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

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 fromcoproheme 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

Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme <i>b</i>. 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 <i>b</i> 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 <i>b</i> at excess of hydrogen peroxide

Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

Chlorite dismutases (Clds) are heme <i>b</i>-containing prokaryotic oxidoreductases that catalyze the reduction of chlorite to chloride with the concomitant release of molecular oxygen. Over time, they are irreversibly inactivated. To elucidate the mechanism of inactivation and investigate the role of the postulated intermediate hypochlorite, the pentameric chlorite dismutase of “Candidatus Nitrospira defluvii” (NdCld) and two variants (having the conserved distal arginine 173 exchanged with alanine and lysine) were recombinantly produced in <i>Escherichia coli</i>. Exchange of the distal arginine boosts the extent of irreversible inactivation. In the presence of the hypochlorite traps methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3<i>H</i>-xanthen-9-yl]­benzoic acid, the extent of chlorite degradation and release of molecular oxygen is significantly increased, whereas heme bleaching and oxidative modifications of the protein are suppressed. Among other modifications, hypochlorite-mediated formation of chlorinated tyrosines is demonstrated by mass spectrometry. The data obtained were analyzed with respect to the proposed reaction mechanism for chlorite degradation and its dependence on pH. We discuss the role of distal Arg173 by keeping hypochlorite in the reaction sphere for O–O bond formation

From Dynamic Combinatorial Chemistry to <i>in Vivo</i> Evaluation of Reversible and Irreversible Myeloperoxidase Inhibitors

The implementation of dynamic combinatorial libraries allowed the determination of highly active reversible and irreversible inhibitors of myeloperoxidase (MPO) at the nanomolar level. Docking experiments highlighted the interaction between the most active ligands and MPO, and further kinetic studies defined the mode of inhibition of these compounds. Finally, <i>in vivo</i> evaluation showed that one dose of irreversible inhibitors is able to suppress the activity of MPO after inducing inflammation

Posttranslational Modification of Heme <i>b</i> in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium <i>Lyngbya</i> sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in <i>Escherichia coli</i>, fully reconstituted to the respective heme <i>b</i> proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme <i>b</i> and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme <i>b</i> nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases

Chemistry and Molecular Dynamics Simulations of Heme <i>b</i>‑HemQ and Coproheme-HemQ

Recently, a novel pathway for heme <i>b</i> 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 <i>b</i>. 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 <i>Listeria monocytogenes</i> (LmHemQ) and <i>Staphylococcus aureus</i> (SaHemQ) recombinantly produced as apoproteins in <i>Escherichia coli.</i> 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 <i>b</i>-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 <i>b</i> 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 <i>b</i> release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria

Evaluation of New Scaffolds of Myeloperoxidase Inhibitors by Rational Design Combined with High-Throughput Virtual Screening

Myeloperoxidase (MPO) is a major player of the innate immune defense system of human neutrophils and catalyzes the production of strong oxidizing and halogenating antimicrobial products. Because of its role in pathogenesis of many (inflammatory) diseases, there is great interest in the development of efficient and specific inhibitors. Here, using the X-ray structure of MPO, high-throughput molecular docking of 1350000 compounds was performed. From this virtual screening process, 81 were tested for inhibition of the chlorination activity of MPO, finally ending up with eight inhibiting candidates of different chemical structures. These were tested for inhibiting MPO-mediated low-density lipoprotein oxidation and for interacting with the relevant redox intermediates of MPO. The best inhibitors were bis-2,2′-[(dihydro-1,3­(2<i>H</i>,4<i>H</i>)-pyrimidinediyl)­bis­(methylene)]­phenol and 8-[(2-aminoethyl)­amino]-3,7-dihydro-3-methyl-7-(3-phenoxypropyl)-1<i>H</i>-purine-2,6-dione. Both did not irreversibly inactivate the enzyme but efficiently trapped it in its compound II state. We discuss the mechanism of inactivation as well as pros and cons of the performed selection process

Redox Thermodynamics of High-Spin and Low-Spin Forms of Chlorite Dismutases with Diverse Subunit and Oligomeric Structures

Chlorite dismutases (Clds) are heme <i>b</i>-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from <i>Nitrobacter winogradskyi</i> (NwCld) and <i>Candidatus</i> “Nitrospira defluvii” (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The <i>E</i>°′ values for free and cyanide-bound proteins were determined to be −119 and −397 mV for NwCld and −113 and −404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism

Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry, and Reactivity

Chlorite dismutases (Clds) are heme <i>b</i> containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “<i>Candidatus</i> Nitrospira defluvii” (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV–vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an OO bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (<i>E</i>°′ = −113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation

Eukaryotic Catalase-Peroxidase: The Role of the Trp-Tyr-Met Adduct in Protein Stability, Substrate Accessibility, and Catalysis of Hydrogen Peroxide Dismutation

Recently, it was demonstrated that bifunctional catalase-peroxidases (KatGs) are found not only in archaea and bacteria but also in lower eukaryotes. Structural studies and preliminary biochemical data of the secreted KatG from the rice pathogen <i>Magnaporthe grisea</i> (<i>Mag</i>KatG2) suggested both similar and novel features when compared to those of the prokaryotic counterparts studied so far. In this work, we demonstrate the role of the autocatalytically formed redox-active Trp140-Tyr273-Met299 adduct of <i>Mag</i>KatG2 in (i) the maintenance of the active site architecture, (ii) the catalysis of hydrogen peroxide dismutation, and (iii) the protein stability by comparing wild-type <i>Mag</i>KatG2 with the single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of disruption of the covalent bonds between the adduct residues on the spectral signatures and heme cavity architecture was small. By contrast, loss of its integrity converts bifunctional <i>Mag</i>KatG2 to a monofunctional peroxidase of significantly reduced thermal stability. It increases the accessibility of ligands due to the increased flexibility of the KatG-typical large loop 1 (LL1), which contributes to the substrate access channel and anchors at the adduct Tyr. We discuss these data with respect to those known from prokaryotic KatGs and in addition present a high-resolution structure of an oxoiron compound of <i>Mag</i>KatG2