The hydrogen bonding network of coproheme in coproheme decarboxylase from Listeria monocytogenes: Effect on structure and catalysis

Abstract

Coproheme decarboxylase (ChdC) catalyzes the oxidative decarboxylation of coproheme to heme b , i.e. the last step in the recently described coproporphyrin-dependent pathway. Coproheme decarboxylation from Listeria monocytogenes is a robust enzymatic reaction of low catalytic efficiency. Coproheme acts as both substrate and redox cofactor activated by H 2 O 2 . It fully depends on the catalytic Y147 close to the propionyl group at position 2. In the present study we have investigated the effect of disruption of the comprehensive and conserved hydrogen bonding network between the four propionates and heme cavity residues on (i) the conformational stability of the heme cavity, (ii) the electronic configuration of the ferric redox cofactor/substrate, (iii) the binding of carbon monoxide and, (iv) the decarboxylation reaction mediated by addition of H 2 O 2 . Nine single, double and triple mutants of ChdC from Listeria monocytogenes were produced in E. coli . The respective coproheme- and heme b -complexed proteins were studied by UV–Vis, resonance Raman, circular dichroism spectroscopy, and mass spectrometry. Interactions of propionates 2 and 4 with residues in the hydrophobic cavity are crucial for maintenance of the heme cavity architecture, for the mobile distal glutamine to interact with carbon monoxide, and to keep the heme cavity in a closed conformation during turnover. By contrast, the impact of substitution of residues interacting with solvent exposed propionates 6 and 7 was negligible. Except for Y147A and K151A all mutant ChdCs exhibited a wild-type-like catalytic activity. The findings are discussed with respect to the structure-function relationships of ChdCs.

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].…”

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

“…Except for Y147A and K151A, all LmChdC variants exhibit a wild-type-like catalytic activity. 18 During decarboxylation of the first propionate substituent, H 2 O 2 oxidizes coproheme-LmChdC to compound I [oxoiron(IV) Por •+ ], which rapidly converts to compound I* [oxoiron(IV) • Y147]. This species attacks the Cβ atom of the corresponding propionate.…”

“…This could play an important role in compound I formation. 10,18 Upon disrupting the H-bonding network between p2 and p4 or elimination of Q187, formation of an open conformation has been observed and catalytic activity was diminished. 10…”

Understanding molecular enzymology of porphyrin-binding α + β barrel proteins - One fold, multiple functions

“…9 ). In ChdCs, a tyrosine at the edge of the distal pocket, in close proximity to propionate 2 (p2) in the resting state is essential for catalysis [ 62 , 90 , 91 ]. For ChdCs from Actinobacteria, a distal histidine residue was identified to be important for deprotonation of the incoming hydrogen peroxide, which is part of the same flexible loop described above [ 64 ].…”

“…It establishes a sulfonium-ion linkage, similar as in human myeloperoxidase [ 94 – 98 ] to the formed vinyl group of the product heme b , when hydrogen peroxide is present in excess [ 93 ]. The integrity of the active site and the maintenance of the wild-type like spin-state of the heme iron is further guaranteed by the hydrogen bonding network spanning from residues interacting with p2 (arginine, serine) to residues interacting with p4 (lysine) [ 91 ]. Disturbance of this H-bonding network leads to a collapse of the active site, as was demonstrated by electronic circular dichroism spectroscopy [ 91 ].…”