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.

Characterization and origin of heme precursors in amniotic fluid: lessons from normal and pathological pregnancies.

BACKGROUND: Heme is the prosthetic group of numerous proteins involved in vital processes such as oxygen transport, oxidative stress, and energetic mitochondrial metabolism. Free heme also plays a significant role at early stages of development and in cell differentiation processes. The metabolism of heme by the fetal placenta unit is not well-established in humans. METHODS: In a retrospective study, we measured heme precursors in the amniotic fluid (AF) of 51 healthy women, and 10 AF samples from pregnancies with either upper or lower intestinal atresia or ileus were also analyzed. RESULTS: We showed that the porphyrin precursors aminolevulinic acid, porphobilinogen, and protoporphyrin IX are present at the limit of detection in the AF. Total porphyrin levels decreased progressively from week 13 to week 33 (p < 0.01). Interestingly, uroporphyrin, initially detected as traces, increased with maturation, in contrast to coproporphyrin. Uro- and coproporphyrins were type I immature isomers (>90%), suggesting a lack of maturity in the fetal compartment of the heme pathway. Finally, the differential analysis of AF from normal and pathological pregnancies demonstrated the predominant hepatic origin of fetal porphyrins excreted in the AF. CONCLUSION: This study gives the first insight into heme metabolism in the AF during normal and pathological pregnancies.

The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron-binding site.

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.

Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III.

1. Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are polyspecific transporters that mediate the transport of organic acids into hepatocytes. Inactivating mutations of both OATP1B1 and OATP1B3 alleles lead to Rotor syndrome, a disease characterized by coproporphyrinuria, an elevated urinary excretion of coproporphyrins I and III. It was hypothesized that transport of coproporphyrins I and III was mediated by OATP1B1 and OATP1B3. 2. This hypothesis was tested using cells transfected with OATP1B1 and OATP1B3. OATP1B-mediated transport of coproporphyrin was time-dependent and concentration-dependent. OATP1B1-mediated transport of coproporphyrins I and III (Km = 0.13 and 0.22 µM, respectively), as did OATP1B3 (Km = 3.25 and 4.61 µM, respectively). The OATP1B-mediated transport of each coproporphyrin was inhibited by rifampicin. 3. The specificity of coproporphyrin transport was also investigated where OATP2B1 demonstrated meaningful transport of coproporphyrin III (Km = 0.31 µM), while OCT1, OCT2, OAT1, OAT3 and NTCP were negative for coproporphyrin transport. 4. The identification of coproporphyrins as OATP substrates in vitro more clearly defines the role of OATPs in the hepatic disposition and renal excretion of coproporphyrins I and III and provides compelling evidence for future in vivo exploration of coproporphyrins as biomarkers of OATP activity.

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.

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.

Cell-penetrating conjugates of coproporphyrins with oligoarginine peptides: rational design and application for sensing intracellular O2.

A panel of phosphorescent oligoarginine conjugates of tetracarboxylic Pt(II)-coproporphyrin I dye (PtCP), monosubstituted with long peptides or tetra-substituted with short peptides and having different linkers and peripheral groups, is described. Their photophysical properties, cell loading efficiency, and mechanisms of transport into the cell were investigated and compared. The conjugates were seen to rely on endocytotic mechanisms of cell entry, which are different from that of the unconjugated oligoarginine peptide, and show diverse patterns of intracellular distribution. On the basis of this study, the tetra-substituted PtCP conjugate displaying whole cell distribution was selected for the sensing of intracellular O(2). This probe has been tested in biological experiments on a fluorescence plate reader, including the monitoring of in situ oxygenation of respiring cells and their responses to metabolic stimulation. Similar conjugates of the phosphorescent Pd(II)-coprorphyrin and fluorescent coproporphyrin-ketone were also synthesized and assessed for the sensing of low levels intracellular O(2) and ratiometric pH-sensing, respectively. The results produced and the structure-activity relationships determined can facilitate the rational design of new bioconjugates of porphyrin dyes tailored to specific applications.

Evaluation of the derivates of phosphorescent Pt-coproporphyrin as intracellular oxygen-sensitive probes.

Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH(2) derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH(2) demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0-200 µM O(2)). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH(2) represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.

Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase.

Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram-positive) bacteria within the recently detected coproporphyrin-dependent (CPD) heme biosynthesis pathway. Here, we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme-bound CpfC. Comparison of crystal structures of apo-LmCpfC and coproheme-LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo-, coproporphyrin III-, and coproheme-LmCpfC underline the pronounced noncovalent interaction of both coproporphyrin and coproheme with the protein (ΔTm  = 11 °C compared to apo-LmCpfC), which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53, and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo-LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III-LmCpfC that immediately leads to formation of ferric coproheme-LmCpfC (kcat /KM  = 4.7 × 105  m-1 ·s-1 ). We compare the crystal structure of coproheme-LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the CPD heme biosynthesis pathway. ENZYME: EC 4.99.1.9 DATABASE: pdb-codes of structural data in this work: 6RWV, 6SV3.

Histamine interaction with Zn2+ and Cu2+ porphyrins.

Histamine may be present in biological fluids and in pharmaceutical dosage forms such as antiallergenic agents; when in excess, it causes a disorder called histaminosis. Many techniques have been developed to determine the concentration of this compound but the application of such methods is complicated and laborious, requiring expensive equipment and long times. A better alternative is to design chemical sensors. In the work reported here, six metalloporphyrins (Cu2+ or Zn2+) with different peripheral groups - benzoate, tosylate and carboxylate - were studied. The stability constants for these compounds were determined with histamine at different temperatures. Histamine is strongly bound to metallic porphyrins containing Cu2+ and Zn2+; however, the binding force does not depend exclusively on the metal center. Stabilization of the complex is strongly influenced, in some cases, by the lateral chains of the porphyrin. This possibility implies that this system can be very selective for this biogenic amine.

Phototoxicity of coproporphyrin as a novel photodynamic therapy was enhanced by liposomalization.

This study attempted the liposomalization of coproporphyrin I (CPI) with hydrophobic properties. Liposomalization of CPI was not successful at any pH when using lactate buffer. In contrast, when using 9% sucrose/10mM phosphate buffer (pH 7.8), CPI liposomes (Lipo-CPI) and polyethyleneglycol (PEG) modified liposomes (PEG-CPI) were prepared with a high entrapment ratio of CPI and small particle size. Plasma CPI concentration at 6h after PEG-CPI injection were 6.5-fold greater than that after the injection of Lipo-CPI. In tumors, the CPI concentration was higher after PEG-CPI injection than after Lipo-CPI or CPI solution. Therefore, PEG-CPI was likely to increase blood circulation and achieve greater accumulation of CPI in the tumor. When loaded into tumor cells, photosensitizers generate singlet oxygen during laser irradiation, resulting in the induction of necrosis in the cells. The order of magnitude of CPI tumor cells uptake was PEG-CPI>Lipo-CPI>CPI solution. Thus, the PEG modification of CPI liposomes improved its tumor cell uptake. Furthermore, it is likely that the order of the ability to produce singlet oxygen was PEG-CPI [symbol: see text] Lipo-CPI>CPI solution. The cytotoxicity of PEG-CPI was significantly greater than the other formulations, suggesting that the cytotoxicity reflected the CPI concentration in tumor cells. In conclusion, PEG-CPI was confirmed to show effective tissue distribution, elevated CPI concentration in the tumor cells, to produce singlet oxygen, and cytotoxicity by PDT.

A fully automated and validated human plasma LC-MS/MS assay for endogenous OATP biomarkers coproporphyrin-I and coproporphyrin-III.

AIM: A validated LC-MS/MS assay for the quantitation of coproporphyrin-I and -III (CP-I, CP-III) in human plasma has been developed to understand the utility of both as possible endogenous biomarkers for organic anion-transporting polypeptides (OATP)-mediated drug-drug interactions (DDIs). MATERIALS AND METHODS:  Human plasma extracts were analyzed for CP-I and CP-III using a Sciex API 6500+ mass spectrometer. Results: The assay was utilized for plasma samples from a clinical DDI study involving a new chemical entity that presented as an OATP inhibitor in vitro. A formal DDI study, with a probe drug (atorvastatin), was also included as part of the clinical study. CONCLUSION: Changes in CP-I area under the plasma concentration versus time curve (AUC0-48 h) were observed, which were similar to the AUC ratio obtained with atorvastatin. These results support the idea that plasma CP-I may have utility in Phase I by supporting the rapid assessment of OATP inhibition risk.

UHPLC-MS/MS bioanalysis of human plasma coproporphyrins as potential biomarkers for organic anion-transporting polypeptide-mediated drug interactions.

AIM: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. RESULTS: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. CONCLUSION: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.

Compositional analysis of endogenous porphyrins from Helicobacter pylori.

Bacteria able to accumulate porphyrins can be inactivated by visible light irradiation thanks to the photosensitizing properties of this class of aromatic pigments (photodynamic therapy, PDT). Since the bacterial resistance to antibiotic is growing, PDT is becoming a valid alternative. In this context, the pathogen Helicobacter pylori (Hp) is a suitable target for PDT since it spontaneously produces and accumulates porphyrins. It is then important to understand the spectroscopic behavior of these endogenous species to exploit them as photosensitizers, thus improving the results given by the application of PDT in the treatment of Hp infections. In this work we extracted porphyrins from both a laboratory-adapted and a virulent strain of Hp, and we performed spectroscopic and chromatographic experiments to collect information about the composition and the spectrophotometric features of the extracts. The main components of the porphyrin mixtures were identified and their relative contribution to the global red fluorescence was examined.

Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins.

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.

The main pigment of the dormant Mycobacterium smegmatis is porphyrin.

Upon transition of Mycobacterium smegmatis into the dormant state, accumulation of a dark brown fluorescent pigment was observed. This pigment gave bright red fluorescence in both cells and the culture medium. Based on 1H-NMR, MALDI and UV spectra, the fluorescent compounds, extracted from the culture medium as well as from the dormant cells, were concluded to be a mixture of free coproporphyrin III and uroporphyrin III and their corresponding methyl esters. A possible significance of porphyrin pigment accumulation in the dormant cells is discussed.

Modification of monoclonal and polyclonal IgG with palladium (II) coproporphyrin I: stimulatory and inhibitory functional effects induced by two different methods.

Antibodies conjugated with porphyrins and metalloporphyrins have a great potential for applications in fluorescence or phosphorescence immunoassays as well as in photodynamic therapy, radioimaging and internal radiation therapy of cancer. Here we describe how the new preactivated metalloporphyrin, palladium (II) coproporphyrin I-tetra-N-hydroxysuccinimide ester, can be covalently attached to mouse monoclonal and rabbit anti-human ferritin antibodies. The advantages of the proposed reagent over the previously reported carboxylic porphyrins coupled through carbodiimide activation are indicated. Conformational changes in antibodies caused by each of the two methods were assessed from their binding to the antigen (a probe for the antibody Fv domain) and anti-IgG antibodies probing the global conformation of the CH2 domain in the Fc fragment. Porphyrin coupling through carbodiimide activation resulted in a decrease in both functional activities of modified antibodies even at low levels of modification. In contrast, when the N-hydroxysuccinimide (NHS) derivative of porphyrin was used, enhancement of the antigen-binding affinity of porphyrin-antibody conjugates occurred due to an increase in the conformational mobility (flexibility) of the modified antibodies. The stimulatory effect of conjugation was maximal when one porphyrin molecule was coupled to an antibody molecule. Coupling of NHS-activated porphyrin at pH 7.4, 7.8 and pH 8.5 suggested that the high efficiency of the reaction at pH 8.5 could be attributed predominantly to the formation of antibody aggregates, only 50% of which were covalently cross-linked. The lowest percentage of aggregates in porphyrin-antibody conjugates was found at pH 7.4 and a molar reagent-to-protein ratio in the 10:1-40:1 range. Thus, the use of the NHS-activated carboxylic porphyrin provides a mild, simple and convenient procedure for preparing antibody conjugates with enhanced antigen-binding affinity.

Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

Phosphorescent metalloporphyrins as labels in time-resolved luminescence microscopy: effect of mounting on emission intensity.

In this study, we present an investigation of the effects of mounting media on the phosphorescence of metalloporphyrin stained microscopy samples. The samples were: (1) Platinum(II) coproporphyrin (=PtCP) stained porous Sephadex beads; (2) compact polystyrene microspheres coated with IgG-PtCP conjugate; and (3) immunocytochemically labeled human peripheral blood neutrophils. The human neutrophils in a mixed leukocyte population were fixed, permeabilized, and then immunolabeled with PtCP conjugate of monoclonal mouse IgG directed to the intracellular antigen myeloperoxidase. The samples were mounted in twelve different mounting media and studied with quantitative time-resolved luminescence imaging microscopy with respect to the intensity and stability of the phosphorescence signal. The results indicate that microscopy samples stained with PtCP exhibit the brightest phosphorescence emission in non-mounted form or when mounted in non-aqueous permanent mounting media.

Investigations on the formation of urinary coproporphyrin isomers I-IV in 5-aminolevulinic acid dehydratase deficiency porphyria, acute lead intoxication and after oral 5-aminolevulinic acid loading.

OBJECTIVES: Investigation of the metabolism of the four urinary coproporphyrin isomers I-IV in the extremely rare 5-aminolevulinic acid dehydratase (ALAD) deficiency porphyria (syn.: Doss porphyria), in acute lead intoxication, and after oral 5-aminolevulinic acid (ALA) loading. DESIGN AND METHODS: We analyzed the excretion of total urinary coproporphyrins and the composition of the respective isomers I-IV with ion-pair HPLC methods in these conditions. RESULTS: The concentration of total coproporphyrins was about 30-fold increased in patients with ALAD deficiency porphyria and acute lead intoxication as compared with controls. In addition, the proportion of coproporphyrin III as well as that of the atypical isomers II and IV were significantly elevated at the expense of isomer I. After oral ALA administration to normal volunteers, a 10- to 15-fold increase in the maximal concentration of total urinary coproporphyrins was observed within 12 to 24 h. Urinary levels were back to normal after another 24 h. The excretion pattern of the individual urinary coproporphyrin isomers I-IV after ALA ingestion revealed a dynamic process: initially isomer III was preferentially formed, followed by a 3-fold increase of isomers II and IV via non-enzymatic rearrangement of isomer III, and finally normalization of all four isomers occurred within 48 h. CONCLUSIONS: These results demonstrate that oral ALA loading can be used as an in vivo model to study the metabolism of the four urinary coproporphyrin isomers I-IV especially in ALAD deficiency porphyria and in acute lead poisoning.