Reaction intermediate rotation during the decarboxylation of coproheme to heme b in C. diphtheriae

Abstract

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H 2 O 2 . Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.

Reorienting Mechanism of Harderoheme in Coproheme Decarboxylase—A Computational Study

“…Since the experiment results indicated that Tyr145 also engages in p4 decarboxylation, it is rational to speculate that, after the decarboxylation of p2, harderoheme experiences reorientation to place p4 toward Tyr145. Such a process could be accomplished either through the intermediate harderoheme rotation along the Fe–N ε(His171) axis or through a harderoheme release–rebinding mechanism [ 23 , 24 , 25 ]. In a recent study, by mutating His118, which is located between the p6 and p7 side chains, to a bulkier phenylalanine, Hofbauer found that the His118Phe mutant of CdChdC was unable to perform the second decarboxylation reaction of p4 [ 25 ].…”

“…Such a process could be accomplished either through the intermediate harderoheme rotation along the Fe–N ε(His171) axis or through a harderoheme release–rebinding mechanism [ 23 , 24 , 25 ]. In a recent study, by mutating His118, which is located between the p6 and p7 side chains, to a bulkier phenylalanine, Hofbauer found that the His118Phe mutant of CdChdC was unable to perform the second decarboxylation reaction of p4 [ 25 ]. However, the His118Ala mutation did not interrupt the production of heme b [ 25 ].…”

“…In a recent study, by mutating His118, which is located between the p6 and p7 side chains, to a bulkier phenylalanine, Hofbauer found that the His118Phe mutant of CdChdC was unable to perform the second decarboxylation reaction of p4 [ 25 ]. However, the His118Ala mutation did not interrupt the production of heme b [ 25 ]. Hofbauer therefore suggested that Phe118 might inhibit the second decarboxylation step by hindering the reorientation process of the reaction intermediate harderoheme and proposed a rotation mechanism of harderoheme in the active site of CdChdC [ 25 , 26 ].…”

“…However, the His118Ala mutation did not interrupt the production of heme b [ 25 ]. Hofbauer therefore suggested that Phe118 might inhibit the second decarboxylation step by hindering the reorientation process of the reaction intermediate harderoheme and proposed a rotation mechanism of harderoheme in the active site of CdChdC [ 25 , 26 ]. However, this model does not fully exclude the possibility that harderoheme accomplishes reorientation through a release–rebinding mechanism.…”

Initial Steps to Engineer Coproheme Decarboxylase to Obtain Stereospecific Monovinyl, Monopropionyl Deuterohemes

“…H118, unique in actinobacterial ChdCds, is part of a flexible loop, linking the N- and the C-terminal domain of one subunit in ChdCs ( Hofbauer et al, 2021 ). In this variant, the catalytic tyrosine (Y135) of Cd ChdC is positioned correctly to facilitate decarboxylation of p2 but due to steric hindrance, the reorientation of the three-propionate intermediate is prohibited and therefore no decarboxylation of p4 occurs ( Sebastiani et al, 2021 ). In this study, we designed variants where another tyrosine is introduced at the site of p4 (W183Y).…”