Spectroscopic evidence of the effect of hydrogen peroxide excess on the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae

Abstract

The actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae catalyzes the final reaction to generate heme b via the “coproporphyrin‐dependent” heme biosynthesis pathway in the presence of hydrogen peroxide. The enzyme has a high reactivity toward H2O2 used for the catalytic reaction and in the presence of an excess of H2O2 new species are generated. Resonance Raman data, together with electronic absorption spectroscopy and mass spectrometry, indicate that an excess of hydrogen peroxide for both the substrate (coproheme) and product (heme b) complexes of this enzyme causes a porphyrin hydroxylation of ring C or D, which is compatible with the formation of an iron chlorin‐type heme d species. A similar effect has been previously observed for other heme‐containing proteins, but this is the first time that a similar mechanism is reported for a coproheme enzyme. The hydroxylation determines a symmetry lowering of the porphyrin macrocycle, which causes the activation of A2g modes upon Soret excitation with a significant change in their polarization ratios, the enhancement and splitting into two components of many Eu bands, and an intensity decrease of the non‐totally symmetric modes B1g, which become polarized. This latter effect is clearly observed for the isolated ν10 mode upon either Soret or Q‐band excitations. The distal His118 is shown to be an absolute requirement for the conversion to heme d. This residue also plays an important role in the oxidative decarboxylation, because it acts as a base for deprotonation and subsequent heterolytic cleavage of hydrogen peroxide.

Reactivity of Coproheme Decarboxylase with Monovinyl, Monopropionate Deuteroheme

“…The second decarboxylation of p4 to yield the final product, heme b , was proposed to yield Compound I starting from MMD, which is now reported with experimental evidence in this work ( Figure 5 ). The lack of the distal H118 slows down the Compound I formation rate significantly and therefore also the decarboxylation reaction [ 9 ] and the side reaction of coproheme d formation [ 19 ]. H118 is found on the loop which connects the N-terminal and the C-terminal ferredoxin-like folds of a ChdC subunit [ 24 ].…”

“…The active site of ChdCs is built to stabilize the substrate via H-bonding interactions, especially interacting with propionate 2 (p2) and propionate 4 (p4), which are the ones to be cleaved off [ 14 , 15 , 16 ]. Coproheme iron is coordinated by a proximal histidine (H158), which is not involved in any hydrogen bonding network [ 14 , 17 , 18 , 19 ] ( Figure 2 C). This lack of H-bonding on the proximal side not only modulates the redox potential of the Fe(III)/Fe(II) couple [ 20 , 21 ], but also has two other major implications: (i) the final substrate, heme b , is not tightly bound to ChdC and can be easily delivered to heme-accepting apo-proteins [ 22 , 23 ]; and (ii) it is a necessary precondition for the proposed reorientation of MMD after the first decarboxylation in the active site, giving the appropriate amount of freedom to enable reorientation [ 12 , 13 ].…”

The role of the distal cavity in carbon monoxide stabilization in the coproheme decarboxylase enzyme from C. diphtheriae

“…For the wild-type protein, the heme b complex was also obtained by hemin reconstitution, as already described in Ref. [17], to avoid the presence of additional iron chlorintype heme d [23], or residual MMD species. For the H118A variant the heme b complex was prepared both by H 2 O 2 titration (heme b) and hemin reconstitution (reversed) [17].…”

“…WT heme d -and Y135A coproheme d -complexes were obtained with an excess of hydrogen peroxide as described in Ref. [23]. In the following figures and tables, both species are indicated as heme d for the sake of simplicity.…”

“…Moreover, in the presence of H118 residue, a stoichiometric excess of hydrogen peroxide leads to the formation of iron chlorin-type heme d species [20][21][22]. Heme d formation was correlated with a potential protection mechanism from oxidative damage [23]. 2) The ferric resting state of coproheme CdChdC is oxidized by activated (i.e.…”