Entrance channels to coproheme in coproporphyrin ferrochelatase probed by exogenous imidazole binding.

Iron insertion into porphyrins is an essential step in heme biosynthesis. In the coproporphyrin-dependent pathway, specific to monoderm bacteria, this reaction is catalyzed by the monomeric enzyme coproporphyrin ferrochelatase. In addition to the mechanistic details of the metalation of the porphyrin, the identification of the substrate access channel for ferrous iron to the active site is important to fully understand this enzymatic system. In fact, whether the iron reaches the active site from the distal or the proximal porphyrin side is still under debate. In this study we have thoroughly addressed this question in Listeria monocytogenes coproporphyrin ferrochelatase by X-ray crystallography, steady-state and pre-steady-state imidazole ligand binding studies, together with a detailed spectroscopic characterization using resonance Raman and UV-vis absorption spectroscopies in solution. Analysis of the X-ray structures of coproporphyrin ferrochelatase-coproporphyrin III crystals soaked with ferrous iron shows that iron is present on both sides of the porphyrin. The kinetic and spectroscopic study of imidazole binding to coproporphyrin ferrochelatase­iron coproporphyrin III clearly indicates the presence of two possible binding sites in this monomeric enzyme that influence each other, which is confirmed by the observed cooperativity at steady-state and a biphasic behavior in the pre-steady-state experiments. The current results are discussed in the context of the entire heme biosynthetic pathway and pave the way for future studies focusing on protein-protein interactions.

Proximal ligand tunes active site structure and reactivity in bacterial L. monocytogenes coproheme ferrochelatase.

Ferrochelatases catalyze the insertion of ferrous iron into the porphyrin during the heme b biosynthesis pathway, which is fundamental for both prokaryotes and eukaryotes. Interestingly, in the active site of ferrochelatases, the proximal ligand coordinating the porphyrin iron of the product is not conserved, and its catalytic role is still unclear. Here we compare the L. monocytogenes bacterial coproporphyrin ferrochelatase native enzyme together with selected variants, where the proximal Tyr residue was replaced by a His (i.e. the most common ligand in heme proteins), a Met or a Phe (as in human and actinobacterial ferrochelatases, respectively), in their Fe(III), Fe(II) and Fe(II)-CO adduct forms. The study of the active site structure and the activity of the proteins in solution has been performed by UV-vis electronic absorption and resonance Raman spectroscopies, biochemical characterization, and classical MD simulations. All the mutations alter the H-bond interactions between the iron porphyrin propionate groups and the protein, and induce effects on the activity, depending on the polarity of the proximal ligand. The overall results confirm that the weak or non-existing coordination of the porphyrin iron by the proximal residue is essential for the binding of the substrate and the release of the final product.

Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants.

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey et al. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here, we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely, His182Ala (H182A) and Glu263Gln (E263Q), involving two key active site residues. Interestingly, both variants are active only toward the physiological substrate coproporphyrin III but inactive toward protoporphyrin IX. In addition, E263 exchange impairs the final oxidation step from ferrous coproheme to ferric coproheme. The characteristics of the active site in the context of the residues involved and the substrate binding properties are discussed here using structural and functional means, providing a further contribution to the deciphering of this enigmatic reaction mechanism.

The Molecular Evolution, Structure, and Function of Coproporphyrinogen Oxidase and Protoporphyrinogen Oxidase in Prokaryotes.

Coproporphyrinogen oxidase (CgoX) and protoporphyrinogen oxidase (PgoX) catalyze the oxidation of the flexible cyclic tetrapyrrole of porphyrinogen compounds into fully conjugated, planar macrocyclic porphyrin compounds during heme biosynthesis. These enzymes are activated via different pathways. CgoX oxidizes coproporphyrinogen III to coproporphyrin III in the coproporphyrin-dependent pathway, whereas PgoX oxidizes protoporphyrinogen IX to protoporphyrin IX in the penultimate step of the protoporphyrin-dependent pathway. The phylogenetic analysis presented herein demonstrates a clear differentiation between the two enzyme classes, as evidenced by the clustering of sequences in distinct clades, and it shows that, at the origin of porphyrinogen-type oxidase evolution, PgoXs from cyanobacteria were found, which were noticeably separated from descendant PgoX representatives of Deltaproteobacteria and all later PgoX variants, leading to many eukaryotic clades. CgoX sequences originating from the monoderm Actinomycetota and Bacillota were well separated from the predecessor clades containing PgoX types and represent a peculiar type of gene speciation. The structural similarities and differences between these two oxidases are discussed based on their protein sequence alignment and a structural comparison.

Iron insertion into coproporphyrin III-ferrochelatase complex: Evidence for an intermediate distorted catalytic species.

Understanding the reaction mechanism of enzymes at the molecular level is generally a difficult task, since many parameters affect the turnover. Often, due to high reactivity and formation of transient species or intermediates, detailed information on enzymatic catalysis is obtained by means of model substrates. Whenever possible, it is essential to confirm a reaction mechanism based on substrate analogues or model systems by using the physiological substrates. Here we disclose the ferrous iron incorporation mechanism, in solution, and in crystallo, by the coproporphyrin III-coproporphyrin ferrochelatase complex from the firmicute, pathogen, and antibiotic resistant, Listeria monocytogenes. Coproporphyrin ferrochelatase plays an important physiological role as the metalation represents the penultimate reaction step in the prokaryotic coproporphyrin-dependent heme biosynthetic pathway, yielding coproheme (ferric coproporphyrin III). By following the metal titration with resonance Raman spectroscopy and x-ray crystallography, we prove that upon metalation the saddling distortion becomes predominant both in the crystal and in solution. This is a consequence of the readjustment of hydrogen bond interactions of the propionates with the protein scaffold during the enzymatic catalysis. Once the propionates have established the interactions typical of the coproheme complex, the distortion slowly decreases, to reach the almost planar final product.

Structural aspects of enzymes involved in prokaryotic Gram-positive heme biosynthesis.

The coproporphyrin dependent heme biosynthesis pathway is almost exclusively utilized by Gram-positive bacteria. This fact makes it a worthwhile topic for basic research, since a fundamental understanding of a metabolic pathway is necessary to translate the focus towards medical biotechnology, which is very relevant in this specific case, considering the need for new antibiotic targets to counteract the pathogenicity of Gram-positive superbugs. Over the years a lot of structural data on the set of enzymes acting in Gram-positive heme biosynthesis has accumulated in the Protein Database (www.pdb.org). One major challenge is to filter and analyze all available structural information in sufficient detail in order to be helpful and to draw conclusions. Here we pursued to give a holistic overview of structural information on enzymes involved in the coproporphyrin dependent heme biosynthesis pathway. There are many aspects to be extracted from experimentally determined structures regarding the reaction mechanisms, where the smallest variation of the position of an amino acid residue might be important, but also on a larger level regarding protein-protein interactions, where the focus has to be on surface characteristics and subunit (secondary) structural elements and oligomerization. This review delivers a status quo, highlights still missing information, and formulates future research endeavors in order to better understand prokaryotic heme biosynthesis.

Reactivity of Coproheme Decarboxylase with Monovinyl, Monopropionate Deuteroheme.

Coproheme decarboxylases (ChdCs) are terminal enzymes of the coproporphyrin-dependent heme biosynthetic pathway. In this reaction, two propionate groups are cleaved from the redox-active iron-containing substrate, coproheme, to form vinyl groups of the heme b product. The two decarboxylation reactions proceed sequentially, and a redox-active three-propionate porphyrin, called monovinyl, monopropionate deuteroheme (MMD), is transiently formed as an intermediate. While the reaction mechanism for the first part of the redox reaction, which is initiated by hydrogen peroxide, has been elucidated in some detail, the second part of this reaction, starting from MMD, has not been studied. Here, we report the optimization of enzymatic MMD production by ChdC and purification by reversed-phase chromatography. With the obtained MMD, we were able to study the second part of heme b formation by actinobacterial ChdC from Corynebacterium diphtheriae, starting with Compound I formation upon the addition of hydrogen peroxide. The results indicate that the second part of the decarboxylation reaction is analogous to the first part, although somewhat slower, which is explained by differences in the active site architecture and its H-bonding network. The results are discussed in terms of known kinetic and structural data and help to fill some mechanistic gaps in the overall reaction catalyzed by ChdCs.

The role of the distal cavity in carbon monoxide stabilization in the coproheme decarboxylase enzyme from C. diphtheriae.

This work focuses on the carbon monoxide adducts of the wild-type and selected variants of the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae complexed with coproheme, monovinyl monopropionyl deuteroheme (MMD), and heme b. The UV - vis and resonance Raman spectroscopies together with the molecular dynamics simulations clearly show that the wild-type coproheme-CO adduct is characterized by two CO conformers, one hydrogen-bonded to the distal H118 residue and the other showing a weak polar interaction with the distal cavity. Instead, upon conversion to heme b, i.e. after decarboxylation of propionates 2 and 4 and rotation by 90o of the porphyrin ring inside the cavity, CO probes a less polar environment. In the absence of the H118 residue, both coproheme and heme b complexes form only the non-H-bonded CO species. The unrotated MMD-CO adduct as observed in the H118F variant, confirms that decarboxylation of propionate 2 only, does not affect the heme cavity. The rupture of both the H-bonds involving propionates 2 and 4 destabilizes the porphyrin inside the cavity with the subsequent formation of a CO adduct in an open conformation. In addition, in this work we present data on CO binding to reversed heme b, obtained by hemin reconstitution of the H118A variant, and to heme d, obtained by addition of an excess of hydrogen peroxide. The results will be discussed and compared with those reported for the representatives of the firmicute clade.

The Role of the Hydrogen Bond Network in Maintaining Heme Pocket Stability and Protein Function Specificity of C. diphtheriae Coproheme Decarboxylase.

Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H2O2, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen Corynebacterium diphtheriae ChdC complexed with coproheme and heme b. The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product.

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.

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.

Spectroscopic evidence of the effect of hydrogen peroxide excess on the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae.

The actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae catalyzes the final reaction to generate heme b via the "coproporphyrin-dependent" heme biosynthesis pathway in the presence of hydrogen peroxide. The enzyme has a high reactivity toward H2O2 used for the catalytic reaction and in the presence of an excess of H2O2 new species are generated. Resonance Raman data, together with electronic absorption spectroscopy and mass spectrometry, indicate that an excess of hydrogen peroxide for both the substrate (coproheme) and product (heme b) complexes of this enzyme causes a porphyrin hydroxylation of ring C or D, which is compatible with the formation of an iron chlorin-type heme d species. A similar effect has been previously observed for other heme-containing proteins, but this is the first time that a similar mechanism is reported for a coproheme enzyme. The hydroxylation determines a symmetry lowering of the porphyrin macrocycle, which causes the activation of A2g modes upon Soret excitation with a significant change in their polarization ratios, the enhancement and splitting into two components of many Eu bands, and an intensity decrease of the non-totally symmetric modes B1g, which become polarized. This latter effect is clearly observed for the isolated ν10 mode upon either Soret or Q-band excitations. The distal His118 is shown to be an absolute requirement for the conversion to heme d. This residue also plays an important role in the oxidative decarboxylation, because it acts as a base for deprotonation and subsequent heterolytic cleavage of hydrogen peroxide.

An active site at work - the role of key residues in C. diphteriae coproheme decarboxylase.

Coproheme decarboxylases (ChdCs) are utilized by monoderm bacteria to produce heme b by a stepwise oxidative decarboxylation of the 2- and 4-propionate groups of iron coproporphyrin III (coproheme) to vinyl groups. This work compares the effect of hemin reconstitution versus the hydrogen peroxide-mediated conversion of coproheme to heme b in the actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) and selected variants. Both ferric and ferrous forms of wild-type (WT) CdChdC and its H118A, H118F, and A207E variants were characterized by resonance Raman and UV-vis spectroscopies. The heme b ligand assumes the same conformation in the WT active site for both the reconstituted and H2O2-mediated product, maintaining the same vinyl and propionate interactions with the protein. Nevertheless, it is important to note that the distal His118, which serves as a distal base, plays an important role in the stabilization of the cavity and for the heme b reconstitution. In fact, while the access of heme b is prevented by steric hindrance in the H118F variant, the substitution of His with the small apolar Ala residue favors the insertion of the heme b in the reversed conformation. The overall data strongly support that during decarboxylation, the intermediate product, a monovinyl-monopropionyl deuteroheme, rotates by 90o within the active site. Moreover, in the ferrous forms the frequency of the ν(Fe-Nδ(His)) stretching mode provides information on the strength of the proximal Fe-His bond and allows us to follow its variation during the two oxidative decarboxylation steps.

Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.

Substrate specificity and complex stability of coproporphyrin ferrochelatase is governed by hydrogen-bonding interactions of the four propionate groups.

Coproporpyhrin III is the substrate of coproporphyrin ferrochelatases (CpfCs). These enzymes catalyse the insertion of ferrous iron into the porphyrin ring. This is the penultimate step within the coproporphyrin-dependent haeme biosynthesis pathway. This pathway was discovered in 2015 and is mainly utilised by monoderm bacteria. Prior to this discovery, monoderm bacteria were believed to utilise the protoporphyrin-dependent pathway, analogously to diderm bacteria, where the substrate for the respective ferrochelatase is protoporphyrin IX, which has two propionate groups at positions 6 and 7 and two vinyl groups at positions 2 and 4. In this work, we describe for the first time the interactions of the four-propionate substrate, coproporphyrin III, and the four-propionate product, iron coproporphyrin III (coproheme), with the CpfC from Listeria monocytogenes and pin down differences with respect to the protoporphyrin IX and haeme b complexes in the wild-type (WT) enzyme. We further created seven LmCpfC variants aiming at altering substrate and product coordination. The WT enzyme and all the variants were comparatively studied by spectroscopic, thermodynamic and kinetic means to investigate in detail the H-bonding interactions, which govern complex stability and substrate specificity. We identified a tyrosine residue (Y124 in LmCpfC), coordinating the propionate at position 2, which is conserved in monoderm CpfCs, to be highly important for binding and stabilisation. Importantly, we also describe a tyrosine-serine-threonine triad, which coordinates the propionate at position 4. The study of the triad variants indicates structural differences between the coproporphyrin III and the coproheme complexes. ENZYME: EC 4.99.1.9.

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.

Pseudoperoxidase activity, conformational stability, and aggregation propensity of the His98Tyr myoglobin variant: implications for the onset of myoglobinopathy.

The autosomal dominant striated muscle disease myoglobinopathy is due to the single point mutation His98Tyr in human myoglobin (MB), the heme protein responsible for binding, storage, and controlled release of O2 in striated muscle. In order to understand the molecular basis of this disease, a comprehensive biochemical and biophysical study on wt MB and the variant H98Y has been performed. Although only small differences exist between the active site architectures of the two proteins, the mutant (a) exhibits an increased reactivity toward hydrogen peroxide, (b) exhibits a higher tendency to form high-molecular-weight aggregates, and (c) is more prone to heme bleaching, possibly as a consequence of the observed H2 O2 -induced formation of the Tyr98 radical close to the metal center. These effects add to the impaired oxygen binding capacity and faster heme dissociation of the H98Y variant compared with wt MB. As the above effects result from bond formation/cleavage events occurring at the distal and proximal heme sites, it appears that the molecular determinants of the disease are localized there. These findings set the basis for clarifying the onset of the cascade of chemical events that are responsible for the pathological symptoms of myoglobinopathy.

Reaction intermediate rotation during the decarboxylation of coproheme to heme b in C. diphtheriae.

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H2O2. Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.