Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

Abstract

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme b. This work compares the structure and function of wild-type (WT) coproheme decarboxylase from Listeria monocytogenes and its M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance Raman, and electron paramagnetic resonance spectroscopies clearly show that the ferric form of the WT protein is a pentacoordinate quantum mechanically mixed-spin state, which is very unusual in biological systems. Exchange of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a 6-coordinate low spin species with the amide nitrogen atom of the Q187 residue bound to the heme iron. The interaction between M149 and propionyl 2 is found to play an important role in keeping the Q187 residue correctly positioned for closure of the distal cavity. This is confirmed by the observation that in the M149A variant two CO conformers are present corresponding to open (A0) and closed (A1) conformations. The CO of the latter species, the only conformer observed in the WT protein, is H-bonded to Q187. In the absence of the Q187 residue or in the adducts of all the heme b forms of ChdC investigated herein (containing vinyls in positions 2 and 4), only the A0 conformer has been found. Moreover, M149 is shown to be involved in the formation of a covalent bond with a vinyl substituent of heme b at excess of hydrogen peroxide.

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

“…Therefore, we concluded that in the A 1 conformation Q187 is H-bonded to bound carbon monoxide, as previously observed in SaChdC [9]. Interestingly, the open form (A 0 ) has also been observed in all CO adducts of the heme b products (containing two vinyl substituents in positions 2 and 4) of Lm ChdC [7]. These data indicate that in the coproheme complex, the interaction between the M149 residue and the propionate in position 2 has an important role in keeping glutamine 187 correctly positioned for the closure of the distal cavity and for the radicalic decarboxylation mediated by Y147.…”

“…Site-directed mutagenesis to obtain LmChdC M149A, Q187A and M149A/Q187A variants using the QuikChange Lightning Kit (Agilent Technologies) was described previously [7]. Generation of LmChdC Y147A, R133A, R179A, K151A, Y113A and Y113A/K151A, Y147A/R220A/S225A variants were produced following the same protocol with the primers listed in Table S1.…”

“…1 depicts the active site of ChdC with histidine as proximal ligand of coproheme and a conserved mobile glutamine in the distal cavity. We used the structure of GsChdC with Mn-coproheme (pdb-code: 5T2K), instead of that of LmChdC with Fe-coproheme since, as stated previously, the former represents the more appropriate interpretation of the structural data of coproheme decarboxylase [7].…”

“…Recently, we have shown that the native ferric form of coproheme decarboxylase from Listeria monocytogenes ( Lm ChdC) is a pentacoordinate quantum mechanically mixed-spin state (5cQS) which is very unusual in biological systems [7]. However, mutation of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a hexacoordinate low-spin (6cLS) species with the amide nitrogen atom of flexible Q187 bound to the heme iron (Fig.…”

“…1). Interestingly, the heme b product formed upon reaction of the M149A mutant with H 2 O 2 consists of two 6cLS species with different resonance Raman (RR) bands [7]. One form (RR ν 3 and ν 2 bands at 1507 and 1588 cm −1 , respectively), absent in the Q187A single (Q187A) and double (M149A/Q187A) mutants, is due to the coordination with Q187 at the sixth position.…”