Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ

Abstract

Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of −205 ± 3 mV for LmHemQ and −207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 104 M–1 s–1). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria.

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b

“…5). The spectrum measured at 0s was largely high spin (S=5/2) and rhombic, similar to spectra previously reported for HemQcoproheme complexes from S. aureus and Listeria monocytogenes (23). At 30s, no obvious new signals due to iron species appeared.…”

Hydrogen peroxide‐mediated conversion of coproheme to heme b by HemQ—lessons from the first crystal structure and kinetic studies

“…). The interactions of Y147 and S225 with p4 and Q187 with p2 were previously correctly predicted in silico using MD simulations .…”

“…However, this requires a detailed knowledge of structure–function relationships that at present is lacking; only apo‐structures of HemQs are available in the Protein Data Bank (http://www.pdb.org) . The interaction of apo‐HemQ from Listeria monocytogenes with coproheme was previously evaluated by means of molecular dynamics simulations of a coproheme‐bound model of LmHemQ, based on the apo‐structure (http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4WWS), suggesting hydrogen bonds between Y147 and S225 and p4 as well as between Q187 and p2 . Coproheme binding to apo‐HemQ was shown to be biphasic resulting in the formation of mainly high‐spin ferric protein with a standard reduction potential of the Fe(III)/Fe(II) couple of −205 mV at pH 7.0 .…”

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ)

“…S7) or energy-minimized predictions of the structure of the decarboxylase-coproheme complex. 31 This orientation positions the two unreactive propionates (6 and 7, Scheme 1) pointing outward from the protein toward the solvent, where they occupy positions on the distal side of the heme plane (Fig. 2B).…”

“…31 Unique peripheral substituent environments of ferric heme b in decarboxylases and Clds are supported by their propionate and vinyl (δ(C β C a C b )) bending mode frequencies. The decarboxylase complexes exhibit δ(C β C c C d ) and δ(C β C a C b ) frequencies 8–11 cm −1 and 4–8 cm −1 lower, respectively, relative to those modes in WT Da Cld and Da Cld Arg183Gln mutant (Fig.…”