Absorbance at 375 nm was plotted versus the protein concentration and fitted to a hyperbola function to derive K D values.…”

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy

“… 13 − 15 More recently, a tyrosyl radical was shown to play a central role during conversion of coproheme to heme b in coproheme decarboxylases (ChdC). 16 − 18 Crucially, the radical sites described above are located on highly conserved, catalytically active residues within the protein core and in the proximity of the heme cofactor.…”

The His-tag as a decoy modulating preferred orientation in cryoEM

“…Coproheme decarboxylases (formerly HemQ) from Geobacillus stearothermophilus was one of the targets of the Midwest Center for Structural Genomics (MCSG; target APC35880). We solved its Xray crystallographic structure in 2004 (PDB code: 1T0T.pdb), while others determined its function later (Dailey & Gerdes, 2015, Milazzo et al, 2019, Pfanzagl et al, 2018. We recently characterized this protein using cryoEM SPR (EMPIAR-10363, EMD-21373 and EMPIAR-10362, EMD-21376) (Bromberg et al, 2020) and noticed during cryoEM experiments that batches of the protein purified at different times showed different patterns of preferred orientation during cryoEM SPR data collection.…”