Biosynthesis of Tetrapyrrole Cofactors by Bacterial Community Inhabiting Porphyrine-Containing Shale Rock (Fore-Sudetic Monocline).

This study describes for the first time the comprehensive characterization of tetrapyrrole cofactor biosynthetic pathways developed for bacterial community (BC) inhabiting shale rock. Based on the genomic and proteomic metadata, we have detailed the biosynthesis of siroheme, heme, cobalamin, and the major precursor uroporphyrinogen III by a deep BC living on a rock containing sedimentary tetrapyrrole compounds. The obtained results showed the presence of incomplete heme and cobalamin biosynthesis pathways in the studied BC. At the same time, the production of proteins containing these cofactors, such as cytochromes, catalases and sulfite reductase, was observed. The results obtained are crucial for understanding the ecology of bacteria inhabiting shale rock, as well as their metabolism and potential impact on the biogeochemistry of these rocks. Based on the findings, we hypothesize that the bacteria may use primary or modified sedimentary porphyrins and their degradation products as precursors for synthesizing tetrapyrrole cofactors. Experimental testing of this hypothesis is of course necessary, but its evidence would point to an important and unique phenomenon of the tetrapyrrole ring cycle on Earth involving bacteria.

Light-induced depigmentation in planarians models the pathophysiology of acute porphyrias.

Porphyrias are disorders of heme metabolism frequently characterized by extreme photosensitivity. This symptom results from accumulation of porphyrins, tetrapyrrole intermediates in heme biosynthesis that generate reactive oxygen species when exposed to light, in the skin of affected individuals. Here we report that in addition to producing an ommochrome body pigment, the planarian flatworm Schmidtea mediterranea generates porphyrins in its subepithelial pigment cells under physiological conditions, and that this leads to pigment cell loss when animals are exposed to intense visible light. Remarkably, porphyrin biosynthesis and light-induced depigmentation are enhanced by starvation, recapitulating a common feature of some porphyrias - decreased nutrient intake precipitates an acute manifestation of the disease. Our results establish planarians as an experimentally tractable animal model for research into the pathophysiology of acute porphyrias, and potentially for the identification of novel pharmacological interventions capable of alleviating porphyrin-mediated photosensitivity or decoupling dieting and fasting from disease pathogenesis.

Structure of REV-ERBß ligand-binding domain bound to a porphyrin antagonist.

REV-ERBα and REV-ERBß are members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors that play important roles in the regulation of circadian physiology, metabolism, and immune function. Although the REV-ERBs were originally characterized as orphan receptors, recent studies have demonstrated that they function as receptors for heme. Here, we demonstrate that cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP) are ligands that bind directly to the REV-ERBs. However, instead of mimicking the agonist action of heme, CoPP and ZnPP function as antagonists of REV-ERB function. This was unexpected because the only distinction between these ligands is the metal ion that is coordinated. To understand the structural basis by which REV-ERBß can differentiate between a porphyrin agonist and antagonist, we characterized the interaction between REV-ERBß with heme, CoPP, and ZnPP using biochemical and structural approaches, including x-ray crystallography and NMR. The crystal structure of CoPP-bound REV-ERBß indicates only minor conformational changes induced by CoPP compared with heme, including the porphyrin ring of CoPP, which adopts a planar conformation as opposed to the puckered conformation observed in the heme-bound REV-ERBß crystal structure. Thus, subtle changes in the porphyrin metal center and ring conformation may influence the agonist versus antagonist action of porphyrins and when considered with other studies suggest that gas binding to the iron metal center heme may drive alterations in REV-ERB activity.

Binding free energies of inhibitors to iron porphyrin complex as a model for Cytochrome P450.

The binding free energies of the inhibitor-heme model complexes are calculated using the density functional methods and the implicit solvation models in water, where the 16 structurally diverse compounds with a spectrum of IC(50) values from 0.05 (clotrimazole) to 1000 (piroxicam) µM are chosen as inhibitors for Cytochrome P450 3A4 (CYP3A4). CYP3A4 is the most predominant constituent of the human hepatic CYP enzymes that play a role in metabolizing structurally diverse xenobiotics. The observed free energy change for each inhibitory binding, ΔG inh0, is obtained from its IC(50) value. The total binding free energy (ΔG b0) of each inhibitor-heme model complex is calculated by the sum of its relative free energy (ΔG(0) ) in the gas phase and solvation free energy to the water-heme model complex. The UB3LYP/LanL2DZ level of theory provides the correct relative stabilities of the high- and low-spin states for the penta- and hexa-coordinated ferric complexes, respectively. The optimized distances of the inhibitor nitrogen (or water oxygen) and the methyl mercaptide S to the ferric iron of the inhibitor-heme model complexes at the same level of theory are consistent with the values of the corresponding X-ray structures, except for the econazole complex. The correlation coefficient r(2) values of 0.91 and 0.75 are obtained from the ΔG b0-ΔG inh0 and ΔG(0) -ΔG inh0 plots, respectively, at the UM06/LanL2DZ:CPCM_UB3LYP/LanL2DZ//UB3LYP/LanL2DZ level of theory in water. This indicates that the total binding free energies calculated for the inhibitor-heme model complexes can be a good descriptor in interpreting the inhibitor binding to CYP3A4 and the relative free energies in the gas phase are mainly responsible for the total binding free energies in water, although the desolvation can be a factor to affect the binding affinity of the inhibitors to CYP3A4. From the theozyme analysis of the X-ray structures for ketoconazole- and metyrapone-CYP3A4 complexes, the interaction free energy of the neighboring residues with each inhibitor in the active site is calculated to be about -3 kcal mol(-1) in water, whose the interaction energy and the desolvation free energy change are about -5 and 2 kcal mol(-1) , respectively.

Topical use of liposomal copper palmitate formulation blocks porphyrin-induced photosensitivity in rats.

Photodynamic therapy (PDT) is a new treatment modality that uses porphyrin derivatives and visible light, especially for the treatment of cancer. However, PDT with certain photosensitisers can cause prolonged skin photosensitization. This is particularly true for Photofrin II (Photofrin)-mediated PDT where patients are required to avoid direct exposure to sunlight for a period of 4-6 weeks. This is the only long-term adverse reaction to the drug. Recent studies have shown that topical copper treatment avoids this type of inflammatory reaction. In this study, we have tested the efficiency of the liposomal formulation of copper palmitate on porphyrin-photosensitized rats. Initially, adult male Sprague-Dawley rats were rendered photosensitive either by administration of Photofrin or aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX). Prior to this, their dorsal skin was shaved and treated topically with a cream consisting of either empty or copper palmitate-encapsulated liposomal formulation. After being kept in a dimmed light environment, the rats were exposed to visible light, and inflammatory responses were inspected. Histological studies revealed that no inflammatory cells were present at the skin sites treated with liposomal cream containing copper palmitate in the Photofrin-sensitized group while no reduction in the number of inflammatory cells was observed at the skin samples treated with the empty liposomes. In conclusion, the data demonstrate the significant protective effect of topically-applied liposome-encapsulated copper palmitate against both Photofrin and ALA-induced PpIX photosensitivity.

Effect of albumin on the photodynamic inactivation of microorganisms by a cationic porphyrin.

BACKGROUND: Photodynamic inactivation (PDI) employs visible light and a photosensitizer to inactivate cells. The technique is currently clinically used for the treatment of several malignancies. However, the PDI of microorganisms still remains in the research phase. PURPOSE: To study the effect of human blood plasma and human serum albumin (HSA) on the PDI of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. METHODS: PDI experiments were performed using white light (30 mW cm-2) and the cationic 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) as photosensitizer. RESULTS: The microorganisms could be successfully photoinactivated by TriP[4] when suspended in phosphate buffered saline (PBS). In this medium, P. aeruginosa was the most resistant microorganism. Changing the suspending medium from PBS to human blood plasma reduced the PDI of all three microorganisms. In human blood plasma C. albicans was the most resistant microorganism. The same results were obtained with 4.5% and 7% HSA/PBS suspensions. CONCLUSIONS: Albumin inhibits the PDI of S. aureus, P. aeruginosa and C. albicans in a dose dependent manner. However, our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans.

Ultrasonically induced cell damage and active oxygen generation by 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid: on the mechanism of sonodynamic activation.

Ultrasonically induced cell damage and active oxygen generation with 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid (ATX-S10) were compared in the same in vitro insonation setup. Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound at 2 MHz for up to 60 s in the presence and absence of ATX-S10. The viability was determined by Trypan blue exclusion test. Ultrasonically induced active oxygen generation in the presence and absence of ATX-S10 in air-saturated aqueous solutions of 50 mM 2,2,6,6-tetramethyl-4-piperidone was detected by electron spin resonance (ESR). Significant enhancement of the rates of both ultrasonically induced cell damage and nitroxide generation was demonstrated with 40-160 microM ATX-S10. Both rates correlated very well. The enhancement of both rates with ATX-S10 was suppressed by 10 mM histidine. These results suggest that ultrasonically generated active oxygen plays a primary role in the ultrasonically induced cell damage in the presence of ATX-S10.

Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes.

It is well known that porphyrins cause a toxic light-mediated effect due to their capability to generate free radicals. Several reports have proved that melatonin is a potent free radical scavenger. The aim of this work has been to study the ability of melatonin to prevent the cell damage caused by porphyrins in the Harderian gland of female Syrian hamsters. Cell injury was evaluated estimating the percentage of damaged cells found in the gland and analyzing the degree of this damage at ultrastructural level. To explain the mechanism by which this hormone could prevent the cell damage caused by porphyrins, its capability to both decrease porphyrin synthesis and increase the mRNA levels for antioxidant enzymes was evaluated. Our results demonstrate that melatonin administration decreases the percentage of damaged cells, porphyrin synthesis, and aminolevulinate synthase (ALA-S) mRNA levels and increases the mRNA levels for manganese superoxide-dismutase and copper-zinc superoxide dismutase. When observed under an electron microscope, the lesions in the clear cells of the treated females were much less severe than in the corresponding cells of the control animals. Melatonin exerts a cytoprotective effect by inhibiting the ALA-S gene expression (and so porphyrin synthesis) and by raising the mRNA levels for several antioxidant enzymes.

Ligands specific to peripheral benzodiazepine receptors for treatment of porphyrias.

Accumulating data indicate that porphyrins are physiologically endogenous ligands to mitochondrial peripheral benzodiazepine receptors. An isoquinoline carboxamide derivative that likewise binds to peripheral benzodiazepine receptors could prove therapeutically useful in porphyrias by displacing porphyrins from these receptors in mitochondria.

Influence of middle molecules on the anemia of uremic patients.

To evaluate their toxicity at the cellular level, middle molecules from uremic serum were incubated with erythrocytes from healthy subjects and the activity of the enzyme Delta-aminolevulinic acid dehydrase (D-ALA-D) and peroxidative hemolysis were investigated. Uremic middle molecules caused a significant decrease of the D-ALA-D activity of normal erythrocytes which was not due to differences in the concentrations of Pb, Cd or Zn. The decreased enzyme activity could be restored by adding reduced glutathione (GSH; 5 mmol/L) together with the middle molecules to the assay system. Uremic middle molecules caused a significant increase of peroxidative hemolysis in normal erythrocytes. Uremic middle molecules contribute to the anemia of uremic patients by impeding hemoglobin synthesis and by increasing peroxidative hemolysis, possibly by affecting SH-groups. H2O2-producing compounds should be avoided in uremic patients.

Repression of the overproduction of porphyrin precursors in acute intermittent porphyria by intravenous infusions of hematin.

In a patient with a severe attack of acute intermittent porphyria, hematin given intravenously caused marked diminution of serum delta-aminolevulinic acid and porphobilinogen. The decline of aminolevulinate was more rapid than that of porphobilinoge. After 2 days of hematin administration, about 5 days were required for delta-aminolevulinic acid, and 11 days for porphobilinogen to return to the concentrations that were detected before treatment. Urinary excretion of both compounds also decreased after hematin administration. Considerable amounts of porphobilinogen were also found in the cerebrospinal fluid of the patient.