Search Results (45)

NADPH–cytochrome P450 reductase (CPR) is an essential redox partner for a wide range of metal-containing proteins, mediating the stepwise transfer of two electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to the redox centers of its partner proteins. This Perspective summarizes recent advances in understanding the mechanisms underlying the CPR-mediated electron transfer (ET) cycle. Emphasis is placed on human and other mammalian CPRs, which provide critical insights into human biology and drug metabolism. Recent experimental and computational approaches that have deepened our mechanistic understanding of CPR function are highlighted. Selected studies are reviewed to illustrate progress in elucidating the interflavin ET within CPR, the interplay between its redox states and structural dynamics, and its protein–protein interactions with redox partners, along with the associated ET pathways. Finally, the remaining challenges and future research directions are outlined. Full article

The activity of cytochrome P450 enzymes decreases in older adults, which can lead to toxic effects from polypharmacy. Cytochromes P450 are the most significant enzymes involved in the metabolism of foreign compounds, including pharmaceutical substances. Vitamin B2, or riboflavin (RF), is a potent antioxidant that is vital for the body and participates in numerous enzyme-catalyzed redox reactions. RF is phosphorylated intracellularly to form flavin mononucleotide (FMN), which is further metabolized into flavin adenine dinucleotide (FAD). The active site of the NADPH-dependent cytochrome P450 reductase (CPR), a redox partner of CYP enzymes, is necessary for the catalytic functions of cytochromes P450. The active site of reductase is a complex formed by two types of vitamin B2, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). In our study, we investigated the impact of the phosphorylated form of vitamin B2, FAD, and FMN on the catalytic activity of cytochrome P450 2C9 (CYP2C9) towards non-steroidal anti-inflammatory medications diclofenac and naproxen. It was shown that FAD significantly enhanced the catalytic efficiency of CYP2C9. The 4-hydroxylation of diclofenac was enhanced by 148 ± 10%. The O-demethylation of naproxen showed an increase of 120 ± 14%. Based on these data, we can assume that intake of vitamin B2 (riboflavin) improves catalytic efficiency of CYP2C9. This finding is essential for the modulation of catalytic activity of CYP2C9. The proposed electroanalytic approach is a sensitive and robust method for drug metabolism assay. Full article

Vibrio parahaemolyticus is a major foodborne pathogen worldwide, responsible for seafood-associated poisoning. Among its toxin genes, tdh2 is the most critical. To investigate the role of tdh2 in V. parahaemolyticus under gastrointestinal conditions, we constructed tdh2 deletion and complementation strains and compared their survival under acid (pH 3 and 4) and bile stress (2%). The results showed that tdh2 expression was significantly upregulated under cold (4 °C) and bile stress (0.9%). Survival assays and PI staining revealed that the tdh2 mutant strain (VP: △tdh2) was more sensitive to acid and bile stress than the wild-type (WT), and this sensitivity was rescued by tdh2 complementation. These findings suggest that tdh2 plays a protective role in enhancing V. parahaemolyticus tolerance to acid and bile stress. In the VP: △tdh2 strain, seven genes were significantly upregulated and six were downregulated as a result of tdh2 deletion. These genes included VPA1332 (vtrA), VPA1348 (vtrB), VP2467 (ompU), VP0301 and VP1995 (ABC transporters), VP0527 (nhaR), and VP2553 (rpoS), among others. Additionally, LC-MS/MS analysis identified 12 differential metabolites between the WT and VP: △tdh2 strains, including phosphatidylserine (PS) (17:2 (9Z,12Z) /0:0 and 20:1 (11Z) /0:0), phosphatidylglycerol (PG) (17:0/0:0), flavin mononucleotide (FMN), and various nucleotides. The protective mechanism of tdh2 may involve preserving cell membrane permeability through regulation of ompU and ABC transporters and enhancing electron transfer efficiency via regulation of nhaR. The resulting reduction in ATP, DNA, and RNA synthesis—along with changes in membrane permeability and electron transfer due to decreased FMN—likely contributed to the reduced survival of the VP: △tdh2 strain. Meanwhile, the cells actively synthesized phospholipids to repair membrane damage, leading to increased levels of PS and PG. This study provides important insights into strategies for preventing and controlling food poisoning caused by tdh⁺ V. parahaemolyticus. Full article

Ex situ machine perfusion has emerged as a pivotal technique for organ preservation and pre-transplant viability assessment, where the real-time monitoring of mitochondrial biomarkers—flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH)—could significantly mitigate ischemia-reperfusion injury risks. This study develops a non-invasive optical method combining fluorescence and UV-visible spectrophotometry to quantify FMN and NADH in hypothermic oxygenated perfusion media. Calibration curves revealed linear responses for both biomarkers in absorption and fluorescence (FMN: λ_ex = 445 nm, λ_em = 530–540 nm; NADH: λ_ex = 340 nm, λ_em = 465 nm) at concentrations < 100 μg mL⁻¹. However, NADH exhibited nonlinear fluorescence above 100 μg mL⁻¹, requiring shifted excitation to 365 nm for reliable detection. Spectroscopic analysis further demonstrated how perfusion solution composition alters FMN/NADH fluorescence properties, with consistent reproducibility across media. The method’s robustness was validated through comparative studies in clinically relevant solutions, proposing a strategy for precise biomarker quantification without invasive sampling. These findings establish a foundation for real-time, optical biosensor development to enhance organ perfusion monitoring. By bridging spectroscopic principles with clinical needs, this work advances translational sensor technologies for transplant medicine, offering a template for future device integration. Full article

Background: The function and bioavailability of water-soluble vitamins in human milk (HM) is contingent upon their specific molecular configurations. This study aims to investigate the concentrations of different forms of thiamine, riboflavin, and folate in HM and to elucidate the temporal variations of these nutrients across different stages of lactation. Methods: A cohort of 35 healthy mother–infant pairs from Beijing was recruited, and 214 HM samples were collected. The concentrations of water-soluble vitamins in these samples were analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A mixed linear regression model was employed to examine the relationship between HM vitamin levels and lactation stages. Results: This study analyzed the concentrations of free thiamine, thiamine monophosphate (TMP), thiamine pyrophosphate (TPP), free riboflavin, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), 5-methyl-tetrahydrofolate (5-MTHF), tetrahydrofolate (THF), 5-formyl-tetrahydrofolate (5-fTHF), 5,10-methenyl-tetrahydrofolate (5,10-MTHF), and unmetabolized folic acid (UMFA) at various lactation stages (0–7 days, 15 days, 30 days, 60 days, 90 days, 120 days, 150 days, and 180 days). Free thiamine concentrations increased from colostrum to 180 days, while total thiamine rose during the first month and then stabilized. Free and total riboflavin levels remained relatively constant throughout lactation. Free and total folate concentrations peaked at 90 days and subsequently declined. Significant correlations were observed between follow-up time and changes in free thiamine, free folate, and total folate concentrations over 180 days. Conclusions: This study provides detailed data on the concentrations and trends of free and total thiamine, riboflavin, and folate in HM from 0 to 180 days postpartum, highlighting the dynamic nature of vitamin concentrations in HM. No deficiencies in these HM vitamins were detected in the surveyed population. Future further research will be conducted to reveal the correlation between different forms of water-soluble vitamins in HM and dietary factors. Full article

Background/Objectives: The rising use of liver grafts from donation after circulatory death (DCD) has been enabled by advances in normothermic regional perfusion (NRP) and machine perfusion (MP) technologies. We aimed to identify predictive biomarkers in DCD grafts subjected to NRP, followed by randomization to either normothermic machine perfusion (NMP) or dual hypothermic oxygenated perfusion (D-HOPE). Methods: Among 57 DCD donors, 32 liver grafts were transplanted, and recipients were monitored for one week post-transplant. Biomarkers linked with oxidative stress, hepatic injury, mitochondrial dysfunction, inflammation, regeneration, and autophagy were measured during NRP, end-ischemic MP, and one week post-transplant. Results: Arginase-1 (ARG-1) levels were consistently higher in discarded grafts and in recipients who later developed early allograft dysfunction (EAD). Specifically, ARG-1 levels at the end of MP correlated with markers of hepatic injury. Receiver operating characteristic analysis indicated that ARG-1 at the end of MP had a good predictive accuracy for EAD (AUC = 0.713; p = 0.02). Lipid peroxidation (TBARS) elevated at the start of NRP, declined over time, with higher levels in D-HOPE than in NMP, suggesting a more oxidative environment in D-HOPE. Metabolites like flavin mononucleotide (FMN) and NADH exhibited significant disparities between perfusion types, due to differences in perfusate compositions. Inflammatory biomarkers rose during NRP and NMP but normalized post-transplantation. Regenerative markers, including osteopontin and hepatocyte growth factor, increased during NRP and NMP and normalized post-transplant. Conclusions: ARG-1 demonstrates strong potential as an early biomarker for assessing liver graft viability during perfusion, supporting timely and effective decision-making in transplantation. Full article

X-ray-induced photodynamic therapy (X-PDT) represents a promising new method of cancer treatment. A novel type of nanoscintillator based on cerium fluoride (CeF₃) nanoparticles (NPs) modified with flavin mononucleotide (FMN) has been proposed. A method for synthesizing CeF₃-FMN NPs has been developed, enabling the production of colloidal, spherical NPs with an approximate diameter of 100 nm, low polydispersity, and a high fluorescence quantum yield of 0.42. It has been demonstrated that CeF₃-FMN NPs exhibit pH-dependent radiation-induced redox activity when exposed to X-rays. This activity results in the generation of reactive oxygen species, which is associated with the scintillation properties of cerium and the transfer of electrons to FMN. The synthesized NPs have been demonstrated to exhibit minimal cytotoxicity towards normal cells (NCTC L929 fibroblasts) but are more toxic to tumor cells (epidermoid carcinoma A431). Concurrently, the synthesized NPs (CeF₃ and CeF₃-FMN NPs) demonstrate a pronounced selective radiosensitizing effect on tumor cells at concentrations of 10⁻⁷ and 10⁻³ M, resulting in a significant reduction in their clonogenic activity, increasing radiosensitivity for cancer cells by 1.9 times following X-ray irradiation at a dose of 3 to 6 Gy. In the context of normal cells, these nanoparticles serve the function of antioxidants, maintaining a high level of clonogenic activity. Functional nanoscintillators on the basis of cerium fluoride can be used as part of the latest technologies for the treatment of tumors within the framework of X-PDT. Full article

Background and objectives: Riboflavin in the form flavin mononucleotide (FMN) acts as a cofactor for the pyridoxine phosphate oxidase required to generate pyridoxal 5′-phosphate (PLP), the active form of vitamin B6 in tissues. Few human studies have investigated this metabolic interaction between riboflavin and vitamin B6. The primary objective of this study was to examine the response of plasma PLP to riboflavin supplementation in individuals with the MTHFR 677TT genotype. A secondary objective was to consider whether the dose of riboflavin (1.6 mg/d vs. 10 mg/d) affects the PLP response. Methods: Data from four randomised controlled trials (RCTs) of riboflavin supplementation previously conducted at this centre were accessed to identify 209 participants of 19–60 years meeting the inclusion criteria (≤60 years, MTHFR 677TT genotype, not taking a vitamin B6 supplement). In the original RCTs, participants were randomly assigned to receive a placebo (n = 85) or 1.6 mg/d of riboflavin (n = 87) for 16 weeks. In one trial only, a higher riboflavin dose, 10 mg/d (n = 37), was administered. Plasma PLP was measured via reversed phase HPLC with fluorescence detection. Riboflavin status was assessed using the functional assay, erythrocyte glutathione reductase activation coefficient (EGRac). Results: riboflavin supplementation resulted in a decrease (p < 0.001) in the mean EGRac values, from 1.34 (1.32, 1.37) to 1.21 (1.19, 1.22). Correspondingly, PLP increased (p = 0.027), an effect driven by those with a sub-optimal riboflavin status at baseline (EGRac > 1.26), whereby PLP increased by 5.2 nmol/L, from 44.9 (40.3, 49.4) to 50.1 (44.6, 55.6) nmol/L (p = 0.042), while with the optimal baseline riboflavin (EGRac ≤ 1.26), there was no significant PLP response to the intervention. Although 10 mg/d vs. 1.6 mg/d of riboflavin resulted in a greater EGRac response (p = 0.012), there was no significant effect of riboflavin dose on the PLP response. Discussion: These results provide randomised trial evidence that optimising riboflavin status leads to an increase in plasma PLP, confirming the metabolic dependency of vitamin B6 on FMN. The findings indicate that riboflavin intake may need to be considered when setting dietary recommendations for vitamin B6 in adults. Further work is needed to explore the impact of the common MTHFR C677T polymorphism of the interrelationship of these B vitamins. Full article

Bacillus subtilis ferredoxin:NADP⁺ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be −0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH^• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures. Full article

5-Deazaflavins are analogs of naturally occurring flavin cofactors. They serve as substitutes for natural flavin cofactors to investigate and modify the reaction pathways of flavoproteins. Demethylated 5-deazaflavins are potential candidates for artificial cofactors, allowing us to fine-tune the reaction kinetics and absorption characteristics of flavoproteins. In this contribution, demethylated 5-deazariboflavin radicals are investigated (1) to assess the influence of the methyl groups on the electronic structure of the 5-deazaflavin radical and (2) to explore their photophysical properties with regard to their potential as artificial cofactors. We determined the proton hyperfine structure of demethylated 5-deazariboflavins using photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, as well as density functional theory (DFT). To provide context, we compare our findings to a study of flavin mononucleotide (FMN) derivatives. We found a significant influence of the methylation pattern on the absorption properties, as well as on the proton hyperfine coupling ratios of the xylene moiety, which appears to be solvent-dependent. This effect is enhanced by the replacement of N5 by C5-H in 5-deazaflavin derivatives compared to their respective flavin counterparts. Full article

Antibiotic resistance remains a pressing global concern, with most antibiotics targeting the bacterial ribosome or a limited range of proteins. One class of underexplored antibiotic targets is bacterial riboswitches, structured RNA elements that regulate key biosynthetic pathways by binding a specific ligand. We developed a methodology termed Fluorescent Ligand Equilibrium Displacement (FLED) to rapidly discover small molecules that bind the flavin mononucleotide (FMN) riboswitch. FLED leverages intrinsically fluorescent FMN and the quenching effect on RNA binding to create a label-free, in vitro method to identify compounds that can bind the apo population of riboswitch in a system at equilibrium. The response difference between known riboswitch ligands and controls demonstrates the robustness of the method for high-throughput screening. An existing drug discovery library that was screened using FLED resulted in a final hit rate of 0.67%. The concentration response of each hit was determined and revealed a variety of approximate effective concentration values. Our preliminary screening data support the use of FLED to identify small molecules for medicinal chemistry development as FMN riboswitch-targeted antibiotic compounds. This robust, label-free, and cell-free method offers a strong alternative to other riboswitch screening methods and can be adapted to a variety of laboratory setups. Full article

Post-translational modifications (PTMs) of proteins are paramount in health and disease. Phosphoproteome analysis by enrichment techniques is becoming increasingly attractive for biomedical research. Recent findings show co-enrichment of other phosphate-containing biologically relevant PTMs, but these results were obtained by closed searches focused on the modifications sought. Open searches are a breakthrough in high-throughput PTM analysis (OS-PTM), identifying practically all PTMs detectable by mass spectrometry, even unknown ones, with their modified sites, in a hypothesis-free and deep manner. Here we reanalyze liver phosphoproteome by OS-PTM, demonstrating its extremely complex nature. We found extensive Lys glycerophosphorylations (pgK), as well as modification with glycerylphosphorylethanolamine on Glu (gpetE) and flavin mononucleotide on His (fmnH). The functionality of these metabolite-derived PTMs is demonstrated during metabolic dysfunction-associated steatotic liver disease (MASLD) development in mice. MASLD elicits specific alterations in pgK, epgE and fmnH in the liver, mainly on glycolytic enzymes and mitochondrial proteins, suggesting an increase in glycolysis and mitochondrial ATP production from the early insulin-resistant stages. Thus, we show new possible mechanisms based on metabolite-derived PTMs leading to intrahepatic lipid accumulation during MASLD development and reinforce phosphoproteome enrichment as a valuable tool with which to study the functional implications of a variety of low-abundant phosphate-containing PTMs in cell physiology. Full article

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H⁺/Na⁺-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively. Full article

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes. Full article

Flavin mononucleotides (FMNs) and flavin adenine nucleotide (FAD) play vital roles in the electron-transfer processes in diverse enzymatic reactions. Owing to the isoalloxazine chromophore, flavins are easily detectable by surface-enhanced Raman spectroscopy (SERS), a surface-sensitive technique. However, the details of the adsorption of flavins on SERS-active materials have never been investigated. In this study, a comprehensive SERS analysis of flavins containing lumichrome and lumiflavin on silver nanoparticles was conducted. With the aid of density-functional-theory calculations, our results suggested that the flavin molecules were adsorbed on the silver nanoparticles via the N₃ site of the isoalloxazine moiety, which had a stronger adsorption ability than the adenine moiety in the FAD. The SERS spectra of the flavins at different pH values also supported this conclusion. This study demonstrated the feasibility of SERS for the structural characterization of flavins, paving the way for the functional exploration of flavin-labeled detection sensors and flavoprotein researches. Full article