Compound I Formation and Reactivity in Dimeric Chlorite Dismutase: Impact of pH and the Dynamics of the Catalytic Arginine.

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III: Propionate interactions and porphyrin core deformation.

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV-vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.

Posttranslational modification and heme cavity architecture of human eosinophil peroxidase-insights from first crystal structure and biochemical characterization.

Eosinophil peroxidase (EPO) is the most abundant granule protein exocytosed by eosinophils, specialized human phagocytes. Released EPO catalyzes the formation of reactive oxidants from bromide, thiocyanate, and nitrite that kill tissue-invading parasites. However, EPO also plays a deleterious role in inflammatory diseases, making it a potential pharmacological target. A major hurdle is the high similarity to the homologous myeloperoxidase (MPO), which requires a detailed understanding of the small structural differences that can be used to increase the specificity of the inhibitors. Here, we present the first crystal structure of mature leukocyte EPO at 1.6 Å resolution together with analyses of its posttranslational modifications and biochemical properties. EPO has an exceptionally high number of positively charged surface patches but only two occupied glycosylation sites. The crystal structure further revealed the existence of a light (L) and heavy (H) chain as a result of proteolytic cleavage. Detailed comparison with the structure of human MPO allows us to identify differences that may contribute to the known divergent enzymatic properties. The crystal structure revealed fully established ester links between the prosthetic group and the protein, the comparably weak imidazolate character of the proximal histidine, and the conserved structure of the catalytic amino acids and Ca2+-binding site. Prediction of the structure of unprocessed proeosinophil peroxidase allows further structural analysis of the three protease cleavage sites and the potential pro-convertase recognition site in the propeptide. Finally, EPO biosynthesis and its biochemical and biophysical properties are discussed with respect to the available data from the well-studied MPO.

The staphylococcal inhibitory protein SPIN binds to human myeloperoxidase with picomolar affinity but only dampens halide oxidation.

The heme enzyme myeloperoxidase (MPO) is one of the key players in the neutrophil-mediated killing of invading pathogens as part of the innate immune system. MPO generates antimicrobial oxidants, which indiscriminately and effectively kill phagocytosed pathogens. Staphylococcus aureus, however, is able to escape this fate, in part by secreting a small protein called SPIN (Staphylococcal Peroxidase Inhibitor), which specifically targets and inhibits MPO in a structurally complex manner. Here, we present the first crystal structures of the complex of SPIN-aureus and a truncated version (SPIN-truncated) with mature dimeric leukocyte MPO. We unravel the contributions of the two domains to the kinetics and thermodynamics of SPIN-aureus binding to MPO by using a broad array of complementary biochemical and biophysical methods. The C-terminal "recognition" domain is shown to mediate specific binding to MPO, while interaction of the N-terminal "inhibitory" domain is guided mainly by hydrophobic effects and thus is less sequence dependent. We found that inhibition of MPO is achieved by reducing substrate migration, but SPIN-aureus cannot completely block MPO activity. Its' effectiveness is inversely related to substrate size, with no discernible dependence on other factors. Thus, SPIN-aureus is an extremely high-affinity inhibitor and highly efficient for substrates larger than halogens. As aberrant MPO activity is implicated in a number of chronic inflammatory diseases, SPIN-aureus is the first promising protein inhibitor for specific inhibition of human MPO.

Impact of the dynamics of the catalytic arginine on nitrite and chlorite binding by dimeric chlorite dismutase.

Chlorite dismutases (Clds) are heme b containing oxidoreductases able to decompose chlorite to chloride and molecular oxygen. This work analyses the impact of the distal, flexible and catalytic arginine on the binding of anionic angulate ligands like nitrite and the substrate chlorite. Dimeric Cld from Cyanothece sp. PCC7425 was used as a model enzyme. We have investigated wild-type CCld having the distal catalytic R127 hydrogen-bonded to glutamine Q74 and variants with R127 (i) being arrested in a salt-bridge with a glutamate (Q74E), (ii) being fully flexible (Q74V) or (iii) substituted by either alanine (R127A) or lysine (R127K). We present the electronic and spectral signatures of the high-spin ferric proteins and the corresponding low-spin nitrite complexes elucidated by UV-visible, circular dichroism and electron paramagnetic resonance spectroscopies. Furthermore, we demonstrate the impact of the dynamics of R127 on the thermal stability of the respective nitrite adducts and present the X-ray crystal structures of the nitrite complexes of wild-type CCld and the variants Q74V, Q74E and R127A. In addition, the molecular dynamics (MD) and the binding modi of nitrite and chlorite to the ferric wild-type enzyme and the mutant proteins and the interaction of the oxoanions with R127 have been analysed by MD simulations. The findings are discussed with respect to the role(s) of R127 in ligand and chlorite binding and substrate degradation.

In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase-The Interplay of Key Amino Acids, pH, Buffer and Glycerol.

Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from Klebsiella pneumoniae (KpDyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs.

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

Arresting the Catalytic Arginine in Chlorite Dismutases: Impact on Heme Coordination, Thermal Stability, and Catalysis.

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that can decompose chlorite to chloride and molecular oxygen. They are divided in two clades that differ in oligomerization, subunit architecture, and the hydrogen-bonding network of the distal catalytic arginine, which is proposed to switch between two conformations during turnover. To understand the impact of the conformational dynamics of this basic amino acid on heme coordination, structure, and catalysis, Cld from Cyanothece sp. PCC7425 was used as a model enzyme. As typical for a clade 2 Cld, its distal arginine 127 is hydrogen-bonded to glutamine 74. The latter has been exchanged with either glutamate (Q74E) to arrest R127 in a salt bridge or valine (Q74V) that mirrors the setting in clade 1 Clds. We present the X-ray crystal structures of Q74V and Q74E and demonstrate the pH-induced changes in the environment and coordination of the heme iron by ultraviolet-visible, circular dichroism, and electron paramagnetic resonance spectroscopies as well as differential scanning calorimetry. The conformational dynamics of R127 is shown to have a significant role in heme coordination during the alkaline transition and in the thermal stability of the heme cavity, whereas its impact on the catalytic efficiency of chlorite degradation is relatively small. The findings are discussed with respect to (i) the flexible loop connecting the N-terminal and C-terminal ferredoxin-like domains, which differs in clade 1 and clade 2 Clds and carries Q74 in clade 2 proteins, and (ii) the proposed role(s) of the arginine in catalysis.

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

There is a high functional diversity within the structural superfamily of porphyrin-binding dimeric α + ß barrel proteins. In this review we aim to analyze structural constraints of chlorite dismutases, dye-decolorizing peroxidases and coproheme decarboxylases in detail. We identify regions of structural variations within the highly conserved fold, which are most likely crucial for functional specificities. The loop linking the two ferredoxin-like domains within one subunit can be of different sequence lengths and can adopt various structural conformations, consequently defining the shape of the substrate channels and the respective active site architectures. The redox cofactor, heme b or coproheme, is oriented differently in either of the analyzed enzymes. By thoroughly dissecting available structures and discussing all available results in the context of the respective functional mechanisms of each of these redox-active enzymes, we highlight unsolved mechanistic questions in order to spark future research in this field.

X-ray-induced photoreduction of heme metal centers rapidly induces active-site perturbations in a protein-independent manner.

Since the advent of protein crystallography, atomic-level macromolecular structures have provided a basis to understand biological function. Enzymologists use detailed structural insights on ligand coordination, interatomic distances, and positioning of catalytic amino acids to rationalize the underlying electronic reaction mechanisms. Often the proteins in question catalyze redox reactions using metal cofactors that are explicitly intertwined with their function. In these cases, the exact nature of the coordination sphere and the oxidation state of the metal is of utmost importance. Unfortunately, the redox-active nature of metal cofactors makes them especially susceptible to photoreduction, meaning that information obtained by photoreducing X-ray sources about the environment of the cofactor is the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species by X-ray radiation. We show that a dose of ∼40 kilograys already yields 50% ferrous iron in a heme protein crystal. We also demonstrate that the kinetics of photoreduction are completely independent from variables unique to the different samples tested. The photoreduction-induced structural rearrangements around the metal cofactors have to be considered when biochemical data of ferric proteins are rationalized by constraints derived from crystal structures of reduced enzymes.

Actinobacterial Coproheme Decarboxylases Use Histidine as a Distal Base to Promote Compound I Formation.

Coproheme decarboxylases (ChdCs) catalyze the final step in heme b biosynthesis of monoderm and some diderm bacteria. In this reaction, coproheme is converted to heme b via monovinyl monopropionate deuteroheme (MMD) in two consecutive decarboxylation steps. In Firmicutes decarboxylation of propionates 2 and 4 of coproheme depend on hydrogen peroxide and the presence of a catalytic tyrosine. Here we demonstrate that ChdCs from Actinobacteria are unique in using a histidine (H118 in ChdC from Corynebacterium diphtheriae, CdChdC) as a distal base in addition to the redox-active tyrosine (Y135). We present the X-ray crystal structures of coproheme-CdChdC and MMD-CdChdC, which clearly show (i) differences in the active site architecture between Firmicutes and Actinobacteria and (ii) rotation of the redox-active reaction intermediate (MMD) after formation of the vinyl group at position 2. Distal H118 is shown to catalyze the heterolytic cleavage of hydrogen peroxide (k app = (4.90 ± 1.25) × 104 M-1 s-1). The resulting Compound I is rapidly converted to a catalytically active Compound I* (oxoiron(IV) Y135•) that initiates the radical decarboxylation reactions. As a consequence of the more efficient Compound I formation, actinobacterial ChdCs exhibit a higher catalytic efficiency in comparison to representatives from Firmicutes. On the basis of the kinetic data of wild-type CdChdC and the variants H118A, Y135A, and H118A/Y135A together with high-resolution crystal structures and molecular dynamics simulations, we present a molecular mechanism for the hydrogen peroxide dependent conversion of coproheme via MMD to heme b and discuss differences between ChdCs from Actinobacteria and Firmicutes.

Reaction of human peroxidasin 1 compound I and compound II with one-electron donors.

Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M-1 s-1 (urate) to (4.8 ± 0.1) × 105 M-1 s-1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M-1 s-1 (tyrosine) to (7.7 ± 0.1) × 103 M-1 s-1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

Monomeric and homotrimeric solution structures of truncated human peroxidasin 1 variants.

Human peroxidasin 1 is a multidomain peroxidase situated in the basement membrane. The iron enzyme with covalently bound heme oxidizes bromide to hypobromous acid which facilitates the formation of distinct sulfilimine cross-links in the collagen IV network and therefore contributes to its mechanical stability. Additional to the catalytically active peroxidase domain peroxidasin comprises a leucine rich repeat domain, four Ig domains and a C-terminal von Willebrand factor type C module (VWC). Peroxidasin has been shown to form homotrimers involving two redox-sensitive cysteine residues and to undergo posttranslational C-terminal proteolytic cleavage. The present study on several recombinantly produced truncated peroxidasin variants showed that the VWC is not required for trimer formation whereas the alpha-helical linker region located between the peroxidase domain and the VWC is crucial for trimerization. Our data furthermore implies that peroxidasin oligomerization occurs intracellularly before C-terminal cleavage. For the first time we present overall solution structures of monomeric and trimeric truncated peroxidasin variants which were determined by rotary shadowing combined with transmission electron microscopy and by small-angle X-ray scattering (SAXS). A triangular arrangement of the peroxidase domains to each other within the homotrimer was revealed and this structure was confirmed by a model of trimeric peroxidase domains. Our SAXS data showed that the Ig domains are highly flexible and interact with the peroxidase domain and that within the homotrimer each alpha-helical linker region interacts with the respective adjacent peroxidase domain. The implications of our findings on the structure-function relationship of peroxidasin are discussed.

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

Coproheme decarboxylase (ChdC) catalyzes the last step in the heme biosynthesis pathway of monoderm bacteria with coproheme acting both as redox cofactor and substrate. Hydrogen peroxide mediates the stepwise decarboxylation of propionates 2 and 4 of coproheme. Here we present the crystal structures of coproheme-loaded ChdC from Listeria monocytogenes (LmChdC) and the three-propionate intermediate, for which the propionate at position 2 (p2) has been converted to a vinyl group and is rotated by 90° compared to the coproheme complex structure. Single, double, and triple mutants of LmChdC, in which H-bonding interactions to propionates 2, 4, 6, and 7 were eliminated, allowed us to obtain the assignment of the coproheme propionates by resonance Raman spectroscopy and to follow the H2O2-mediated conversion of coproheme to heme b. Substitution of H2O2 by chlorite allowed us to monitor compound I formation in the inactive Y147H variant which lacks the catalytically essential Y147. This residue was demonstrated to be oxidized during turnover by using the spin-trap 2-methyl-2-nitrosopropane. Based on these findings and the data derived from molecular dynamics simulations of cofactor structures in distinct poses, we propose a reaction mechanism for the stepwise decarboxylation of coproheme that includes a 90° rotation of the intermediate three-propionate redox cofactor.

Redox thermodynamics of B-class dye-decolorizing peroxidases.

With >5000 annotated genes dye-decolorizing peroxidases (DyPs) represent a heme b peroxidase family of broad functional diversity. Bacterial B-class DyPs are poor peroxidases of unknown physiological function. Hydrogen peroxide efficiently mediates the rapid formation of Compound I in B-class DyPs, which, however, is stable and shows modest reactivity towards organic and inorganic electron donors. To understand these characteristics, we have investigated the redox thermodynamics of the one-electron reduction of the ferric high-spin form of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) and the variants D143A, R232A and D143A/R232A. These distal amino acids are fully conserved in all DyPs and play important roles in Compound I formation and maintenance of the heme cavity architecture and substrate access route(s). The E°' values of the respective redox couples Fe(III)/Fe(II) varied from -350 mV (wild-type KpDyP) to -299 mV (D143A/R232A) at pH 7.0. Variable-temperature spectroelectrochemical experiments revealed that the reduction reaction of B-class DyPs is enthalpically unfavored but entropically favored with significant differences in enthalpic and entropic contributions to E°' between the four proteins. Molecular dynamics simulations demonstrated the impact of solvent reorganization on the entropy change during reduction reaction and revealed the dynamics and restriction of substrate access channels. Obtained data are discussed with respect to the poor peroxidase activities of B-class DyPs and compared with heme peroxidases from other (super)families as well as with chlorite dismutases, which do not react with hydrogen peroxide but share a similar fold and heme cavity architecture.

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

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 H2O2. 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 H2O2. 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.

A constitutive active allele of the transcription factor Msn2 mimicking low PKA activity dictates metabolic remodeling in yeast.

In yeast, protein kinase A (PKA) adjusts transcriptional profiles, metabolic rates, and cell growth in accord with carbon source availability. PKA affects gene expression mostly via the transcription factors Msn2 and Msn4, two key regulators of the environmental stress response. Here we analyze the role of the PKA-Msn2 signaling module using an Msn2 allele that harbors serine-to-alanine substitutions at six functionally important PKA motifs (Msn2A6) . Expression of Msn2A6 mimics low PKA activity, entails a transcription profile similar to that of respiring cells, and prevents formation of colonies on glucose-containing medium. Furthermore, Msn2A6 leads to high oxygen consumption and hence high respiratory activity. Substantially increased intracellular concentrations of several carbon metabolites, such as trehalose, point to a metabolic adjustment similar to diauxic shift. This partial metabolic switch is the likely cause for the slow-growth phenotype in the presence of glucose. Consistently, Msn2A6 expression does not interfere with growth on ethanol and tolerated is to a limited degree in deletion mutant strains with a gene expression signature corresponding to nonfermentative growth. We propose that the lethality observed in mutants with hampered PKA activity resides in metabolic reprogramming that is initiated by Msn2 hyperactivity.

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage.

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide-dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

Posttranslational modification of heme in peroxidases - Impact on structure and catalysis.

Four heme peroxidase superfamilies arose independently in evolution. Only in the peroxidase-cyclooxygenase superfamily the prosthetic group is posttranslationally modified (PTM). As a consequence these peroxidases can form one, two or three covalent bonds between heme substituents and the protein. This may include ester bonds between heme 1- and 5-methyl groups and glutamate and aspartate residues as well as a sulfonium ion link between the heme 2-vinyl substituent and a methionine. Here the phylogeny and physiological roles of representatives of this superfamily, their occurrence in all kingdoms of life, the relevant sequence motifs for definite identification and the available crystal structures are presented. We demonstrate the autocatalytic posttranslational maturation process and the impact of the covalent links on spectral and redox properties as well as on catalysis, including Compound I formation and reduction by one- and two-electron donors. Finally, we discuss the evolutionary advantage of these PTMs with respect to the proposed physiological functions of the metalloenzymes that range from antimicrobial defence in innate immunity to extracellular matrix formation and hormone biosynthesis.

Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e. g. Nitrospirae) and also in Archaea. In order to test phylogenetic prediction ChdC representatives from all clades were expressed and examined for their coproheme decarboxylase activity. Based on available biochemical data and phylogenetic analyses a sequence motif (-Y-P-M/F-X-K/R-) is defined for ChdCs. We show for the first time that in diderm bacteria an active coproheme decarboxylase is present and that the archaeal ChdC homolog from Sulfolobus solfataricus is inactive and its physiological role remains elusive. This shows the limitation of phylogenetic prediction of an enzymatic activity, since the identified sequence motif is equally conserved across all previously defined clades.