Related Tetrapyrroles Research Articles

Production of copropophyrin III, biliverdin and bilirubin by the rufomycin producer, Streptomyces atratus.

Heme is best known for its role as a versatile prosthetic group in prokaryotic and eukaryotic proteins with diverse biological functions including gas and electron transport, as well as a wide array of redox chemistry. However, free heme and related tetrapyrroles also have important roles in the cell. In several bacterial strains, heme biosynthetic precursors and degradation products have been proposed to function as signaling molecules, ion chelators, antioxidants and photoprotectants. While the uptake and degradation of heme by bacterial pathogens is well studied, less is understood about the physiological role of these processes and their products in non-pathogenic bacteria. Streptomyces are slow growing soil bacteria known for their extraordinary capacity to produce complex secondary metabolites, particularly many clinically used antibiotics. Here we report the unambiguous identification of three tetrapyrrole metabolites from heme metabolism, coproporphyrin III, biliverdin and bilirubin, in culture extracts of the rufomycin antibiotic producing Streptomyces atratus DSM41673. We propose that biliverdin and bilirubin may combat oxidative stress induced by nitric oxide production during rufomycin biosynthesis, and indicate the genes involved in their production. This is, to our knowledge, the first report of the production of all three of these tetrapyrroles by a Streptomycete.

Iron phthalocyanine on Cu(111): Coverage-dependent assembly and symmetry breaking, temperature-induced homocoupling, and modification of the adsorbate-surface interaction by annealing.

We have examined the geometric and electronic structures of iron phthalocyanine assemblies on a Cu(111) surface at different sub- to mono-layer coverages and the changes induced by thermal annealing at temperatures between 250 and 320 °C by scanning tunneling microscopy, x-ray photoelectron spectroscopy, and x-ray absorption spectroscopy. The symmetry breaking observed in scanning tunneling microscopy images is found to be coverage dependent and to persist upon annealing. Further, we find that annealing to temperatures between 300 and 320 °C leads to both desorption of iron phthalocyanine molecules from the surface and their agglomeration. We see clear evidence of temperature-induced homocoupling reactions of the iron phthalocyanine molecules following dehydrogenation of their isoindole rings, similar to what has been observed for related tetrapyrroles on transition metal surfaces. Finally, spectroscopy indicates a modified substrate-adsorbate interaction upon annealing with a shortened bond distance. This finding could potentially explain a changed reactivity of Cu-supported iron phthalocyanine in comparison to that of the pristine compound.

Porpholactone and Metal Complexes: Effect of Lactonization of ß-Pyrrole

The development of “porphyrin-like” molecules (porphyrinoids) and related tetrapyrroles has attracted considerable attention for the potential as optical materials and photosensitizers for photodynamic therapy (PDT). Porpholactones (TPPLs) are an important but relatively unstudied class of tetrapyrroles, which combine some of the features of porphyrins and chlorins. To explore the effect ß-Lactonization on the electronic structures and spectral properties is of importance to expand the porpholactone chemistry. In recent years, we focused on the synthesis, characterization and the application of porpholactone and their metal complexes. Based on the skeleton of porpholactones, the β-lactone moiety provides an access to tune their NIR optical characteristics, amphilicity and cellular behaviour. More importantly, the relative orientation of the β-dilactone moieties significantly influences their electronic structures and photophysical properties, for example, the Qy band of trans-porphodilactone is red shifted 19 nm compared to that of the cis-isomer with a 2-fold increase in the absorption intensity. Trans-isomer exhibit much higher singlet oxygen quantum yield and the related PDT efficiency than cis-isomer upon irradiation at their Qv bands, respectively. Thus, these results reveal the importance of relative orientation of b-substituents on optical properties at NIR regions.

Bilirubin and related tetrapyrroles inhibit food-borne mutagenesis: a mechanism for antigenotoxic action against a model epoxide.

Bilirubin exhibits antioxidant and antimutagenic effects in vitro. Additional tetrapyrroles that are naturally abundant were tested for antigenotoxicity in Salmonella. Un-/conjugated bilirubin (1 and 2), biliverdin (4), bilirubin and biliverdin dimethyl esters (3 and 5), stercobilin (6), urobilin (7), and protoporphyrin (8) were evaluated at physiological concentrations (0.01–2 μmol/plate; 3.5–714 μM) against the metabolically activated food-borne mutagens aflatoxin B1 (9) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (10). Compound 8 most effectively inhibited the mutagenic effects of 9 in strain TA102 and 10 in TA98. Compound 7 inhibited 9-induced mutagenesis in strain TA98 most effectively, while 1 and 4 were promutagenic in this strain. This is likely due to their competition with mutagens for phase-II detoxification. Mechanistic investigations into antimutagenesis demonstrate that tetrapyrroles react efficiently with a model epoxide of 9, styrene epoxide (11), to form covalent adducts. This reaction is significantly faster than that of 11 with guanine. Hence, the evaluated tetrapyrroles inhibited genotoxicity induced by poly-/heterocyclic amines found in foods, and novel evidence obtained in the present investigation suggests this may occur via chemical scavenging of genotoxic metabolites of the mutagens investigated. This may have important ramifications for maintaining health, especially with regard to cancer prevention.

In vitro DNA-damaging effects of intestinal and related tetrapyrroles in human cancer cells

Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17μM for 24h. Addition of 300μM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system.

In vitro antioxidant capacity and antigenotoxic properties of protoporphyrin and structurally related tetrapyrroles

The antioxidant properties of protoporphyrin IX and related tetrapyrroles are poorly characterized. Therefore, eight tetrapyrroles, five of which are produced in vivo, were tested to assess their antioxidant capacities in the Salmonella reverse mutation, TEAC, FRAP and ORAC assays. Tertiary-butyl hydroperoxide (tert-BOOH) in the presence or absence of metabolic activation (±S9) was added to Salmonella strain TA102 together with the test compounds. In the absence of metabolic activation, the order of effectiveness was protoporphyrin > biliverdin > bilirubin ditaurate > bilirubin > biliverdin dimethyl ester > stercobilin > bilirubin dimethyl ester > urobilin. In the presence of S9, the effectiveness was reversed, with urobilin > biliverdin dimethyl ester > bilirubin dimethyl ester > stercobilin > biliverdin > bilirubin > bilirubin ditaurate > protoporphyrin. In the antioxidant capacity assays FRAP, TEAC and ORAC, mainly bilirubin, bilirubin ditaurate, biliverdin and protoporphyrin showed antioxidant activity. This study reports that previously untested tetrapyrroles of related structure prevent oxidatively induced genotoxicity, and for some, novel underlying mechanisms of antioxidant action were revealed. These results support the physiological importance and biological relevance of tetrapyrroles including protoporphyrin that might act as antioxidants, protecting from oxidatively induced DNA damage, particularly in the tissues/organs where they accumulate.

Heme and HO-1 Inhibition of HCV, HBV, and HIV

Hepatitis C virus, human immunodeficiency virus, and hepatitis B virus are chronic viral infections that cause considerable morbidity and mortality throughout the world. In the decades following the identification and sequencing of these viruses, in vitro experiments demonstrated that heme oxygenase-1, its oxidative products, and related compounds of the heme oxygenase system inhibit replication of all 3 viruses. The purpose of this review is to critically evaluate and summarize the seminal studies that described and characterized this remarkable behavior. It will also discuss more recent work that discovered the antiviral mechanisms and target sites of these unique antiviral agents. In spite of the fact that these viruses are diverse pathogens with quite profound differences in structure and life cycle, it is significant that heme and related compounds show striking similarity for viral target sites across all three species. Collectively, these findings strongly indicate that we should move forward and develop heme and related tetrapyrroles into versatile antiviral agents that could be used therapeutically in patients with single or multiple viral infections.

Self-assembled porphyrin nanostructures

Porphyrins and related tetrapyrroles have been extensively studied because of their importance in biological processes and they are often used in the development of artificial photosynthesis, catalysis, and sensor systems. Challenges in the development of functional nanoscale porphyrin systems are many, including the need to organize the porphyrins (e.g., to facilitate processes such as energy- and electron transfer) and to couple the porphyrin nanostructures to other nanoscale components (e.g., catalytic elements and conductors) to produce multifunctional nanoscale systems. This article summarizes recent advances in the synthesis of discrete self-assembled porphyrin nanostructures with well-defined shapes and sizes. A novel method for growing metal on the porphyrin nanostructures to produce nanocomposites with metal catalysts or interconnects is also described. Current and potential applications of these nanostructures and porphyrin-metal nanocomposites are discussed.

Chlorophyll, chlorophyllin and related tetrapyrroles are significant inducers of mammalian phase 2 cytoprotective genes

Plant chlorophylls and carotenoids are highly colored, conjugated polyenes that play central roles in photosynthesis. Other porphyrins (tetrapyrroles), such as cytochromes, which are structurally related to chlorophyll, participate in redox reactions in many living systems. An unexpected new property of tetrapyrroles, including tetramethyl coproporphyrin III, tetrabenzoporphine, copper chlorin e4 ethyl ester, and of carotenoids including zeaxanthin and alpha-cryptoxanthin is their ability to induce mammalian phase 2 proteins that protect cells against oxidants and electrophiles. The capacity of these compounds to induce the phase 2 response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase 2 inducers, which show few other structural similarities. Pseudo second-order rate constants of these inducers are correlated with their potency in inducing the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma cells. One of the most potent inducers was isolated from chlorophyllin, a semisynthetic water-soluble chlorophyll derivative. Although chlorophyll itself is low in inducer potency, it may nevertheless account for some of the disease-protective effects attributed to diets rich in green vegetables because it occurs in much higher concentrations in those plants than the widely studied 'phytochemicals'.

Chlorophyll: a symptom and a regulator of plastid development

SUMMARYThe metabolism of chlorophylls and related tetrapyrroles directly influences, and is influenced by, the proteins and cell structures with which they are associated. During net accumulation, de‐greening and at the steady state chlorophyll and its derivatives are important elements in the post‐translational regulation of the expression of genes for chloroplast proteins. At the same time, they represent potential photodynamic hazards against which green cells need to have protective mechanisms. This review deals with genetic, chemical and environmental perturbations of chlorophyll biosynthesis that impact on protein stability, membrane organization and susceptibility to photodamage. NADPH‐protochlorophyllide oxidoreductase is considered in detail as a pigment– protein regulating, and regulated by, chlorophyll metabolism. The question of the extent and significance of chlorophyll turnover at the steady state is addressed, with particular emphasis on the dynamics of the photosystem II reaction centre. The pathway of chlorophyll catabolism is described, along with its interrelationship with protein mobilization in chloroplast senescence. Finally, the structural basis of pigment‐protein interaction and stability is examined, and the discussion ends by expressing some general thoughts about the control of protein lifetimes in the living cell. CONTENTS Summary 163 I. Introduction 164 II. Chlorophyll and related tetrapyrroles 164 III. Chlorophyll‐protein complexes 164 IV. Chlorophyll biosynthesis 166 V. NADPH‐protochlorophyllide oxidoreductase 168 VI. Assembling chlorophyll‐proteins 171 VII. Chlorophyll turnover at the steady state 172 VIII. The plastid family tree 173 IX. The pathway of chlorophyll catabolism 173 X. Chlorophyll and associated proteins in senescence 175 XI. How does chlorophyll stabilize associated proteins ? 176 XII. Conclusion 177 Acknowledgements 177

Metal dependence of the nonplanar distortion of octaalkyltetraphenylporphyrins

The biological activity of porphyrins and related tetrapyrroles in proteins may be modulated by nonplanar conformational distortions; consequently, two aspects of nonplanarity have been investigated in the highly nonplanar octaalkyltetraphenylporphyrins (OATPPs). In the first part, the effect of the central metal ion (M = Ni(II), Co(II), Cu(II), Zn(II), Co(III), Fe(III)) on the conformation of the OATPP macrocycle has been determined. Crystallographic studies reveal that the sterically encumbered, nonplanar porphyrin 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin (OETPP) remains sufficiently flexible to show a small decrease in nonplanarity for large metal ions. This decrease in nonplanarity for the OETPP metal complexes is predicted by using a molecular mechanics force field derived from structural and vibrational data for planar metalloporphyrins. A detailed analysis of the crystal structures of the Co(II) and Cu(II) complexes of OETPP reveals that the metal-dependent changes in bond lengths and bond angles are qualitatively similar to the changes observed for the OEP complexes. As the metal size increases, both OEPs and OETPPs exhibit expansion of the meso bridges (increases in the C[sub [alpha]]-C[sub m] bond length and the C[sub [alpha]]-C[sub m]-C[sub [alpha]] bond angle) and a movement of the coordinating nitrogen atoms away from the metal atom (increases in the M-N bond length andmore » the C[sub [alpha]]-N-C[sub [alpha]] bond angle and a decrease in the N-C[sub [alpha]] bond length). In the second part, a combination of molecular mechanics and INDO/CI molecular orbital calculations successfully predicts the optical spectra of a series of highly substituted OATPPs with increasing nonplanar distortion. The successes of these calculations indicates the importance of including both the macrocycle conformation and the peripheral substituents in the INDO calculations. 62 refs., 10 figs., 6 tabs.« less

Resonance Raman spectroscopy of non‐planar nickel porphyrins

AbstractNon‐planar conformational distortions have recently been implicated in the biological activity of porphyrins and related tetrapyrroles in proteins, and several studies have used highly substituted porphyrins to model these nonplanar conformational distortions. Several aspects of non‐planarity in the highly substituted metalloporphyrins are discussed, focusing on resonance Raman spectroscopy as a technique for investigating these structural issues. First, different non‐planar distortions cause characteristic changes in the Raman spectrum. Specifically, the decreases in frequency of several Raman lines when compared with planar porphyrin analogs are shown to be similar for several classes of non‐planar highly substituted porphyrins. Second, the effect of the central metal ion [ M = Ni(II), Co(II), Cu(II), Zn(II), Co(III), Fe(III)] on the conformation of the sterically constrained non‐planar porphyrin octaethyltetraphenylporphyrin is considered. Responding to the conformational adjustments resulting from different metal size, the frequency of the structure‐sensitive Raman line v2 correlates with several structural parameters obtained from either mechanics calculations or crystallographic studies. The parameters include CβCβ bond length, core size and CαNCα angle. Finally, an effect of electron‐withdrawing substituents on the Raman frequencies is evident for the different classes of highly substituted porphyrins.

On the Chemical Reactivity of the Phytochrome Chromophore in the Pr and Pfr Form

The reactivity of the phytochrome chromophore and related tetrapyrroles towards ozone and tetranitromethane was investigated. Both oxidizing reagents cause bleaching of the main absorp­tion band of the pigment. The rate constants for this bleaching were determined under conditions of pseudo first order reaction kinetics. The rate constants for the reaction with ozone are similar for native phytochrome and for freely accessible tetrapyrroles (biliverdin, small chromopeptides from phytochrome) indicating that accessibility is not the limiting factor for the reaction with ozone. Under a variety of conditions, the Pfr chromophore reacts by about 10% faster than the Pr chromophore. This may reflect the true difference in reactivity. The rate constants for the reaction with tetranitromethane are much larger for biliverdin, bilirubin and small chromopeptides from phytochrome than for native phytochrome. The limiting factor for this reaction in native phytochrome therefore is the accessibility of the chromophore by the reagent. Previous conclusions on the difference in exposure of the tetrapyrrole chromophore in Pr and Pfr are confirmed.

Applications of mass spectrometry in studies of porphyrins and related tetrapyrroles

Electron impact mass spectrometry has played a vital role in the structure determination of a variety of porphyrin and chlorophyll derivatives, especially those occurring in small quantities, and which are related to important intermediates in both normal and abnormal metabolism. More recently, other newer mass spectral techniques have also become of increasing importance, i.e. soft ionization methods and g.c.-m.s. of degradation products. Field desorption mass spectrometry is particularly useful for porphyrins because of their low volatility and it is now very useful for preliminary analyses of crude mixtures of porphyrins, as well as for direct analysis of individual fractions obtained in high pressure liquid chromatography h.p.l.c. separations. Examples include porphyrins excreted by porphyric patients, petro-porphyrins, chlorophyll derivatives, and synthetic porphyrins. The coupling of a high pressure liquid chromatograph by means of a moving belt system also appears to be a promising technique for the direct mass spectromatic analysis of porphyrins in liquid chromatography fractions, and preliminary results are described. Chromic acid oxidation, or hydriodic acid reduction, of porphyrins to monopyrrolic fragments (the classical methods used for porphyrin structure determination) have now been refined by use of g.c.-m.s. and can be applied to sub-milligram quantities of porphyrins, or bile pigments; examples include isocoproporphyrin, ( d -urobilin and stercobilin. Biosynthetic studies have also been facilitated by degradative techniques combined with mass spectrometry. The final section of the paper describes preliminary studies of some complex metastable fragmentation processes occurring with chlorophyll derivatives with the use of the 'Dadi’ and linked B/E scan methods, together with field desorption and collisional activation.

Singlet oxygen reactivity of bilirubin and related tetrapyrroles

The rates of chemical reactivity (kR) and physical quenching (kQ) of singlet oxygen by bilirubin IXα, mesobilirubin IXα, bilirubin IXα dimethyl ester, aetiobilirubin IVγ, biliverdin IXα, biliverdin IXα dimethyl ester, aetiobiliverdin IVγ and an oxodipyrromethene have been determined. The kR and kQ values approach the diffusion threshold for the bilirubin-like substrates, but kR<kQ by about a factor of 103 for the verdins. A reaction mechanism involving superoxide ion is suggested. Bilirubin appears to quench singlet oxygen by an electron-transfer mechanism.