Slow Turnover Research Articles

Labile, recalcitrant and stable soil organic carbon: comparison of agronomic management in a vineyard of Trentino (Italy)

The intensive exploitation of agricultural land has caused a depletion of soil organic carbon (SOC) and a decline in soil fertility, with a consequent decrease in the productivity of agroecosystems, also contributing to atmospheric GHG emissions. The ability of soil to storage organic carbon (OC) depends on its persistence and susceptibility to decomposition. SOC is generally partitioned into fractions differing in protection mechanisms and in turn decomposability. This study aimed to compare the OC pool repartition in soils managed with three different fertilization practices (mineral – CONV, manure – ORGM and green manure – ORG-GM) during a medium-term trial in vineyard, in order to understand the best solution in increasing C sequestration. The OC fractionation by acid hydrolysis allowed separating three fractions of OC: a labile, quickly mineralizable pool, a recalcitrant pool with a slower turnover and a stable pool protected by microbial attack. The results showed that the periodic application of organic matrices (manure and green manure), although did not increase total OC, enriched the soil along time with readily available OC, thus promoting the release of nutrients. Green manure has also been shown to promote the accumulation of stabilized OC, able to improve the structure of the soil and, therefore, its fertility.

Diversity of reptile sex chromosome evolution revealed by cytogenetic and linked-read sequencing.

Reptile sex determination is attracting much attention because the great diversity of sex-determination and dosage compensation mechanisms permits us to approach fundamental questions about mechanisms of sex chromosome turnover. Recent studies have made significant progress in better understanding diversity and conservation of reptile sex chromosomes, with however no reptile master sex determination genes identified. Here we describe an integrated genomics and cytogenetics pipeline, combining probes generated from the microdissected sex chromosomes with transcriptome and genome sequencing to explore the sex chromosome diversity in non-model Australian reptiles. We tested our pipeline on a turtle, two species of geckos, and a monitor lizard. Genes identified on sex chromosomes were compared to the chicken genome to identify homologous regions among the four species. We identified candidate sex determining genes within these regions, including conserved vertebrate sex-determining genes pdgfa, pdgfra amh and wt1, and demonstrated their testis or ovary-specific expression. All four species showed gene-by-gene rather than chromosome-wide dosage compensation. Our results imply that reptile sex chromosomes originated by independent acquisition of sex-determining genes on different autosomes, as well as translocations between different ancestral macro- and microchromosomes. We discuss the evolutionary drivers of the slow differentiation and turnover of reptile sex chromosomes.

Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins.

Here, we present a detailed protocol for fluorescence recovery after photobleaching (FRAP) to measure the dynamics of junctional populations of proteins in living tissue. Specifically, we describe how to perform FRAP in Drosophila pupal wings on fluorescently tagged core planar polarity proteins, which exhibit relatively slow junctional turnover. We provide a step-by-step practical guide to performing FRAP, and list a series of controls and optimizations to do before conducting a FRAP experiment. Finally, we describe how to present the FRAP data for publication.

D2O labelling reveals synthesis of small, water-soluble metabolites in soil

Small, water-soluble metabolites (e.g. amino acids, sugars) occupy central positions in biogeochemical cycling, yet for most metabolites we do not know rates of de novo synthesis. The aim of this study was to estimate synthesis of water-soluble metabolites by developing a workflow based on D2O labelling and mass spectrometry quantification of 2H-enrichment. Initial tests established that labelling up to 87 atom% soil water with D2O did not affect soil respiration, suggesting that the smaller (typically ≤60 atom% D2O) labelling of most experiments was unlikely to affect microbial activity. To examine 2H incorporation from D2O, four contrasting soils were spiked with 99.9% D2O leading to 46–61 atom% water present as D2O. Soils were incubated from ten to 240 days before extraction, then isotopologues of a broad range of water-soluble metabolites were quantified by gas chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. There were dramatic differences among metabolites in the rate of 2H-enrichment. Mannitol, Asp, Asn, Glu, Gln, approached the theoretical maximum 2H enrichment after ten days, indicating that turnover of the metabolite pool due to synthesis occurred at a time scale of around ten days; whereas muramic acid was less than 20% of maximum 2H enrichment after 240 days suggesting turnover due to de novo synthesis was likely on the time scale of several years. Previous studies of the same and similar water-soluble metabolites reported rapid metabolite fluxes into and out of the soil solution led to residence time in solution of 1 h or less. The slow turnover of metabolite pools due to synthesis indicates the fluxes of metabolites in and out of cells is dominated by community-level recycling.

Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage.

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans—decorin and biglycan—and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue’s ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies –Eteplirsen, Golodirsen, Viltolarsen, and Casimersen –for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Reawakening the Intrinsic Cardiac Regenerative Potential: Molecular Strategies to Boost Dedifferentiation and Proliferation of Endogenous Cardiomyocytes.

Despite considerable efforts carried out to develop stem/progenitor cell-based technologies aiming at replacing and restoring the cardiac tissue following severe damages, thus far no strategies based on adult stem cell transplantation have been demonstrated to efficiently generate new cardiac muscle cells. Intriguingly, dedifferentiation, and proliferation of pre-existing cardiomyocytes and not stem cell differentiation represent the preponderant cellular mechanism by which lower vertebrates spontaneously regenerate the injured heart. Mammals can also regenerate their heart up to the early neonatal period, even in this case by activating the proliferation of endogenous cardiomyocytes. However, the mammalian cardiac regenerative potential is dramatically reduced soon after birth, when most cardiomyocytes exit from the cell cycle, undergo further maturation, and continue to grow in size. Although a slow rate of cardiomyocyte turnover has also been documented in adult mammals, both in mice and humans, this is not enough to sustain a robust regenerative process. Nevertheless, these remarkable findings opened the door to a branch of novel regenerative approaches aiming at reactivating the endogenous cardiac regenerative potential by triggering a partial dedifferentiation process and cell cycle re-entry in endogenous cardiomyocytes. Several adaptations from intrauterine to extrauterine life starting at birth and continuing in the immediate neonatal period concur to the loss of the mammalian cardiac regenerative ability. A wide range of systemic and microenvironmental factors or cell-intrinsic molecular players proved to regulate cardiomyocyte proliferation and their manipulation has been explored as a therapeutic strategy to boost cardiac function after injuries. We here review the scientific knowledge gained thus far in this novel and flourishing field of research, elucidating the key biological and molecular mechanisms whose modulation may represent a viable approach for regenerating the human damaged myocardium.

Biokinetic assessment of the nitrogen transformation cycle and turnover of nitrogen pools in the soil–plant system of Asian Russia: experience and results

Given research presents the results of long-term experiments on the cycle of nitrogen transformation with the application of a stable nitrogen isotope (15N). Its modeling is a renewed attempt to make a biokinetic hypothesis of the nitrogen transformation cycle in the Asian part of Russia. The fundamental concept “kinetics” is used in the form of classical exponential equations to evaluate the velocity characteristics of the cycle and turnover of nitrogen pools. The differences in kinetics of the easy, stable and slow turnover rate of nitrogen pools in terms of the velocity constants (k) served as the criteria for the formation of the nitrogen biokinetic cycle. The exponential character of the nitrogen transformation cycle indicates a high sensitivity of the turnover of nitrogen pools in harsh cryogenic conditions. The response of the system is characterized by the excitation processes of the mineral nitrogen pools’ formation (k = 0.867 – 0.666 day−1), absorption (k = 0.637 – 0.374 day−1) and immobilization (k = 0.449 – 0.105 day). The results show a new possibility of estimating the nitrogen cycle by calculating the velocity constant (k). This assessment enables to formulate a new original hypothesis as a theoretical kinetic model of the nitrogen cycle and the turnover of nitrogen pools.

Financial Analysis of SUNING.COM in China Based on the Harvard Analysis Framework

The number of internet users in China continues to rise and the country has begun promoting e-commerce development since 2011. Various supportive policies have created an ideal environment for the development of e-commerce. Under their combined effect, Chinese e-commerce market has developed rapidly. The COVID-19 pandemic’s impact has compelled retail enterprises that insist on operating offline chain stores to realize the defects of the traditional model and the need to transform to the e-commerce model. SUNING.COM is an enterprise that started its transition to e-commerce earlier than its competitors. It has made certain achievements in e-commerce but also exposed many problems. This paper takes SUNING.COM as the research object, analyzes its problems, proposes countermeasures, and provides a certain reference significance for the development of e-commerce enterprises. Considering the limitations of traditional financial analysis methods, this study adopts the more systematic and rigorous Harvard analysis framework to carry out a detailed analysis from four aspects: strategy, accounting, finance, and prospect. The analysis finds that SUNING.COM has both opportunities and risks, slow inventory turnover, high operational risks, poor solvency, and insufficient profitability. In response to the problem, the suggestions are as follows: First, SUNING.COM should give full play to the advantages of its offline retail stores, build a community-based e-commerce platform, and reshape its own brand image. Second, they should speed up the implementation of corporate projects and accelerate the release of restricted funds. Third, they must optimize the main business structure, eliminate non-essential businesses, and improve the online and offline dual line business model. Fourth, they must optimize the debt structure of enterprises, reduce current liabilities, and reduce the risk of bad debts.

Self-renewal of peripheral nerve resident macrophage: does it represent a unique activation status?

Self-renewal of peripheral nerve resident macrophage: does it represent a unique activation status?

Abstract MP230: Nuclear Rupture In A Mouse Model Of Lmna -related Cardiomyopathy Causes Cytoplasmic Exposure Of The Proinflammatory Signaling Protein Hmgb1

Cardiomyopathies caused by mutations in LMNA, encoding nuclear Lamin A/C, are highly malignant and prevalent. How LMNA mutations cause cardiomyopathies remains unknown. We characterized cellular, molecular, and pathological evolution of mouse models of LMNA -related cardiomyopathy and provide evidence for a model in which nuclear rupture generates nuclear-localized proinflammatory signaling as a candidate molecular mechanism underlying disease pathogenesis. We observed that cardiomyocyte-specific, tamoxifen-inducible deletion of Lmna in adult mice ( Lmna CMKO ) caused a gradual reduction of Lamin A/C protein at the nuclear lamina, reflecting the slow turnover of Lamin A/C. A modest reduction of Lamin A/C in Lmna CMKO was sufficient to cause extensive fibrosis, reduced ejection fraction, and chamber dilation by 3 weeks after Lmna gene deletion. Lmna CMKO cardiomyocytes exhibited localized rupture of the nuclear envelope 2 weeks prior to the development of fibrosis and reduction of ejection fraction. Nuclear rupture in Lmna CMKO was immediately followed by an extensive upregulation of pro-inflammatory gene expression programs. We hypothesized that nuclear rupture might expose nuclear DNA to the cytoplasm thereby activating the pro-inflammatory cGas-STING cytosolic DNA sensing pathway. However, we did not observe localization of the cytosolic DNA sensor cGas to cytoplasmic DNA protruded from the ruptured nuclei in Lmna CMKO cardiomyocytes. Instead, we found that HMGB1, a potent proinflammatory protein normally sequestered in the nucleus, was released from the ruptured nuclei in Lmna CMKO cardiomyocytes. Mass spectrometry identified a strong interaction between Lamin A/C and HMGB1 in normal human fibroblast cells. Our data suggested that Lamin A/C tethers HMGB1 to the nuclear periphery by direct interaction and that reduction of Lamin A/C unleashes HMGB1 to the cytoplasm upon nuclear rupture. Future work will examine the hypothesis that cytoplasmic HMGB1 triggers pathogenic sterile inflammation leading to dilated cardiomyopathies in Lmna CMKO mice. In conclusion, we identified the nuclear rupture-induced cytoplasmic release of HMGB1 as a candidate mechanism underlying LMNA -related cardiomyopathies.

Brick by brick: Governing industry decarbonization in the face of uncertainty and risk

Basic materials such as steel and cement have been instrumental to modern society, but the production requires high energy inputs and is associated with significant CO2 emissions. Long investment cycles, slow turnover rate of the capital stock, and high capital intensity mean that basic materials industries are among the economic activities that will be the most difficult to decarbonize. Although the importance of addressing industrial emissions has been increasingly recognized, the development of measures and technologies that could deliver deeper emissions cuts has been relatively slow. In this paper, we present a conceptual framework focusing on four categories of barriers—market, technology, regulatory, and coordination—that hamper deep decarbonization in the basic materials industries. We use the Swedish basic materials industries and policy context to illustrate how the proposed framework can be understood and applied and broadly discuss policies that can facilitate the climate transition.

Senescent Progenitor Cells in the Skin of Patients with Cutaneous Lupus Erythematosus

Senescent Progenitor Cells in the Skin of Patients with Cutaneous Lupus Erythematosus

Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges.

The CNS is ensheathed by the meninges and cerebrospinal fluid, and recent findings suggest that these CNS-associated border tissues have complex immunological functions. Unlike myeloid lineage cells, lymphocytes in border compartments have yet to be thoroughly characterized. Based on single-cell transcriptomics, we here identified a highly location-specific composition and expression profile of tissue-resident leukocytes in CNS parenchyma, pia-enriched subdural meninges, dura mater, choroid plexus and cerebrospinal fluid. The dura layer of the meninges contained a large population of B cells under homeostatic conditions in mice and rats. Murine dura B cells exhibited slow turnover and long-term tissue residency, and they matured in experimental neuroinflammation. The dura also contained B lineage progenitors at the pro-B cell stage typically not found outside of bone marrow, without direct influx from the periphery or the skull bone marrow. This identified the dura as an unexpected site of B cell residence and potentially of development in both homeostasis and neuroinflammation.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

SUMMARYE-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1’s formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

ADF and cofilin-1 collaborate to promote cortical actin flow and the leader bleb-based migration of confined cells.

Melanoma cells have been shown to undergo fast amoeboid (leader bleb-based) migration, requiring a single large bleb for migration. In leader blebs, is a rapid flow of cortical actin that drives the cell forward. Using RNAi, we find that co-depleting cofilin-1 and actin depolymerizing factor (ADF) led to a large increase in cortical actin, suggesting that both proteins regulate cortical actin. Furthermore, severing factors can promote contractility through the regulation of actin architecture. However, RNAi of cofilin-1 but not ADF led to a significant decrease in cell stiffness. We found cofilin-1 to be enriched at leader bleb necks, whereas RNAi of cofilin-1 and ADF reduced bleb sizes and the frequency of motile cells. Strikingly, cells without cofilin-1 and ADF had blebs with abnormally long necks. Many of these blebs failed to retract and displayed slow actin turnover. Collectively, our data identifies cofilin-1 and ADF as actin remodeling factors required for fast amoeboid migration.

NMDA Receptor Hypofunction in the Aging-Associated Malfunction of Peripheral Tissue.

Glutamatergic transmission through NMDA receptors (NMDARs) is important for the function of peripheral tissues. In the bone, NMDARs and its co-agonist, D-serine participate in all the phases of the remodeling. In the vasculature, NMDARs exerts a tonic vasodilation decreasing blood perfusion in the corpus cavernosum and the filtration rate in the renal glomerulus. NMDARs are relevant for the skin turnover regulating the proliferation and differentiation of keratinocytes and the formation of the cornified envelope (CE). The interference with NMDAR function in the skin leads to a slow turnover and repair. As occurs with the brain and cognitive functions, the manifestations of a hypofunction of NMDARs resembles those observed during aging. This raises the question if the deterioration of the glomerular vasculature, the bone remodeling and the skin turnover associated with age could be related with a hypofunction of NMDARs. Furthermore, the interference of D-serine and the effects of its supplementation on these tissues, suggest that a decrease of D-serine could account for this hypofunction pointing out D-serine as a potential therapeutic target to reduce or even prevent the detriment of the peripheral tissue associated with aging.

Novel factors that activate and deactivate cardiac fibroblasts: A new perspective for treatment of cardiac fibrosis.

Heart disease with attendant cardiac fibrosis kills more patients in developed countries than any other disease, including cancer. We highlight the recent literature on factors that activate and also deactivate cardiac fibroblasts. Activation of cardiac fibroblasts results in myofibroblasts phenotype which incorporates aSMA to stress fibres, express ED-A fibronectin, elevated PDGFRα and are hypersecretory ECM components. These cells facilitate both acute wound healing (infarct site) and chronic cardiac fibrosis. Quiescent fibroblasts are associated with normal myocardial tissue and provide relatively slow turnover of the ECM. Deactivation of activated myofibroblasts is a much less studied phenomenon. In this context, SKI is a known negative regulator of TGFb1 /Smad signalling, and thus may share functional similarity to PPARγ activation. The discovery of SKI's potent anti-fibrotic role, and its ability to deactivate and/or myofibroblasts is featured and contrasted with PPARγ. While myofibroblasts are typically recruited from pools of potential precursor cells in a variety of organs, the importance of activation of resident cardiac fibroblasts has been recently emphasised. Myofibroblasts deposit ECM components at an elevated rate and contribute to both systolic and diastolic dysfunction with attendant cardiac fibrosis. A major knowledge gap exists as to specific proteins that may signal for fibroblast deactivation. As SKI may be a functionally pluripotent protein, we suggest that it serves as a scaffold to proteins other than R-Smads and associated Smad signal proteins, and thus its anti-fibrotic effects may extend beyond binding R-Smads. While cardiac fibrosis is causal to heart failure, the treatment of cardiac fibrosis is hampered by the lack of availability of effective pharmacological anti-fibrotic agents. The current review will provide an overview of work highlighting novel factors which cause fibroblast activation and deactivation to underscore putative therapeutic avenues for improving disease outcomes in cardiac patients with fibrosed hearts.

Soil extracellular oxidases mediated nitrogen fertilization effects on soil organic carbon sequestration in bioenergy croplands

AbstractNitrogen (N) fertilization significantly affects soil extracellular oxidases, agents responsible for decomposition of slow turnover and recalcitrant soil organic carbon (SOC; e.g., lignin), and consequently influences soil carbon sequestration capacity. However, it remains unclear how soil oxidases mediate SOC sequestration under N fertilization, and whether these effects co‐vary with plant type (e.g., bioenergy crop species). Using a spatially explicit design and intensive soil sampling strategy under three fertilization treatments in switchgrass (SG: Panicum virgatum L.) and gamagrass (GG: Tripsacum dactyloides L.) croplands, we quantified the activities of polyphenolic oxidase (PHO), peroxidase (PER), and their sum associated with recalcitrant C acquisition (OX). The fertilization treatments included no N fertilizer input (NN), low N input (LN: 84 kg N ha−1 year−1 in urea), and high N input (HN: 168 kg N ha−1 year−1 in urea). Besides correlations between soil oxidases and SOC (formerly published), both descriptive and geostatistical approaches were applied to evaluate the effects of N fertilization and crop type on soil oxidases activities and their spatial distributions. Results showed significantly negative correlations between soil oxidase activities and SOC across all treatments. The negative relationship of soil oxidases and SOC was also evident under N fertilization. First, LN significantly depressed oxidases in both mean activities and spatial heterogeneity, which corresponded to increased SOC in SG (though by 5.4%). LN slightly influenced oxidases activities and their spatial heterogeneity, consistent with insignificant changes of SOC in GG. Second, HN showed trends of decrease in soil oxidase activities, which aligned with the significantly enhanced SOC in both croplands. Overall, this study demonstrated that soil oxidase activities acted as sensitive and negative mediators of SOC sequestration in bioenergy croplands and optimizing fertilizer use particularly in switchgrass cropland can improve for both carbon sequestration and environmental benefit.

Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase.

The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 Å that are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.