Search Results (41)

Background: Heme is an important cofactor and plays crucial roles in the correct folding of hemoproteins. The synthesis of heme can be enhanced by the plasmid-based expression of heme biosynthetic genes. However, plasmid-based expression is genetically unstable and requires the utilization of antibiotics to maintain high copy numbers of plasmids. Methods: The rate-limiting steps in heme biosynthesis were first analyzed based on previous studies and the accumulation of heme intermediates was achieved by adding heme precursor (5-aminolevulinic acid, ALA). Next, the intracellular accumulation of porphyrin was increased by deleting the porphyrin transporter TolC. Finally, the heme synthetic genes were modified by integrating the hemA and hemL genes into the cheW and yciQ locus, assembling the rate-limiting enzymes HemC and HemD with RIAD-RIDD tags, replacing the promoters of hemE/hemH genes with the constitutive promoter P_J23100, and deleting the heme degradation gene yfeX. Results: An enhanced heme supply HEME2 strain was obtained with a heme titer of 0.14 mg/L, which was 4.60-fold higher than that of the C41(DE3) strain. The HEME2 strain was applied to produce human hemoglobin and leghemoglobin. The titer and peroxidase activity of human hemoglobin were 1.29-fold and 42.4% higher in the HEME2-hHb strain than the values in the control strain C41-hHb. In addition, the peroxidase activity and heme content of leghemoglobin were increased by 39.2% and 53.4% in the HEME2-sHb strain compared to the values in the control strain C41-sHb. Conclusions: A plasmid-free Escherichia coli C41(DE3) strain capable of efficient and stable heme supply was constructed and can be used for the production of high-active hemoglobins. Full article

Being essential intermediates for the biosynthesis of heme, chlorophyll, and several other biologically critical compounds, porphyrins have wide practical applications. However, up till now, their bio-based production remains challenging. In this study, we identified potential metabolic factors limiting the biosynthesis of type-III stereoisomeric porphyrins in Escherichia coli. To alleviate this limitation, we developed bioprocessing strategies by redirecting more dissimilated carbon flux toward the HemD-enzymatic pathway to enhance the production of type-III uroporphyrin (UP-III), which is a key precursor for heme biosynthesis. Our approaches included the use of antioxidant reagents and strain engineering. Supplementation with ascorbic acid (up to 1 g/L) increased the UP-III/UP-I ratio from 0.62 to 2.57. On the other hand, overexpression of ROS-scavenging genes such as sod- and kat-genes significantly enhanced UP production in E. coli. Notably, overexpression of sodA alone led to a 72.9% increase in total porphyrin production (1.56 g/L) while improving the UP-III/UP-I ratio to 1.94. Our findings highlight the potential of both antioxidant supplementation and strain engineering to mitigate ROS-induced oxidative stress and redirect more dissimilated carbon flux toward the biosynthesis of type-III porphyrins in E. coli. This work offers an effective platform to enhance the bio-based production of porphyrins. Full article

The liver, a pivotal organ in human metabolism, serves as a primary site for heme biosynthesis, alongside bone marrow. Maintaining precise control over heme production is paramount in healthy livers to meet high metabolic demands while averting potential toxicity from intermediate metabolites, notably protoporphyrin IX. Intriguingly, our recent research uncovers a disrupted heme biosynthesis process termed ‘porphyrin overdrive’ in cancers that fosters the accumulation of heme intermediates, potentially bolstering tumor survival. Here, we investigate heme and porphyrin metabolism in both healthy and oncogenic human livers, utilizing primary human liver transcriptomics and single-cell RNA sequencing (scRNAseq). Our investigations unveil robust gene expression patterns in heme biosynthesis in healthy livers, supporting electron transport chain (ETC) and cytochrome P450 function without intermediate accumulation. Conversely, liver cancers exhibit rewired heme biosynthesis and a massive downregulation of cytochrome P450 gene expression. Notably, despite diminished drug metabolism, gene expression analysis shows that heme supply to the ETC remains largely unaltered or even elevated with patient cancer progression, suggesting a metabolic priority shift. Liver cancers selectively accumulate intermediates, which are absent in normal tissues, implicating their role in disease advancement as inferred by expression analysis. Furthermore, our findings in genomics establish a link between the aberrant gene expression of porphyrin metabolism and inferior overall survival in aggressive cancers, indicating potential targets for clinical therapy development. We provide in vitro proof-of-concept data on targeting porphyrin overdrive with a drug synergy strategy. Full article

Heme, an iron-containing tetrapyrrole, is essential in almost all organisms. Heme biosynthesis needs to be precisely regulated particularly given the potential cytotoxicity of protoporphyrin IX, the intermediate preceding heme formation. Here, we report on the porphyrin intermediate accumulation within the tumor microenvironment (TME), which we propose to result from dysregulation of heme biosynthesis concomitant with an enhanced cancer survival dependence on mid-step genes, a process we recently termed “Porphyrin Overdrive”. Specifically, porphyrins build up in both lung cancer cells and stromal cells in the TME. Within the TME’s stromal cells, evidence supports cancer-associated fibroblasts (CAFs) actively producing porphyrins through an imbalanced pathway. Conversely, normal tissues exhibit no porphyrin accumulation, and CAFs deprived of tumor cease porphyrin overproduction, indicating that both cancer and tumor-stromal porphyrin overproduction is confined to the cancer-specific tissue niche. The clinical relevance of our findings is implied by establishing a correlation between imbalanced porphyrin production and overall poorer survival in more aggressive cancers. These findings illuminate the anomalous porphyrin dynamics specifically within the tumor microenvironment, suggesting a potential target for therapeutic intervention. Full article

The metal-free porphyrins protonation has gained interest over five decades because its structure modification and hardly monoacid intermediate isolation. Here, upon the hydrogen atom abstraction processes, one step diproptonated H₃STTP(BF₄)₂ (STTP = 5,10,15,20-tetraphenyl-21-thiaporphyrin) (3) and stepwise protonated HS₂TTPSbCl₆ (5) and diprotonated H₂S₂TTP(BF₄)₂ (6) (S₂TTP = 5,10,15,20-tetraphenyl-21,23-thiaporphyrin) compounds were obtained using HSTTP and S₂TTP with oxidants. The closed-shell protonated compounds were fully characterized using XRD, UV-vis, IR and NMR spectra. In addition, the reduced 19π compounds [K(2,2,2)]HSTTP (2) and [K(2,2,2)]S₂TTP (7) were synthesized by the ligands with reductant KC₈ in THF solution. These two open-shell compounds were characterized with UV-vis, IR and EPR spectroscopies. The semiempirical ZINDO/S method was employed to analyze the HOMO/LUMO gap lever and identify the electronic transitions of the UV-vis spectra of the closed- and open-shell porphyrin compounds. Full article

To inhibit the deep conversion of partial oxidation products (POX-products) in C-H bonds’ functionalization utilizing O₂, 5-(4-(chloromethyl)phenyl)-10,15,20-tris(perfluorophenyl)porphyrin cobalt(II) and 5-(4-(chloromethyl)phenyl)-10,15,20-tris(perfluorophenyl)porphyrin copper(II) were immobilized on the surface of hybrid silica to conduct relay catalysis on the surface. Fluorocarbons with low polarity and heterogeneous catalysis were devised to decrease the convenient accessibility of polar POX-products to catalytic centers on the lower polar surface. Relay catalysis between Co and Cu was designed to utilize the oxidation intermediates alkyl hydroperoxides to transform more C-H bonds. Systematic characterizations were conducted to investigate the structure of catalytic materials and confirm their successful syntheses. Applied to C-H bond oxidation, not only deep conversion of POX-products was inhibited but also substrate conversion and POX-product selectivity were improved simultaneously. For cyclohexane oxidation, conversion was improved from 3.87% to 5.27% with selectivity from 84.8% to 92.3%, which was mainly attributed to the relay catalysis on the surface excluding products. The effects of the catalytic materials, product exclusion, relay catalysis, kinetic study, substrate scope, and reaction mechanism were also investigated. To our knowledge, a practical and novel strategy was presented to inhibit the deep conversion of POX-products and to achieve efficient and accurate oxidative functionalization of hydrocarbons. Also, a valuable protocol was provided to avoid over-reaction in other chemical transformations requiring high selectivity. Full article

Substituents at the meso-site of metalloporphyrins profoundly influence the hydrogen evolution reaction (HER) mechanism. This study employs density functional theory (DFT) to computationally analyze Ni^II-porphyrin and its hydrides derived from tetrakis(pentafluorophenyl)porphyrin molecules, presenting stereoisomers in ortho- or para-positions. The results reveal that the spatial resistance effect of meso-substituted groups at the ortho- and para-positions induces significant changes in Ni-N bond lengths, angles, and reaction dynamics. For ortho-position substituents forming complex I, a favorable 88.88 ų spherical space was created, facilitating proton coordination and the formation of H₂ molecules; conversely, para-position substituents forming complex II impeded H₂ formation until bimolecular complexes arose. Molecular dynamics (MD) analysis and comparison were conducted on the intermediation products of I-H₂ and (II-H)₂, focusing on the configuration and energy changes. In the I-H₂ products, H₂ molecules underwent separation after 150 fs and overcame the 2.2 eV energy barrier. Subsequently, significant alterations in the spatial structure were observed as complex I deformed. In the case of (II-H)₂, it was influenced by the distinctive “sandwich” configuration; the spatial structure necessitated overcoming a 6.7 eV energy barrier for H₂ detachment and a process observed after 2400 fs. Full article

Previous biological tests have shown that some resveratrol analogs exhibited significant antioxidative and cholinesterase inhibitory potential, as evidenced by lower IC₅₀ values compared to the established standards, resveratrol and galantamine, respectively. Photochemical transformations were made in parallel on these compounds in the presence of porphyrin photocatalysts in batch and microreactor, showing the significant advantage of flow photochemistry concerning productivity, selectivity, and yields. In this research, the products of photocatalysis and direct irradiation (photolysis) of resveratrol analogs were compared to elucidate how the types and ratios of the products depend on the excitation energy, to reveal the effects of the substituent on the photoinduced reactions and to rationalize experimentally and computationally the nature and ratio of the obtained products. Thus, two main paths were computed in agreement with the experimental results: isomerization with the participation of triplet state intermediates to yield the experimentally detected cis-isomers and subsequent cyclization following a pathway not available for the trans-isomers. The investigation of five model compounds confirmed the advantages of the flow photoreactor in the photochemical reactions of heterocyclic resveratrol analogs. Full article

The photophysical properties of Cu(II) complexes with 5,10,15,20-meso-tetrakis(phenyl)porphyrin and 5,10,15,20-meso-tetrakis(N-methylpyridium-4-yl)porphyrin are examined via the luminescence and femtosecond time-resolved absorbance methods, respectively. These studies are supported by DFT and TD-DFT calculations, which highlight the important role played by ligand-to-metal charge-transfer states in directing the system toward either intersystem crossing to the triplet hypersurface or coordinative expansion to a five-coordinate quasi-stable intermediate. The latter processes occur when the porphyrin is photolyzed in the presence of suitably located Lewis bases. Femtosecond time-resolved absorbance measurements of Cu(II)-5,10,15,20-meso-tetrakis(N-methylpyridium-4-yl)porphyrin confirm that the coordinative expansion in water occurs in approximately 700 fs, while crossing to the triplet hypersurface takes approximately 140 fs in the same solvent. These processes are mutually exclusive, although both can occur simultaneously depending on the environment of the porphyrin. The ratio of the two processes depends on the relative orientation of the Lewis base with respect to the copper atom at the time of excitation. As a consequence, copper porphyrins such as these are excellent probes in the environment of the porphyrin and can be used to identify the location of the porphyrin when interacting with DNA fragments. Full article

The organic dyes used in printing and dyeing wastewater have complex components, diverse structures and strong chemical stability, which make them not suitable for treatment and difficult to degrade in the environment. Porphyrins are macromolecules with 18 π electrons formed by four pyrrole molecules connected with a methylene bridge that has a stable structure. Porphyrin combines with iron to form an active intermediate with a structure similar to the cytochrome P450 enzyme, so they are widely used in the biomimetic field. In the current study, 5,10,15,20-tetra (4-carboxyphenyl) porphine ferric chloride (III) (Fe(III)TCPP) was used as a catalyst and iodosobenzene was used as an oxidant to explore the catalytic degradation of triphenylmethane dyes, such as rhodamine B (RhB) and malachite green (MG). The results of UV-Vis spectral analysis have shown that the conversion rate of the rhodamine B was over 90% when the amount of Fe(III)TCPP was 0.027 mM and the amount of iodosobenzene was eight equivalents. When the catalyst was 0.00681 mM and the amount of the oxidant was five equivalents, the conversion rate of the malachite green reached over 95%. This work provides a feasible method for the degradation of triphenylmethane dyes. Full article

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. Full article

Great efforts of the scientific community are focused on the development of catalysts for the reduction of carbon dioxide (CO₂) to useful molecules such as carbon monoxide, formic acid, methanol, ethanol, methane, ethylene, or acetate. Various metal porphyrin complexes were synthesized and studied to develop highly active and selective catalysts. While the substituents on the porphyrin core (the primary coordination sphere) determine the reactivity of the metal, the introduction of the secondary coordination is important for the binding and activation of CO₂. In this review, selected examples of iron porphyrin catalysts with a secondary coordination sphere capable of stabilizing intermediates of the CO₂ reduction process by hydrogen bonding are presented. Full article

Sustainable functionalization of renewable aromatics is a key step to supply our present needs for specialty chemicals and pursuing the transition to a circular, fossil-free economy. In the present work, three typically stable aromatic compounds, representative of products abundantly obtainable from biomass or recycling processes, were functionalized in one-pot oxidation reactions at room temperature, using H₂O₂ as a green oxidant and ethanol as a green solvent in the presence of a highly electron withdrawing iron porphyrin catalyst. The results show unusual initial epoxidation of the aromatic ring by the green catalytic system. The epoxides were isolated or evolved through rearrangement, ring opening by nucleophiles, and oxidation. Acridine was oxidized to mono- and di-oxides in the peripheral ring: 1:2-epoxy-1,2-dihydroacridine and anti-1:2,3:4-diepoxy-1,2,3,4-tetrahydroacridine, with TON of 285. o-Xylene was oxidized to 4-hydroxy-3,4-dimethylcyclohexa-2,5-dienone, an attractive building block for synthesis, and 3,4-dimethylphenol as an intermediate, with TON of 237. Quinoline was directly functionalized to 4-quinolone or 3-substituted-4-quinolones (3-ethoxy-4-quinolone or 3-hydroxy-4-quinolone) and corresponding hydroxy-tautomers, with TON of 61. Full article

Silicon (Si) and biochar (Bc) are key signaling conditioners that improve plant metabolic processes and promote drought tolerance. However, the specific role of their integrative application under water restrictions on economical plants is not yet well understood. Two field experiments throughout 2018/2019 and 2019/2020 were conducted to examine the physio-biochemical modifications and yield attributes of borage plants mediated by Bc (9.52 tons ha⁻¹) and/or Si (300 mg L⁻¹) under different irrigation regimes (100, 75, and 50% of crop evapotranspiration). Catalase (CAT) and peroxidase (POD) activity; relative water content, water, and osmotic potential; leaf area per plant and yield attributes; and chlorophyll (Chl) content, Chl_a/chlorophyllide_a (Chlid_a), and Chl_b/Chlid_b were considerably reduced within the drought condition. On the other hand, oxidative biomarkers, as well as organic and antioxidant solutes, were increased under drought, associated with membrane dysfunction, superoxide dismutase (SOD) activation, and osmotic adjustment (OA) capacity as well as a hyperaccumulation of porphyrin intermediates. Supplementation of Bc and Si lessens the detrimental impacts of drought on several plant metabolic processes associated with increasing leaf area and yield attributes. Their application under normal or drought conditions significantly elicited the accumulation of organic and antioxidant solutes as well as the activation of antioxidant enzymes, followed by lessening the formation of free radical oxygen and mitigating oxidative injuries. Moreover, their application maintained water status and OA capacity. Si and/or Bc treatment reduced protoporphyrin, magnesium-protoporphyrin, and protochlorophyllide while increasing Chl_a and Chl_b assimilation and boosting the ratio of Chl_a/Chlid_a and Chl_b/Chlid_b, resulting in a rise in leaf area per plant and yield components following these modifications. These findings highlight the significance of Si and/or Bc as (a) stress-signaling molecule(s) in regulating defensive systems in drought-affected borage plants by boosting antioxidant aptitude, regulating water status, and accelerating chlorophyll assimilation, thus leading to increasing leaf area and productivity. Full article

The excited triplet state of a molecule (T₁) is one of the principal intermediate products in various photochemical processes due to its high reactivity and relatively long lifetime. The T₁ quantum yield (${\phi }_{\mathrm{T}}$) is one of the most important characteristics in the study of photochemical reactions. It is of special interest to determine the ${\phi }_{\mathrm{T}}$ of various photoactive compounds (photosensitizer, PS) used in photodynamic therapy (PDT). PDT is an effective medical technique for the treatment of serious diseases, such as cancer and bacterial, fungal and viral infections. This technique is based on the introduction of a PS to a patient’s organism and its further excitation by visible light, producing reactive oxygen species (ROS) via electron or energy transfer from the PS T₁ state to the biological substrate or molecular oxygen. Therefore, information on the ${\phi }_{\mathrm{T}}$ value is fundamental in the search for new and effective PSs. There are various experimental methods to determine ${\phi }_{\mathrm{T}}$ values; however, these methods demonstrate a high discrepancy between ${\phi }_{\mathrm{T}}$ values. This stimulates the analysis of various factors that can affect the determined ${\phi }_{\mathrm{T}}$. In this study, we analyze the effect of the intensity profile of the exciting laser pulse on the calculation of the ${\phi }_{\mathrm{T}}$ value obtained by the Laser Flash Photolysis technique. The φ_T values were determined by analyzing the variation of a sample transient absorption in the function of the exciting laser pulse intensity, in combination with the spectral and kinetic PS characteristics obtained in nonlinear optical experiments by solving the rate equations of a five-level-energy diagram. Well-studied PSs: meso-tetra(4-sulfonatophenyl) (TPPS₄) porphyrins, its zinc complex (ZnTPPS₄) and the zinc complex of meso-tetrakis(N-methylpyridinium-4-yl) (ZnTMPyP) were chosen as test compounds to evaluate the proposed model. The ${\phi }_{\mathrm{T}}$ values were determined through a comparison with the ${\phi }_{\mathrm{T},\mathrm{T}\mathrm{M}\mathrm{P}\mathrm{y}\mathrm{P}}$ = 0.82 of meso-tetrakis(N-methylpyridinium-4-yl) (TMPyP), used as a standard. The obtained results (${\phi }_{\mathrm{T},{\mathrm{T}\mathrm{P}\mathrm{P}\mathrm{S}}_4}=0.75\pm 0.02$, ${\phi }_{\mathrm{T},\mathrm{Z}\mathrm{n}\mathrm{T}\mathrm{M}\mathrm{P}\mathrm{y}\mathrm{P}}=0.90\pm 0.03)$, and ${\phi }_{\mathrm{T},{\mathrm{Z}\mathrm{n}\mathrm{T}\mathrm{P}\mathrm{P}\mathrm{S}}_4}=0.89\pm 0.03$) are highly compatible with the medium ${\phi }_{\mathrm{T}}$ values obtained using the known methods. Full article