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Three-dimensional hepatic cell cultures can provide an important advancement in the toxicity assessment of nanomaterials with respect to 2D models. Here, we describe liver organoids (LOs) obtained by assembling multiple cell lineages in a fixed ratio 1:1:0.2. These are upcyte® human hepatocytes, UHHs, upcyte® liver sinusoidal endothelial cells, LSECs, and human bone marrow-derived mesenchymal stromal cells, hbmMSCs. The structural and functional analyses indicated that LOs reached size stability upon ca. 10 days of cultivation (organoid maturation), showing a surface area of approximately 10 mm2 and the hepatic cellular lineages, UHHs and LSECs, arranged to form both primitive biliary networks and sinusoid structures, alike in vivo. LOs did not show signs of cellular apoptosis, senescence, or alteration of hepatocellular functions (e.g., dis-regulation of CYP3A4 or aberrant production of Albumin) for the entire culture period (19 days since organoid maturation). After that, LOs were repeatedly exposed for 19 days to a single or repeated dose of graphene oxide (GO: 2–40 µg/mL). We observed that the treatment did not induce any macroscopic signs of tissue damage, apoptosis activation, and alteration of cell viability. However, in the repeated dose regimen, we observed a down-regulation of CYP3A4 gene expression. Notably, these findings are in line with recent in vivo data, which report a similar impact on CYP3A4 when mice were repeatedly exposed to GO. Taken together, these findings warn of the potential detrimental effects of GO in real-life exposure (e.g., occupational scenario), where its progressive accumulation is likely expected. More in general, this study highlights that LOs formed by many cell lineages can enable repeated exposure regimens (suitable to mimic accumulation); thus, they can be suitably considered alternative or complementary in vitro systems to animal models. [ABSTRACT FROM AUTHOR]

Cupriavidus necator, despite lacking direct electron transfer capabilities, demonstrates efficient reduction of various redox mediators in oxygen‐free cultivation within bioelectrochemical systems. This study investigates the reduction site of ferricyanide through inhibition and expression rate analysis of oxygen and nitrate respiration chain complexes, comparing aerobic cultivation conditions with fructose as carbon and electron donor to autotrophic (CO2/H2/O2) and anodic cultivation conditions (fructose/anode). Azide inhibition identified cytochrome c oxidase as the primary complex facilitating electron transfer to ferricyanide, with a secondary role proposed for nitrite reductase NirS, demonstrating a 3.9±1.1‐fold higher expression when exposed to anodic conditions. The 2.9±0.6‐fold increase in the expression of the natural porin OmpA under anodic conditions implies its potential involvement in ferricyanide uptake. Additionally, chemically permeabilizing cell membranes with cetyltrimethylammonium bromide doubles ferricyanide reduction rates without an anode present, offering insights for optimizing redox mediation in C. necator based bioelectrochemical systems. This study opens up new possibilities for the targeted optimization of mediated electron transfer in C. necator and other organisms. [ABSTRACT FROM AUTHOR]

The liver plays a key role in the metabolism of lipoproteins, controlling both production and catabolism. To accelerate the development of new lipid‐lowering therapies in humans, it is essential to have a relevant in vitro study model available. The current hepatocyte‐like cells (HLCs) models derived from hiPSC can be used to model many genetically driven diseases but require further improvement to better recapitulate the complexity of liver functions. Here, we aimed to improve the maturation of HLCs using a three‐dimensional (3D) approach using Biomimesys®, a hyaluronic acid‐based hydroscaffold in which hiPSCs may directly form aggregates and differentiate toward a functional liver organoid model. After a 28‐day differentiation 3D protocol, we showed that many hepatic genes were upregulated in the 3D model (liver organoids) in comparison with the 2D model (HLCs). Liver organoids, grown on Biomimesys®, exhibited an autonomous cell organization, were composed of different cell types and displayed enhanced cytochromes P450 activities compared to HLCs. Regarding the functional capacities of these organoids, we showed that they were able to accumulate lipids (hepatic steatosis), internalize low‐density lipoprotein and secrete apolipoprotein B. Interestingly, we showed for the first time that this model was also able to produce apolipoprotein (a), the apolipoprotein (a) specific of Lp(a). This innovative hiPSC‐derived liver organoid model may serve as a relevant model for studying human lipopoprotein metabolism, including Lp(a). [ABSTRACT FROM AUTHOR]

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming "Candidatus Methanoperedens" archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells. Borgs are huge extrachromosomal elements associated with anaerobic methane-consuming archaea. Here, the authors use nanopore sequencing to identify new Borgs from peatland soil and their hosts, uncover their DNA modifications and activities, and elucidate the evolutionary relationships of Borgs. [ABSTRACT FROM AUTHOR]

Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis. Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest. Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase. In the present study, a new colorimetric high-throughput screening assay was successfully developed based on a dualenzyme cascade for the engineering of the P450DA's hydroxylation activity. Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies. Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septupletmutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92% isolated yields and >99% ee. [ABSTRACT FROM AUTHOR]

Pesticides have increased crop yield but severely impacted ecosystems and non-target organisms. Flubendiamide, a new generation pesticide, targets insect larvae but also affects non-target organisms. This study examines the effects of lowest observed effect concentration of technical grade flubendiamide (0.5 µg/µL) flubendiamide on chick liver, focusing on cytochrome P450 (CYP) enzyme expression, oxidative stress, and liver damage. Chick embryos treated with flubendiamide showed significant alterations in CYP mRNA and protein levels, indicating increased toxicant accumulation. Elevated CYP3A4, CYP1A1, CYP1A2, and CYP2C19 levels were noted, suggesting enhanced biotransformation and detoxification processes. However, increased oxidative byproducts led to oxidative stress, as evidenced by decreased glutathione (GSH) levels and elevated superoxide dismutase (SOD) and catalase activities. DCFDA staining confirmed increased hydrogen peroxide (H2O2) levels, indicating heightened reactive oxygen species (ROS). Liver function tests revealed significant increases in serum ALP, ALT, and AST levels, indicating acute liver damage. Histopathological analysis showed structural liver damage, including expanded sinusoidal spaces, impaired portal veins, and compromised hepatocyte architecture. These findings underscore flubendiamide's potential hepatotoxicity in non-target organisms, emphasizing the need for cautious pesticide use to minimize environmental impacts.

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug–drug interactions involving IP6 are low. [ABSTRACT FROM AUTHOR]

Growth hormone (GH) modifies liver gene transcription in a sexually dimorphic manner to meet liver metabolic demands related to sex; thus, GH dysregulation leads to sex-biased hepatic disease. We dissected the steps of the GH regulatory cascade modifying GH-dependent genes involved in metabolism, focusing on the male-predominant genes Lcn13 , Asns , and Cyp7b1 , and the female-predominant genes Hao2 , Pgc1a , Hamp2 , Cyp2a4 , and Cyp2b9. We explored mRNA expression in 2 settings: (i) intact liver GH receptor (GHR) but altered GH and insulin-like growth factor 1 (IGF1) levels (NeuroDrd2KO, HiGH, aHepIGF1kd, and STAT5bCA mouse lines); and (ii) liver loss of GHR, with or without STAT5b reconstitution (aHepGHRkd, and aHepGHRkd + STAT5bCA). Lcn13 was downregulated in males in most models, while Asns and Cyp7b1 were decreased in males by low GH levels or action, or constant GH levels, but unexpectedly upregulated in both sexes by the loss of liver Igf1 or constitutive Stat5b expression. Hao , Cyp2a4 , and Cyp2b9 were generally decreased in female mice with low GH levels or action (NeuroDrd2KO and/or aHepGHRkd mice) and increased in HiGH females, while in contrast, Pgc1a was increased in female NeuroDrd2KO but decreased in STAT5bCA and aHepIGF1kd females. Bioinformatic analysis of RNAseq from aHepGHRkd livers stressed the greater impact of GHR loss on wide gene expression in males and highlighted that GH modifies almost completely different gene signatures in each sex. Concordantly, we show that altering different steps of the GH cascade in the liver modified liver expression of Lcn13 , Asns , Cyp7b1 , Hao2 , Hamp2 , Pgc1a , Cyp2a4 , and Cyp2b9 in a sex- and gene-specific manner. [ABSTRACT FROM AUTHOR]

Paclitaxel is an anticancer therapeutic produced by the yew tree. Over the last two decades, a significant bottleneck in the reconstitution of early paclitaxel biosynthesis has been the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, we structurally characterize four new products of T5αH, many of which appear to be over-oxidation of the primary mono-oxidized products. By tuning the promoter strength for T5αH expression in Nicotiana plants, we observe decreased levels of these proposed byproducts with a concomitant increase in the accumulation of taxadien-5α-ol, the paclitaxel precursor, by three-fold. This enables the reconstitution of a six step biosynthetic pathway, which we further show may function as a metabolic network. Our result demonstrates that six previously characterized Taxus genes can coordinatively produce key paclitaxel intermediates and serves as a crucial platform for the discovery of the remaining biosynthetic genes. Paclitaxel is an important anticancer drug whose biosynthetic pathway reconstruction is hindered by the propensity of heterologously expressed pathway cytochromes P450, including taxadiene 5α-hydroxylase (T5αH), to form multiple products. Here, the authors tune the promoter strength for T5αH expression in Nicotiana plants to increase the levels of paclitaxel precursor taxadien-5α-ol by three-fold and reconstitute the six step early biosynthetic pathway of paclitaxel. [ABSTRACT FROM AUTHOR]

The fossil record demonstrates the preservation of porphyrins (e.g., heme) in organic sediments and the fossilized remains of animals. These molecules are essential components in modern metabolic processes, such as electron transport (cytochromes) and oxygen transport (hemoglobin), and likely originated before the emergence of life. The integration and adaptation of porphyrins and structurally similar molecules (e.g., chlorophylls) are key aspects in the evolution of energy production (i.e., aerobic respiration and photosynthesis) and complex life (i.e., eukaryotes and multicellularity). Here, we discuss the evolution and functional diversity of heme-bound hemoglobin proteins in vertebrates, along with the preservation of these molecules in the fossil record. By elucidating the pivotal role of these molecules in the evolution of life, this review lays the groundwork necessary to explore hemoglobin as a means to investigate the paleobiology of extinct taxa, including non-avian dinosaurs. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease. [ABSTRACT FROM AUTHOR]

Abstract Cupriavidus necator, despite lacking direct electron transfer capabilities, demonstrates efficient reduction of various redox mediators in oxygen‐free cultivation within bioelectrochemical systems. This study investigates the reduction site of ferricyanide through inhibition and expression rate analysis of oxygen and nitrate respiration chain complexes, comparing aerobic cultivation conditions with fructose as carbon and electron donor to autotrophic (CO2/H2/O2) and anodic cultivation conditions (fructose/anode). Azide inhibition identified cytochrome c oxidase as the primary complex facilitating electron transfer to ferricyanide, with a secondary role proposed for nitrite reductase NirS, demonstrating a 3.9±1.1‐fold higher expression when exposed to anodic conditions. The 2.9±0.6‐fold increase in the expression of the natural porin OmpA under anodic conditions implies its potential involvement in ferricyanide uptake. Additionally, chemically permeabilizing cell membranes with cetyltrimethylammonium bromide doubles ferricyanide reduction rates without an anode present, offering insights for optimizing redox mediation in C. necator based bioelectrochemical systems. This study opens up new possibilities for the targeted optimization of mediated electron transfer in C. necator and other organisms.

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights. Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications. Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850 k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers. Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances. Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Sanghuangporus, also known as "Sanghuang" in China, is a well-known genus of traditional Chinese medicinal macrofungi. To make more effective use of Sanghuangporus resources, we completed the first genome assembly and annotation of a monokaryon strain of S. weigelae in the present study. A 33.96-Mb genome sequence was assembled as 13 contigs, leading to prediction of 9377 protein-coding genes. Phylogenetic and average nucleotide identity analyses indicated that the S. weigelae genome is closely related to those of other Sanghuangporus species in evolutionary tree, which clustered in one clade. Collinearity analysis revealed a high level of collinearity of S. weigelae with S. baumii, S. vaninii, and S. sanghuang. Biosynthesis pathways potentially involved in medicinal properties, including terpenoid and polysaccharide synthesis, were identified in S. weigelae, while polysaccharides were identified as the main medicinal metabolites in S. weigelae, with flavonoids more important in Sanghuangporus than other medicinal mushroom groups. Genes encoding 332 carbohydrate-active enzymes were identified in the S. weigelae genome, including major glycoside hydrolases and glycosyltransferases predicted, revealing the robust lignocellulose degradation capacity of S. weigelae. Further, 130 genes, clustered in seven classes were annotated to encode cytochromes P450 in the S. weigelae genome. Overall, our results reveal the remarkably medicinal capacity of S. weigelae and provide new insights that will inform the study of evolution and medicinal application of S. weigelae. The data are a reference resource for the formulation of scientific and rational ecological protection policies for Sanghuangporus species. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of secondary metabolites that have garnered scientific attention due to their complex scaffolds with potential roles in medicine, agriculture, and chemical ecology. RiPPs derive from the cleavage of ribosomally synthesized proteins and additional modifications, catalyzed by various enzymes to alter the peptide backbone or side chains. Of these enzymes, cytochromes P450 (P450s) are a superfamily of heme-thiolate proteins involved in many metabolic pathways, including RiPP biosyntheses. In this review, we focus our discussion on P450 involved in RiPP pathways and the unique chemical transformations they mediate. Previous studies have revealed a wealth of P450s distributed across all domains of life. While the number of characterized P450s involved in RiPP biosyntheses is relatively small, they catalyze various enzymatic reactions such as C–C or C–N bond formation. Formation of some RiPPs is catalyzed by more than one P450, enabling structural diversity. With the continuous improvement of the bioinformatic tools for RiPP prediction and advancement in synthetic biology techniques, it is expected that further cytochrome P450-mediated RiPP biosynthetic pathways will be discovered. Summary The presence of genes encoding P450s in gene clusters for ribosomally synthesized and post-translationally modified peptides expand structural and functional diversity of these secondary metabolites, and here, we review the current state of this knowledge. [ABSTRACT FROM AUTHOR]

Electroactive microorganisms (EAMs) could utilize extracellular electron transfer (EET) pathways to exchange electrons and energy with their external surroundings. Conductive cytochrome proteins and nanowires play crucial roles in controlling electron transfer rate from cytosol to extracellular electrode. Many previous studies elucidated how the c‐type cytochrome proteins and conductive nanowires are synthesized, assembled, and engineered to manipulate the EET rate, and quantified the kinetic processes of electron generation and EET. Here, we firstly overview the electron transfer pathways of EAMs and quantify the kinetic parameters that dictating intracellular electron production and EET. Secondly, we systematically review the structure, conductivity mechanisms, and engineering strategies to manipulate conductive cytochromes and nanowire in EAMs. Lastly, we outlook potential directions for future research in cytochromes and conductive nanowires for enhanced electron transfer. This article reviews the quantitative kinetics of intracellular electron production and EET, and the contribution of engineered c‐type cytochromes and conductive nanowire in enhancing the EET rate, which lay the foundation for enhancing electron transfer capacity of EAMs. [ABSTRACT FROM AUTHOR]

Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal–porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π–interactions, as corroborated by molecular docking. [ABSTRACT FROM AUTHOR]

Background: Vitamins have a great impact on metabolis. Aims: To determine the role of 1,25(OH)2D On Cytochromes CYP27A1 and CYP27B1 in Periodontitis. Material and Method: The investigation was carried out on 45 participants of ages within the range of (30-45 years) who were attending the private dental clinics. Diagnosis of chronic periodontitis was established depending on dental history, clinical examinations (periodontal indices). All participants were examined by the same dentist. They were classified into three groups: Group 1 (control negative): (15) participants with normal serum vitamin D3 level and with pocket depth =3 mm, good oral health and normal periodontal tissues and no previous history of periodontal diseases. Group 2 (control positive): (15) participants with normal serum vitamin D3 level and periodontitis with pocket depth =5 mm, they received placebo medication orally, Group3(treatment): (15) participants with vitamin D3 deficiency (below 30 IU), and periodontitis with pocket depth ≥5 mm, they received oral Vitamin D3 fast acting liquid soft gel capsule 2000 IU/day for 3 months. 3 blood samples were taken from each participant at 0,45,90 days, for research examinations. CYP27A1, CYP27B1 serum levels was measured for each sample in three groups by ELISA kit. Result: there was a highly significant reduction in CYP27A1 serum level in the treatment group at the ninety days of the study while there was no significant elevation CYP27B1 serum level in all groups during 45,90 days of the study. Conclusion: The present study suggested that the 1,25(OH)2D has effects on serum levels of both Cytochromes CYP27A1 and CYP27B1 and this was associated with periodontitis. [ABSTRACT FROM AUTHOR]

We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe+2) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g., from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site. [ABSTRACT FROM AUTHOR]

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights.Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications.Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850 k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers.Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances.Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment.

ABSTRACTHeme trafficking is a fundamental biological process, yet its direct study has been hampered due to heme’s tight intracellular regulation, heme cytotoxicity, and the transient nature of trafficking. The bacterial System I and System II cytochrome c biogenesis pathways are developing into models to interrogate heme trafficking mechanisms, as they function to transport heme from inside to outside the cell for attachment to apocytochrome c. Cytochromes c require heme for folding and to function in the context of electron transport chains for critical cellular functions, such as respiration. We focus on System I, comprised of eight membrane proteins, CcmABCDEFGH, proposed to function in two steps: CcmABCD mediates the transfer of heme and attachment to CcmE. HoloCcmE chaperones heme to CcmFH for attachment to apocytochrome c. While CcmFH is known to be the holocytochrome c synthase, the mechanism of heme interaction and positioning for attachment to apocytochrome c remains to be elucidated. A comprehensive structure-function analysis of the conserved WWD domain in CcmF was undertaken utilizing alanine-scanning and cysteine-scanning, revealing residues critical for CcmF’s synthase function and residues required for interaction with the 2- and 4-vinyls of heme. This analysis demonstrates for the first time that the CcmF WWD domain directly interacts with heme and that heme interactions within this domain are required for attachment to apocytochrome c. This in-depth interrogation of heme binding now allows for comparison across cytochrome c biogenesis proteins CcmF, CcmC, and CcsBA, revealing common mechanisms of heme interaction in these heme trafficking pathways.IMPORTANCEHeme is an essential co-factor for proteins involved with critical cellular functions, such as energy production and oxygen transport. Thus, understanding how heme interacts with proteins and is moved through cells is a fundamental biological question. This work studies the System I cytochrome c biogenesis pathway, which in some species (including Escherichia coli) is composed of eight integral membrane or membrane-associated proteins called CcmA-H that are proposed to function in two steps to transport and attach heme to apocytochrome c. Cytochrome c requires this heme attachment to function in electron transport chains to generate cellular energy. A conserved WWD heme-handling domain in CcmFH is analyzed and residues critical for heme interaction and holocytochrome c synthase activity are identified. CcmFH is the third member of the WWD domain-containing heme-handling protein family to undergo a comprehensive structure-function analysis, allowing for comparison of heme interaction across this protein family.

Geobacter sulfurreducens mediates extracellular electron transfer (EET) reactions with different substrates, such as solid-phase Fe(III)-containing minerals, anodes and the cells of Geobacter metallireducens. To compare their roles in EET, the pilA-N, omcE, omcS, omcT and omcZ genes of G. sulfurreducens were systematically deleted. All mutants showed impaired and varied ability to form biofilms on nonconductive surface. Deletion of omcE also impaired bacterial ability to reduce ferrihydrite, but its impacts on the ability for anode reduction and the co-culture of G. metallireducens-G. sulfurreducens were minimal. The mutant without omcS showed diminished ability to reduce ferrihydrite and to form the co-culture, but was able to regain its ability to reduce anodes. Deletion of omcT, omcZ or pilA-N alone impaired bacterial ability to reduce ferrihydrite and anodes and to form the co-culture. Deletion of all tested genes abolished bacterial ability to reduce ferrihydrite and anodes. Triple-deletion of all omcS, omcT and omcZ abolished the ability of G. sulfurreducens to co-culture with G. metallireducens. However, deletion of only omcZ or pilA-N or both omcS and omcT abolished the ability of G. sulfurreducens without hydrogenase gene hybL to co-culture with G. metallireducens, which show their indispensable roles in direct electron transfer from G. metallireducens to G. sulfurreducens. Thus, the roles of pilA-N, omcE, omcS, omcT and omcZ for G. sulfurreducens in EET vary substantially, which also suggest that possession of PilA-N and multiple cytochromes of different structures enables G. sulfurreducens to mediate EET reactions efficiently with substrates of different properties. [ABSTRACT FROM AUTHOR]

Electroactive microbial cells have evolved unique extracellular electron transfer to conduct the reactions via redox outer-membrane (OM) proteins. However, the electron transfer mechanism at the interface of OM proteins and nanomaterial remains unclear. In this study, the mechanism for the electron transfer at biological/inorganic interface is investigated by integrating molecular modeling with electrochemical and spectroscopic measurements. For this purpose, a model system composed of OmcA, a typical OM protein, and the hexagonal tungsten trioxide (h-WO3) with good biocompatibility is selected. The interfacial electron transfer is dependent mainly on the special molecular configuration of OmcA and the microenvironment of the solvent exposed active center. Also, the apparent electron transfer rate can be tuned by site-directed mutagenesis at the axial ligand of the active center. Furthermore, the equilibrium state of the OmcA/h-WO3 systems suggests that their attachment is attributed to the limited number of residues. The electrochemical analysis of OmcA and its variants reveals that the wild type exhibits the fastest electron transfer rate, and the transient absorption spectroscopy further shows that the axial histidine plays an important role in the interfacial electron transfer process. This study provides a useful approach to promote the site-directed mutagenesis and nanomaterial design for bioelectrocatalytic applications. [ABSTRACT FROM AUTHOR]

The recent reclassification of the strict anaerobe Geobacter sulfurreducens bacterium as aerotolerant brought attention for oxidative stress protection pathways. Although the electron transfer pathways for oxygen detoxification are not well established, evidence was obtained for the formation of a redox complex between the periplasmic triheme cytochrome PpcA and the diheme cytochrome peroxidase MacA. In the latter, the reduction of the high-potential heme triggers a conformational change that displaces the axial histidine of the low-potential heme with peroxidase activity. More recently, a possible involvement of the triheme periplasmic cytochrome family (PpcA-E) in the protection from oxidative stress in G. sulfurreducens was suggested. To evaluate this hypothesis, we investigated the electron transfer reaction and the biomolecular interaction between each PpcA-E cytochrome and MacA. Using a newly developed method that relies on the different NMR spectral signatures of the heme proteins, we directly monitored the electron transfer reaction from reduced PpcA-E cytochromes to oxidized MacA. The results obtained showed a complete electron transfer from the cytochromes to the high-potential heme of MacA. This highlights PpcA-E cytochromes' efficient role in providing the necessary reducing power to mitigate oxidative stress situations, hence contributing to a better knowledge of oxidative stress protection pathways in G. sulfurreducens. [ABSTRACT FROM AUTHOR]

Nivasorexant, a selective orexin‐1‐receptor antagonist, has recently been assessed in the treatment of humans with binge‐eating disorder. Herein, the inhibitory potential of nivasorexant on cytochromes P450 (CYPs) 2C9, 2C19, and 3A4 was evaluated. Human liver microsomes/recombinant CYP enzymes were evaluated in vitro. In vivo, a single‐center, open‐label, fixed‐sequence study was performed in healthy adults to explore the effect of 100 mg nivasorexant administered twice daily (b.i.d.) on the pharmacokinetics (PK) of flurbiprofen (50 mg, CYP2C9), omeprazole (20 mg, CYP2C19), midazolam (2 mg, CYP3A4) making use of a cocktail approach. Plasma PK sampling was performed over 24 h on Day 1 (Cocktail alone), 8 (Cocktail + nivasorexant), and 15 (Cocktail + nivasorexant at steady state). Genotyping of subjects' CYPs was performed while safety and tolerability were also assessed. In vitro, nivasorexant inhibited CYP2C9, 2C19, and 3A4 in competitive inhibition assays with IC50 values of 8.6, 1.6, and 19–44 μM, respectively, while showing a significant time‐dependent CYP2C19 inhibition. In 22 subjects, exposure to flurbiprofen, omeprazole, and midazolam was generally higher during concomitant single‐ (i.e., area under the plasma concentration–time curve [AUC] ratio increased by 1.04‐, 2.05‐, and 1.56‐fold, respectively) and repeated‐dose (i.e., AUC ratio increased by 1.47‐, 6.84‐, and 3.71‐fold, respectively) nivasorexant administration compared with the cocktail substrates administered alone. The most frequently reported adverse event was somnolence. According to regulatory guidance, nivasorexant is classified as a moderate CYP2C19 and weak CYP3A4 inhibitor after 1 day and as a weak CYP2C9, strong CYP2C19, and moderate CYP3A4 inhibitor after 8 days of 100 mg b.i.d. administration. Clinicaltrials.gov ID: NCT05254548. [ABSTRACT FROM AUTHOR]

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments. [ABSTRACT FROM AUTHOR]

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains. [ABSTRACT FROM AUTHOR]

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used. Anaerobic methanotrophic archaea play crucial roles in the methane cycle. Here, Zhang et al. provide experimental evidence supporting that multi-heme cytochromes mediate extracellular electron transfer for the reduction of metals and electrodes in these microorganisms. [ABSTRACT FROM AUTHOR]

Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient‐specific treatment strategies. Herein, novel 3D vascularized HCC on chips (HCCoCs), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness, are created. HCC inflammation is controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450‐3A4 (CYP3A4) is investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allows for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). It is shown that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increases vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, it is shown that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy‐associated endothelial porosity increase. The HCCoCs are shown to have utility in uncovering the impact of the tumor microenvironment during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies. [ABSTRACT FROM AUTHOR]

Mono- and multiheme cytochromes c are post-translationally matured by the covalent attachment of heme. For this, Escherichia coli employs the most complex type of maturation machineries, the Ccm-system (for cytochrome cmaturation). It consists of two membrane protein complexes, one of which shuttles heme across the membrane to a mobile chaperone that then delivers the cofactor to the second complex, an apoprotein:heme lyase, for covalent attachment. Here we report cryo-electron microscopic structures of the heme translocation complex CcmABCD from E. coli, alone and bound to the heme chaperone CcmE. CcmABCD forms a heterooctameric complex centered around the ABC transporter CcmAB that does not by itself transport heme. Our data suggest that the complex flops a heme group from the inner to the outer leaflet at its CcmBC interfaces, driven by ATP hydrolysis at CcmA. A conserved heme-handling motif (WxWD) at the periplasmic side of CcmC rotates the heme by 90° for covalent attachment to the heme chaperone CcmE that we find interacting exclusively with the CcmB subunit. The covalent linkage of hemes to cytochromes c requires a maturation machinery. Here, the authors provide mechanistic insights into how the heme translocase complex CcmABCD flops a heme group, driven by ATP hydrolysis, and delivers it to the chaperone CcmE. [ABSTRACT FROM AUTHOR]

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer. [ABSTRACT FROM AUTHOR]

Improper drug prescription is a main cause of both drug-related harms (inefficacy and toxicity) and ineffective spending and waste of the healthcare system's resources. Nowadays, strategies to support an improved, informed prescription process may benefit from the adequate use of pharmacogenomic testing. Using next-generation sequencing, we analyzed the genomic profile for three major cytochromes P450 (CYP2C9, CYP2C19, CYP2D6) and studied the frequencies of dysfunctional isozymes (e.g., poor, intermediate, or rapid/ultra-rapid metabolizers) in a cohort of 298 Italian subjects. We found just 14.8% of subjects with a fully normal set of cytochromes, whereas 26.5% of subjects had combined cytochrome dysfunction (more than one isozyme involved). As improper drug prescription is more frequent, and more burdening, in polytreated patients, since drug–drug interactions also cause patient harm, we discuss the potential benefits of a more comprehensive PGX testing approach to support informed drug selection in such patients. [ABSTRACT FROM AUTHOR]

I present here a personal perspective of the evolution of the two-light reaction two-pigment scheme for the electron transport in oxygenic photosynthesis - as I have lived through it - first as a graduate student of Robert Emerson, from September 1956-January 1959, and then of Eugene Rabinowitch from February 1959-September 1960. I have provided here some of the key published work in my way and have also provided a few photographs. It is essential to remind ourselves upfront that different individuals may have different recollections of the same event (see e.g., the Japanese movie 'Rashomon' https://en.wikipedia.org/wiki/Rashomon). Thus, I encourage others to write about their perspective. Further, I recognize, upfront, the support and encouragement I have received from Robert Blankenship for writing this perspective, and from Győző Garab for submission of this story to Photosynthetica. I begin my perspective with a dedication to Robert Emerson, whose pioneering work led to the concept of the two-light reaction two-pigment system in oxygenic photosynthesis.

An important role in ensuring the functioning of the respiratory chain belongs to the cytochrome part, which includes complexes III (ubiquinol-cytochrome c oxidoreductase) and IV (cytochrome c oxidase). The key components of these enzymatic complexes are heme-containing cytochromes, the number of which depends on the balance of heme synthesis and catabolism. δ-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway, while heme oxygenase is the key enzyme of heme degradation. It is known that nutritional imbalances drive many risk factors for chronic kidney disease. That is why our research aimed to study the activity of ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase complexes, the level of cytochromes a+a3, b, c, and c1, and the activity of key enzymes of heme metabolism in the mitochondria of rat kidneys under conditions of different content of protein and sucrose in animal diet. The obtained results showed a decreased activity of ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase complexes and reduced levels of mitochondria cytochromes a+a3, b, c, and c1 in the kidney mitochondria under the conditions of nutrient imbalance, with the most pronounced changes found in animals kept on a low-protein/high-sucrose diet. A decrease in δ-aminolevulinate synthase activity with a simultaneous 2-fold increase in heme oxygenase activity was found in kidney mitochondria of animals kept on a low-protein/high-sucrose diet compared to those kept on full-value diet indicating an intensification of heme catabolism along with inhibition of its synthesis. The obtained results testify the energy imbalance under the conditions of low-protein/high-sucrose which in turn can lead to the progression of kidney injury.

Haloalkaliphilic microorganisms are double extremophiles functioning optimally at high salinity and pH. Their typical habitats are soda lakes, geologically ancient yet widespread ecosystems supposed to harbor relict microbial communities. We compared metabolic features and their determinants in two strains of the natronophilic species Dethiobacter alkaliphilus, the only cultured representative of the class "Dethiobacteria" (Bacillota). The strains of D. alkaliphilus were previously isolated from geographically remote Mongolian and Kenyan soda lakes. The type strain AHT1T was described as a facultative chemolithoautotrophic sulfidogen reducing or disproportionating sulfur or thiosulfate, while strain Z-1002 was isolated as a chemolithoautotrophic iron reducer. Here, we uncovered the iron reducing ability of strain AHT1T and the ability of strain Z-1002 for thiosulfate reduction and anaerobic Fe(II) oxidation. Key catabolic processes sustaining the growth of both D. alkaliphilus strains appeared to fit the geochemical settings of two contrasting natural alkaline environments, sulfur-enriched soda lakes and iron-enriched serpentinites. This hypothesis was supported by a meta-analysis of Dethiobacterial genomes and by the enrichment of a novel phylotype from a subsurface alkaline aquifer under Fe(III)-reducing conditions. Genome analysis revealed multiheme c-type cytochromes to be the most probable determinants of iron and sulfur redox transformations in D. alkaliphilus. Phylogeny reconstruction showed that all the respiratory processes in this organism are likely provided by evolutionarily related early forms of unconventional octaheme tetrathionate and sulfite reductases and their structural analogs, OmhA/OcwA Fe(III)-reductases. Several phylogenetically related determinants of anaerobic Fe(II) oxidation were identified in the Z-1002 genome, and the oxidation process was experimentally demonstrated. Proteomic profiling revealed two distinct sets of multiheme cytochromes upregulated in iron(III)- or thiosulfate-respiring cells and the cytochromes peculiar for Fe(II) oxidizing cells. We suggest that maintaining high variation in multiheme cytochromes is an effective adaptive strategy to occupy geochemically contrasting alkaline environments. We propose that sulfurenriched soda lakes could be secondary habitats for D. alkaliphilus compared to Fe-rich serpentinites, and that the ongoing evolution of Dethiobacterales could retrace the evolutionary path that may have occurred in prokaryotes at a turning point in the biosphere's history, when the intensification of the sulfur cycle outweighed the global significance of the iron cycle. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction. [ABSTRACT FROM AUTHOR]

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, andO2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c0 (cyts c0) are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c0-a) and an unrelated family with a large betasheet fold (cyts c0-ß) resembling that of cytochromes P460. A recent structure of cyt c0-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c0-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c0-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins. [ABSTRACT FROM AUTHOR]

Heme is an iron-protoporphyrin complex with an essential physiologic function for all cells, especially for those in which heme is a key prosthetic group of proteins such as hemoglobin, myoglobin, and cytochromes of the mitochondria. However, it is also known that heme can participate in pro-oxidant and pro-inflammatory responses, leading to cytotoxicity in various tissues and organs such as the kidney, brain, heart, liver, and in immune cells. Indeed, heme, released as a result of tissue damage, can stimulate local and remote inflammatory reactions. These can initiate innate immune responses that, if left uncontrolled, can compound primary injuries and promote organ failure. In contrast, a cadre of heme receptors are arrayed on the plasma membrane that is designed either for heme import into the cell, or for the purpose of activating specific signaling pathways. Thus, free heme can serve either as a deleterious molecule, or one that can traffic and initiate highly specific cellular responses that are teleologically important for survival. Herein, we review heme metabolism and signaling pathways, including heme synthesis, degradation, and scavenging. We will focus on trauma and inflammatory diseases, including traumatic brain injury, trauma-related sepsis, cancer, and cardiovascular diseases where current work suggests that heme may be most important. [ABSTRACT FROM AUTHOR]

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes. [ABSTRACT FROM AUTHOR]

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme–albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins. [ABSTRACT FROM AUTHOR]

The effects of two anticancer active copper(II) mixed-ligand complexes of the type [Cu(qui)(mphen)]Y·H2O, where Hqui = 2-phenyl-3-hydroxy- 1H-quinolin-4-one, mphen = bathophenanthroline, and Y = NO3 (complex 1) or BF4 (complex 2) on the activities of different isoenzymes of cytochrome P450 (CYP) have been evaluated. The screening revealed significant inhibitory effects of the complexes on CYP3A4/5 (IC50 values were 2.46 and 4.88 μM), CYP2C9 (IC50 values were 16.34 and 37.25 μM), and CYP2C19 (IC50 values were 61.21 and 77.07 μM). Further, the analysis of mechanisms of action uncovered a non-competitive type of inhibition for both the studied compounds. Consequent studies of pharmacokinetic properties proved good stability of both the complexes in phosphate buffer saline (>96% stability) and human plasma (>91% stability) after 2 h of incubation. Both compounds are moderately metabolised by human liver microsomes (<30% after 1 h of incubation), and over 90% of the complexes bind to plasma proteins. The obtained results showed the potential of complexes 1 and 2 to interact with major metabolic pathways of drugs and, as a consequence of this finding, their apparent incompatibility in combination therapy with most chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c‐type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (∆mmcA‐rnf) or just the MmcA subunit (∆mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA is required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin oxidation that arise in each strain. Transcriptomic data suggest that the ∆mmcA mutant might oxidize ferredoxin by upregulating the cytosolic Wood‐Ljundahl pathway for acetyl‐CoA synthesis, whereas the ∆mmcA‐rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to oxidize ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of rnf or mmcA leads to global transcriptional changes of genes involved in methanogenesis, carbon assimilation and regulation. Overall, our study provides systems‐level insights into the non‐overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are the most important human drug metabolizing enzymes, but their mutual interactions are poorly understood. In this study, we recombinantly co-expressed of each one of the 19 human members of the UGT families 1 and 2 with either CYP2C9, CYP2D6, or CYP4Z1 in fission yeast. Using these strains, we monitored a total of 72 interactions: 57 cases where we tested the influence of UGT co-expression on CYP activity and 15 cases of the opposite approach. In the majority of cases (88%), UGT co-expression had a statistically significant (p < 0.05) effect on P450 activity (58% positive and 30% negative). Strong changes were observed in nine cases, including one case with an activity increase by a factor of 23 (CYP2C9 activity in the presence of UGT2A3) but also four cases with a complete loss of activity. When monitoring the effect of CYP co-expression on the activity of five UGTs, activity changes were generally not so pronounced and, if observed, always detrimental. UGT2B7 activity was not influenced by CYP co-expression, while the other UGTs were affected to varying degrees. These data suggest the notion that mutual influence of CYPs and UGTs on each other's activity is a widespread phenomenon. [ABSTRACT FROM AUTHOR]

Introduction: Most hepatically cleared drugs are metabolized by cytochromes P450 (CYPs), and Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines provide curated clinical references for CYPs to apply individual genome data for optimized drug therapy. However, incorporating novel pharmacogenetic variants into guidelines takes considerable time. Methods: We comprehensively assessed the drug metabolizing capabilities of CYP2C19 variants discovered through population sequencing of two substrates, S-mephenytoin and omeprazole. Results: Based on established functional assays, 75% (18/24) of the variants not yet described in Pharmacogene Variation (PharmVar) had significantly altered drug metabolizing capabilities. Of them, seven variants with inappreciable protein expression were evaluated as protein damaging by all three in silico prediction algorithms, Sorting intolerant from tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and Combined annotation dependent depletion (CADD). The five variants with decreased metabolic capability (<50%) of wild type for either substrates were evaluated as protein damaging by all three in silico prediction algorithms, except CADD exact score of NM_000769.4:c.593T>C that was 19.68 (<20.0). In the crystal structure of the five polymorphic proteins, each altered residue of all those proteins was observed to affect the key structures of drug binding specificity. We also identified polymorphic proteins indicating different tendencies of metabolic capability between the two substrates (5/24). Discussion: Therefore, we propose a methodology that combines in silico prediction algorithms and functional assays on polymorphic CYPs with multiple substrates to evaluate the changes in the metabolism of all possible genomic variants in CYP genes. The approach would reinforce existing guidelines and provide information for prescribing appropriate medicines for individual patients. [ABSTRACT FROM AUTHOR]

Cytochromes are enzymes of the dehydrogenase class with a hemoprotein structure in which the iron in these compounds undergoes oxidation and reduction reactions upon receiving or losing electrons. Quantitatively speaking, CYP3A4 is the most important isoenzyme of cytochrome P450, oxidizing foreign organic molecules such as drugs or toxins to cause them to leave the body. Many drugs and antibiotics can induce or inhibit the activity of cytochrome P450, including dexamethasone. Dexamethasone is a steroidal anti-inflammatory drug used to treat of inflammatory diseases and chronic autoimmunity. This study aimed to investigate the induction effect of dexamethasone in biotransformation pathways by in silico tools. Molegro Virtual Docker software was used to investigate the molecular docking of the enzyme and dexamethasone, which indicated the binding of the drug to the enzyme. The molecular simulation was performed in Linux with the GROMACS program. Root-mean-square distance (RMSD), and radius of gyration (Rg), were evaluated. The results were analyzed with Pymol and VMD software, and the obtained curve was plotted with GRACE software. Docking results show that a cluster with a bond energy of -60.81 was the best cluster, and the bond size between ligand and internal atoms was -23.191in the complex. In addition, the amount of bond between the ligand and water for this pose was zero. The stability of the enzyme-ligand complex and the induction effect of dexamethasone on CYP3A4 were indicated by RMSD and RG results. Results of RMSD and RG of CYP3A4 glucocorticoid obtained from the simulation showed the stability of binding of the drug to the enzyme. Also, RMSD results showed the stability of glucocorticoid and dexamethasone complex during molecular dynamics simulation. It reached relative stability at 0.8 nm after 80,000 ps until the end of the simulation. [ABSTRACT FROM AUTHOR]

I present here a personal perspective of the evolution of the two-light reaction two-pigment scheme for the electron transport in oxygenic photosynthesis – as I have lived through it – first as a graduate student of Robert Emerson, from September 1956–January 1959, and then of Eugene Rabinowitch from February 1959–September 1960. I have provided here some of the key published work in my way and have also provided a few photographs. It is essential to remind ourselves upfront that different individuals may have different recollections of the same event (see e.g., the Japanese movie ‘Rashomon’ https://en.wikipedia.org/wiki/Rashomon). Thus, I encourage others to write about their perspective. Further, I recognize, upfront, the support and encouragement I have received from Robert Blankenship for writing this perspective, and from Győző Garab for submission of this story to Photosynthetica. I begin my perspective with a dedication to Robert Emerson, whose pioneering work led to the concept of the two-light reaction two-pigment system in oxygenic photosynthesis. [ABSTRACT FROM AUTHOR]

Photosynthesis converts light energy to chemical energy to fuel life on earth. Light energy is harvested by antenna pigments and transferred to reaction centers (RCs) to drive the electron transfer (ET) reactions. Here, we present cryo-electron microscopy (cryo-EM) structures of two forms of the RC from the microaerophilic Chloracidobacterium thermophilum (CabRC): one containing 10 subunits, including two different cytochromes; and the other possessing two additional subunits, PscB and PscZ. The larger form contained 2 Zn-bacteriochlorophylls, 16 bacteriochlorophylls, 10 chlorophylls, 2 lycopenes, 2 hemes, 3 Fe4S4 clusters, 12 lipids, 2 Ca2+ ions and 6 water molecules, revealing a type I RC with an ET chain involving two hemes and a hybrid antenna containing bacteriochlorophylls and chlorophylls. Our results provide a structural basis for understanding the excitation energy and ET within the CabRC and offer evolutionary insights into the origin and adaptation of photosynthetic RCs. During photosynthesis, light energy is harvested by an antenna systems and delivered to the reaction centers (RCs) for charge separation and electron transfer (ET). The authors report cryo-EM structures of two forms of RC from the microaerophilic Chloracidobacterium thermophilum (CabRC), providing a structural basis for ET within the CabRC. [ABSTRACT FROM AUTHOR]

Cytochrome P450 is an enzyme involved in the metabolism of phase 1 xenobiotics, toxins, endogenous hormones, and drugs, including those used in COVID-19 treatment. Cytochrome p450 genes are linked to the pathogenesis of some multifactorial traits and diseases, such as cancer, particularly prostate cancer, colorectal cancer, breast cancer, and cervical cancer. Genotyping was performed on 540 supposedly healthy individuals of 5 Finno-Permic populations from the territories of the European part of the Russian Federation. There was a statistically significant difference between Veps and most of the studied populations in the rs4986774 locus of the CYP2D6 gene; data on the rs3892097 locus of the CYP2D6 gene shows that Izhemsky Komis are different from the Mordovian and Udmurt populations. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD) has gained widespread popularity; however, its pharmacological and toxicological profiles in the context of human genetic diversity remain largely unexplored. Here, we investigated the variability in metabolism and toxicity of CBD-rich cannabis extract (CRCE) in genetically diverse mouse models: C57BL/6J, B6C3F 1 /J, and NZO/HlLtJ strains. Mice received a single dose of CRCE containing 57.9% CBD at dosages of 0, 246, 738, and 2460 mg/kg of CBD. At 24 h after treatment, no appreciable histomorphological changes were detected in the liver. Plasma bilirubin levels increased markedly in all strains at the highest CBD dose. Mice in all treatment groups displayed significant but distinct increases in ALT and AST levels. While B6C3F 1 /J and NZO/HlLtJ mice had negligible plasma CBD levels at 738 mg/kg, C57BL/6J mice exhibited levels exceeding 7000 ng/mL. At 2460 mg/kg, high CBD concentrations were found in B6C3F 1 /J and C57BL/6J mice, but markedly lower levels were seen in NZO/HlLtJ mice. Gene expression profiling showed significant increases in Cyp2b10 across all strains but varying responses in Cyp1a1 expression, indicating strain-specific CYP dysregulation. Genetically diverse mice exhibited differential pharmacological and toxicological responses to CRCE, suggesting a high potential for inter-individual variability in the pharmacology and toxicology of CBD in humans. • Metabolism, pharmacology and toxicology of CRCE were studied in three mouse strains. • No histomorphological changes were found in the livers in any experimental group. • CRCE dosing led to altered clinical biochemistry in a strain-dependent manner. • Genetically diverse mice exhibited differential cytochrome induction patterns. • Inter-individual variability in humans in response to CBD must be considered. [ABSTRACT FROM AUTHOR]