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

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 H2O2. 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.