Endometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression.Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins.2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC.Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (logC1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.The online version contains supplementary material available at 10.1007/s13402-023-00778-w. Endometrial cancer (EC) is the most common cancer of female reproductive organs, in which cancerous cells lining the uterus might invade the neighboring myometrium, or metastasize to lymph nodes, or distal organs, mainly vagina, ovaries, and lungs . EndometGlycosylation is one of the most common post-translational modifications of proteins . Since gForward and Reverse SILAC experiments were performed for the identification of changes in the cell extract and secretome proteomes associated to the depletion of C1GALT1 in EC. In total, 5208 and 3616 proteins were identified and quantified in the cell extract and secretome proteomes by LC-MS/MS, respectively. Of them, 100 and 144 proteins showed dysregulation with a log2fold-change ≥ 0.58 or ≤-0.58 in C1GALT1 stably depleted ECC-1 cells’ extracts and secretomes, respectively. Dysregulation of selected proteins was validated by PCR, real-time quantitative PCR (qPCR), western blot (WB), dot blot and immunofluorescence (IF) using cell extracts, secretome and exosomes from the ECC-1 cell lines. Moreover, loss-of-function cell-based assays confirmed the observed association of low C1GALT1 expression with an aggressive phenotype in EC by bioinformatics, demonstrating the usefulness of the cell model mimicking an aggressive phenotype of EC. In addition, ANXA1 silencing reversed partially the aggressive phenotype of shC1GALT1 ECC-1 cells. Finally, the association of LGALS3  dysregulation to C1GALT1 expression levels and EC grade was confirmed by IHC.In this study, we have shown by immunohistochemistry (IHC) that the more aggressive ECs showed low protein expression levels of C1GALT1. These results were confirmed at transcriptomic and proteomic level using the CPTAC data. Thus, we aimed to analyze the effect of C1GALT1 depletion in EC by quantitative proteomics. To this end, ECC-1 cells were used as a model of endometrioid EC, and the effect of C1GALT1 depletion on these cells using short hairpin RNAs (shRNA) was analyzed by SILAC quantitative proteomics analyses. 79 patients diagnosed and surgically treated at University Hospital La Paz between 2003 and 2015 were included in the present study. Formalin fixed paraffin embedded (FFPE) tissue blocks from radical hysterectomy specimens were reviewed by an expert pathologist. From each tissue block 1 to 4 cores from viable tissue areas were obtained to build tissue microarrays (TMA) -L-arginine and [12C6]-L-lysine  or DMEM [13C6]-L-arginine and -L-lysine  supplemented with 10% dialyzed FBS (GeminiBio). Both cell lines were labeled with heavy and light mediums to perform label swapping SILAC forward and reverse experiments as biological and technical replicates, as previously described [6 labelled cells were seeded in 25 cm2 cell culture flasks  and incubated at 37ºC and 5% CO2 in R0K0 or R6K4 supplemented with 10% dialyzed FBS until 90% confluence. Then, cells were washed 3 times with PBS 1x and incubated 1 h with R0K0 or R6K4 medium without FBS to remove all traces of FBS. Next, cells were washed 3 times with PBS 1x and incubated 48 h 37ºC and 5% CO2 with 5 mL of R0K0 or R6K4 DMEM-free serum. Then, conditioned media (secretomes) were collected and centrifuged 5 min at 1200 rpm and RT to remove cell debris. In addition, cells were harvested with 4 mM EDTA-PBS, centrifuged 5 min at 1200 rpm and lysed with RIPA buffer to analyze the total cell extracts.SCRAMBLE and shC1GALT1 ECC-1 cells were grown in 10% FBS DMEM at 37ºC and 5% COescribed . After 8forward and reverse experiments were analyzed. The forward experiment analyzed R6K4 shC1GALT1 ECC-1 cells and R0K0 SCRAMBLE ECC-1 cells, whereas the reverse experiment compared R0K0 shC1GALT1 ECC-1 cells and R6K4 SCRAMBLE ECC-1 R6K4, as biological and technical replicates.Secretomes and cell extracts were separately analyzed. For each proteome analysis, two SILAC 2O Milli-Q, TFA 0.1%, applied to the columns and peptides fractionated according to the manufacturer’s instructions. In total, twelve fractions were obtained for each SILAC experiment, directly dried under vacuum and stored at -80ºC until analysis in LC-MS/MS runs using a Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).For SILAC experiments, 40 µg of each protein extract were pooled together, methanol-chloroform precipitated and the 80 µg of proteins resuspended in 100 µL of 0.1 M TEAB. Then, proteins were reduced with 10 µL 100 mM tris(2-carboxyethyl)phosphine  during 45 min at 37ºC and 600 rpm and alkylated with 11 µL of 0.4 M chloroacetamide (Sigma Aldrich) during 30 min at room temperature (RT) in darkness. Next, proteins were digested in-solution with 3.2 µg of porcine trypsin (Thermo Fisher Scientific) in 20 mM HEPES pH 8.0 at 37ºC and 600 rpm during 14 h. The day after, samples were dried under vacuum prior to separation using High pH Reversed-Phase Peptide Fractionation Kit (Pierce). Desiccated samples were reconstituted in 300 µl HPeptide separations were carried out on an Easy-nLC 1200 nano system (Thermo Fisher Scientific). For each analysis, samples were loaded into a precolumn Acclaim PepMap 100 (Thermo Fisher Scientific) and eluted in a RSLC PepMap C18, 15 cm long, 50 μm inner diameter and 1.9 μm particle size (Thermo Fisher Scientific). The mobile phase flow rate was 300 nl/min using 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient profile was set as follows: 0-35% solvent B for 124 min, 35-90% solvent B for 4 min, 20% solvent B for 17 min. Four microliters of each sample were injected.For ionization, 1800 V of liquid junction voltage and 275 °C capillary temperature was used. The full scan method employed a m/z 350–1700 mass selection, an Orbitrap resolution of 70,000 (at m/z 200), a target automatic gain control (AGC) value of 3e6, and maximum injection times of 100 ms. After the survey scan, the 12 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 27 and MS/MS scans were acquired with a starting mass of m/z 100, AGC target was 2e5, resolution of 17,500 (at m/z 200), intensity threshold of 8e3, isolation window of 2 m/z units and maximum IT was 100 ms. Charge state screening was enabled to reject unassigned, singly charged, and greater than or equal to seven protonated ions. A dynamic exclusion time of 20 s was used to discriminate against previously selected ions.MS data were analyzed with MaxQuant (version 1.6.6.0) using standardized workflows. Mass spectra *.raw files were searched against Uniprot UP000005640_9606.fasta Homo sapiens (human) 2019 database  using standard type. Trypsin/P was specified as cleavage enzyme, allowing a mass tolerance of 20 ppm (Orbitrap). Precursor and reporter mass tolerance were set to 4.5 ppm and 0.003 Da, respectively, allowing 2 missed tryptic cleavages. Carbamidomethylation of cysteines was set as a fixed modification, and methionine oxidation, N-terminal acetylation and Ser, Thr and Tyr phosphorylation were set as variable modifications. Unique and Razor peptides were considered for quantification. Minimal peptide length and maximal peptide mass were fixed to 7 amino acids and 4600 Da, respectively. Identified peptides were filtered by their precursor intensity fraction with a FDR threshold of 0.01. Proteins identified with at least one peptide and an ion score above 99% were considered for evaluation, whereas proteins identified as potential contaminants were excluded from the analysis.2fold-change ≥ 0.58 or ≤-0.58 were selected as dysregulated proteins by the depletion of C1GALT1 in ECC-1 cells . The fold change cutoff for dysregulated proteins was calculated using a permutation-based test [Proteins identified and quantified with one or more peptides and a logsed test , as prevsed test –35.2 cell culture flasks  were seeded per cell line and cells were grown until 90% confluence at 37ºC and under 5% CO2. To avoid exogenous exosome contamination, cells were washed with PBS, incubated for 1 h in FBS-free DMEM, washed with PBS, and incubated with 20 mL of DMEM without FBS for 48 h at 37ºC under 5% CO2. Then, 100 mL of conditioned medium per cell line were centrifuged at 500 g for 5 min at 4ºC to remove the cell debris (pellet) and then centrifuged again at 2000 g for 10 min at 4ºC to remove vesicles greater than 1 μm (pellet). Next, exosomes were purified by differential centrifugation  [Extracellular vesicles released by endometrial ECC-1 cells were isolated via the differential centrifugation of the conditioned media (secretome) . In brie K, USA) . Exosome K, USA) . Exosomeg and 4ºC and the upper phase, containing the RNA, transferred to a new tube. RNA samples were purified using the RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. Purified total RNA was quantified with the Nanodrop 2000 C (Thermo Fisher).For RNA extraction, cell pellets from SCRAMBLE and shC1GALT1 ECC-1 cells were incubated with 500 µL of NZYol (NZYtech) during 5 min at RT for cell disaggregation, incubated with 100 µL of chloroform (Sigma Aldrich) during 3 min at RT and centrifuged 15 min at 12,000 cDNA was obtained from 1 µg of total RNA using the NZY First-Strand cDNA Synthesis Kit (NZYtech) following manufacturer’s instructions, and directly used for the quantification of mRNA levels of selected genes after C1GALT1 depletion. PCR analysis was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and the corresponding specific oligonucleotides  supplemented with 1x protease and phosphatase inhibitors (MedChemExpress) using 16G and 18G needle syringes until homogeneity. Then, samples were centrifuged at 10,000 g and 4ºC during 10 min and protein extracts (supernatants) collected and stored at -80ºC until use. In addition, secretome proteins were methanol-chloroform precipitated and re-suspended in lysis buffer supplemented with 1x protease and phosphatase inhibitors (MedChemExpress) prior to its use.Protein concentration was determined by Trp quantification method . ProteinFor western blot (WB) analysis, 10–15 µg of each protein extract were separated on 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes at 100 V during 90 min. 40–60 µg of secretome proteins in 100 µL PBS were dot blotted onto nitrocellulose membranes using the Bio-Dot 96-Well Microfiltration (Bio-Rad).Then, membranes were blocked with 0.1% Tween PBS 1x containing 3% skimmed milk (blocking buffer) during 1 h at RT and incubated with primary antibodies at optimized dilutions  and 1 × 103 cells per well were seeded in 96-well plates (Corning) in 10% FBS DMEM in quadruplicated. The day after, growth medium was removed and 100 µL of 5% FBS DMEM was added to each well, and incubated during 72 h at 37ºC and 5% CO2. Then, cells were incubated at 37ºC and 5% CO2 for 1 h with 1 mg/mL MTT solution in DMEM. Finally, MTT solution was removed; cells were washed with PBS 1x and lysed with 50 µL of 100% DMSO (Merck). Subsequently absorbance was read at 570 nm using a Spark multimode microplate (TECAN).Cell-based assays of stably depleted C1GALT1 ECC-1 cells in comparison to SCRAMBLE ECC-1 cells were performed as previously described –40. In b2. 96-well plates (Corning) coated with 100 µL 0.1 M NaHCO3 containing Matrigel matrix  were incubated overnight (O/N) at 4ºC. Then, 96-well plates were blocked with 200 µL of sterile DMEM 0.5% BSA (adhesion medium) during 2 h at 37ºC and ECC-1 cells separately stained with 1 mg/mL BCEBF (Sigma) diluted 1:100 in DMEM during 30 min at 37ºC and 5% CO2. Next, cells were harvested with 4 mM EDTA-PBS, and 1 × 105 cells in 50 µL of adhesion medium were transferred to each pre-coated well with Matrigel in quadruplicate. Cells were incubated for 2 h at 37ºC and 5% CO2, and, then wells were washed twice with 100 µL PBS 1x to remove non-attached cells. Finally, attached cells were lysed with 50 µL of 10% SDS in PBS, and fluorescence signal read at 436-535 nm excitation-emission, respectively, with the Spark multimode microplate (TECAN).For cell adhesive properties analysis, ECC-1 cells were incubated in DMEM free serum during 24 h at 37ºC and 5% CO6 cells were re-suspended in 100 µL sterile adhesion medium and transferred to pre-coated transwells, in duplicate. As chemoattractant, 700 µL of 10% FBS DMEM was used. Then, cells were incubated during 22 h at 37ºC and 5% CO2. Non-invading cells and Matrigel were removed from the upper surface of the transwells’ membrane, and invasive cells on the lower membrane were fixed with 1 mL of 4% paraformaldehyde for 1 h at RT. Finally, cells were stained with 1 mL of 0.2% crystal violet 25% methanol during 30 min at RT, washed several times with Milli-Q H2O (H2Omq) to remove dye traces, photographed with the DMi1 Microscope (Leica) and counted with ImageJ program (Fiji).For invasion analyses, 6.5 mm transwells with 8.0 μm Pore Polycarbonate Membrane Inserts (Corning) were placed onto 24-well plates (Corning) and coated with 50 µL of Matrigel matrix diluted in 1:3 in DMEM and incubated at 37ºC for 1 h. ECC-1 cells were harvested with Trypsin-EDTA (Lonza) and 1 × 105 cells were resuspended in 70 µL of 10% FBS DMEM, seeded in each well of the inserts and incubated O/N at 37ºC and 5% CO2. Each cell line was analyzed in duplicate. The next day, inserts were removed, and 1 mL of 10% FBS DMEM was added to each well and the wound monitored on a Thunder imager (Leica) at 37ºC and 5% CO2 by snapshotting the wounds every hour for 48 h. Finally, images were processed with the ImageJ program (Fiji) and the MRI Wound Healing Tool.The migratory capacity of ECC-1 cells was evaluated using 2-well silicone inserts (Ibidi). Inserts were placed on 24-well plate (Corning). Then, ECC-1 cells were harvested with Trypsin-EDTA (Lonza), 2.5 × 102Omq was used. 2 mL of 5% noble agar solution 1:10 diluted in 10% FBS DMEM were solidified on 6-well plates during 15 min at RT. Subsequently, ECC-1 cells were harvested with Trypsin-EDTA (Lonza), and 25,000 cells in 1 mL of 10% FBS DMEM were 1:2 diluted in 0.5% noble agar solution, and placed onto the solidified agar in duplicate. Cells were incubated at RT for 30 min to allow agar solidification, and then incubated at 37ºC and 5% CO2 for 4 weeks for colony formation. 100 µL of 10% FBS DMEM were added to each well once a week. Wells were photographed with the DMi1 Microscope (Leica) and colonies counted with ImageJ program (Fiji).For colony soft agar assay, sterilized 5% and 0.5% noble agar in H2 until 90% confluence in 10% FBS EGM-2 medium, and incubated 24 h in complete EGM-2 medium without FBS. 96-well plates were coated with 30 µL of Matrigel matrix (Sigma Aldrich) per well and incubated 1 h at 37ºC. Then, HUVEC cells were harvested with trypsin-EDTA (Lonza) and 20,000 or 40,000 HUVEC cells were seeded per well in EGM-2 medium without FBS and 1/2 diluted with the secretome of shC1GALT1 or SCRAMBLE ECC-1 cells. Plates were incubated at 37ºC and 5% CO2 during 6 h, and tube formation was monitored each 2 h with the DMi1 Microscope (Leica). Images were processed with the ImageJ program (Fiji) and the angiogenesis analyzer tool.Human umbilical vein endothelial cells (HUVEC) cells were used for tube formation assay using the secretome of shC1GALT1 and SCRAMBLE ECC-1 cells harvested after 48 h of incubation in Endothelial cell growth medium 2 (EGM-2) supplemented with growth factors  without FBS. HUVEC cells were grown at 37ºC and 5% CO5 cells were transfected with 22 pmol siRNA using 2 µl of JetPRIME Transfection reagent and 100 µl of JetPRIME buffer (PolyPlus Transfection). Then, 48 h after transfection, cells were analyzed by PCR, qPCR and WB. Alternatively, 24 h after transfection cells were used for indicated cell-based assays as above.For transient ANXA1 silencing, transfection was performed in 6-well plates using the jetPRIME reagent (PolyPlus Transfection) with, alternatively, ON-TARGETplus Human ANXA1 (301) siRNA  or control siRNAs  on shC1GALT1 and SCRAMBLE ECC-1 cells, according to established protocols , 42. BriA total of 159 out of 178 cores from 79 EC patients, which represents all major histologic EC types, were considered adequate for cytoplasmic C1GALT1 expression assessment by IHC. Out of the 159 cores, 101 (63.5%) belonged to endometrioid histology, 30 (18.9%) were serous and the remaining 28 (17.6%) belonged to clear cell or undifferentiated histology. Among endometrioid carcinoma cores, 65 (64.4%) belonged to low grade (FIGO grade 1 or 2) carcinomas, while 36 (35.6%) were high grade. C1GALT1 score varied across histologic types, with endometrioid tumors displaying the highest protein expression (median C1GALT1 IHC score = 200). On the other hand, aggressive histologic variants  showed lower C1GALT1 IHC scores  , 13 (13.4%) as serous and one as clear cell carcinoma (1%). ent Fig. . Median Finally, morphologic assessment of C1GALT1 IHC staining characteristics revealed three different staining patterns  or ≤-0.58 (downregulated) in shC1GALT1 cells in comparison to SCRAMBLE’s were further analyzed to elucidate the pathways altered in aggressive ECC-1 cells. Proteomic analysis of the secretomes showed 67 proteins upregulated and 77 downregulated, and 14 and 86 proteins in the cell extract of shC1GALT1  were analyzed using STRING and Reactome databases to identify altered pathways. STRING revealed 24 clusters of interaction composed of three or more interacting proteins, highlighting gene expression, cell adhesion, extracellular matrix organization and metabolic processes Fig. . In addiNext, ssGSEA performed with CPTAC data scored 63 pathways, where 35 dysregulated pathways were related to glycosylation, 27 to angiogenesis and 1 to endometrial cancer. Importantly, 33 out of the 63 scored pathways displayed a significant FDR corrected correlation with C1GALT protein expression (FDR < 0.05)  of shC1GALT1 and SCRAMBLE ECC-1 cells were analyzed by dot blot. All upregulated proteins in the secretome of depleted cells by LC/MS-MS were also observed upregulated in shC1GALT1 ECC-1 cells, confirming previous proteomics data Fig. E.Finally, we further analyzed the protein content of exosomes secreted by these cell lines to investigate the potential role of selected proteins in cell communication and metastasis. As previously observed in the secretome and cell extract, ANXA1, COL4A3BP, LGALS3 and MALT1 were found upregulated in exosomes secreted by shC1GALT1 cells. Interestingly, ERCC3 and NCOA1 were overexpressed and PCSK9 levels were decreased in exosomes secreted by silencing cells, in contrast to the dysregulation observed in the cell extracts and secretome of shC1GALT1 cells , whereas no effect on wound closure speed was observed in ANXA1-silenced SCRAMBLE ECC-1 cells in comparison with their control cells . Indeed, ANXA1-silenced shC1GALT1 ECC-1 cells showed a 45% reduced proliferation regarding C1GALT1 depletion-induced proliferation  has been shown to interact with O-glycans in the mucosal epithelium , and conA total of 151 out of 178 cores from 79 EC patients were considered adequate for LGALS3 expression assessment by IHC. Morphologic assessment of LGALS3 IHC staining characteristics revealed different staining patterns  Fig.  C. TheseCollectively, our results reveal that the dysregulation of O-glycosylation processes alters the protein patterns at different cellular compartments and that these changes are involved in vitro in the pathogenesis, development, and progression of the disease; as well as in EC samples.O-glycosylation has emerged as a main regulator of cancer due to its role in tumor development and progression . TruncatAltered O-glycosylation is observed in epithelial cancers and is frequently linked to poor prognosis. Aberrant O-glycosylation can be due to glycosyltransferase expression dysregulation. C1GALT1 is a key glycosyltransferase in the O-glycosylation process synthetizing the T antigen to which branched O-glycans are added. Elevated expression of this enzyme has been associated with tumor progression in lung adenocarcinoma and gastric cancer , 49. On Forward and Reverse SILAC experiments in the secretome and in the cell extract of ECC-1 cells, respectively, with high sensitivity and specificity in living cells [Quantitative proteomics analysis allowed us to identify and quantify 3616 and 5208 proteins in common in ng cells , 52. ProThe nine proteins selected for validation were the most dysregulated proteins between SCRAMBLE and shC1GALT1 ECC-1 cells, and had been previously related to EC or other cancers. MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) had been identified as a key protein in the pathogenesis of different hematological and solid tumors, such as lung or prostate cancers , 54. DueJAG1 (Jagged 1), a ligand of the Notch signaling pathway, has been associated with EC carcinogenesis through the control of multiple cellular processes. It has been described a dual role for the Notch receptor (Notch 1–4)-ligand  pathways as oncogenic or tumor suppressors –65. InteMoreover, COL4A3BP (Ceramide transfer protein or CERT) is a lipid transfer protein that transport ceramide, a pro-apoptotic signal molecule, from the endoplasmic reticulum (ER) to the Golgi apparatus , which hFinally, LGALS3  is a protein that interacts with glycoproteins from the extracellular matrix in a galactose-dependent manner, favoring cell interactions, or with cytosolic or nuclear targets in a glycosylation independent manner . ImportaIn conclusion, quantitative proteomics of a well-characterized cellular model, where upon C1GALT1 depletion a more aggressive phenotype was induced, allow for the identification of proteins dysregulated in aggressive ECs and related pathways that might be of interest for a better understanding of the mechanisms undergoing EC pathogenesis related to O-glycosylation. Proteins here identified by proteomics as dysregulated in aggressive EC were validated by orthogonal techniques using cellular models of non-aggressive (SCRAMBLE) and aggressive (shC1GALT1 ECC-1) endometrial cancer. In addition, a causal effect on the tumorigenic and metastatic properties of C1GALT1 depleted ECC-1 cells through ANXA1 was found, where ANXA1 transient depletion provoked a reversion on proliferation, invasion and migration properties induced because of the absence of C1GALT1. However, as potential limitations of the study, further research would be needed using animal models and a larger cohort of tissue samples from EC patients from different hospitals to further confirm the association between C1GALT1 depletion and EC aggressiveness and protein dysregulation. In addition, the nine most dysregulated proteins here identified by proteomics and validated by orthogonal techniques could be interesting to investigate their role in the pathogenesis and development of aggressive ECs. LGALS3 and C1GALT1 could be used as specific biomarkers of aggressive ECs, which would allow for the earlier treatment of patients with this phenotype associated to more aggressive ECs with higher metastatic potential. Furthermore, the diagnostic value of proteins here identified as upregulated in the secretome of aggressive ECC-1 could be investigated by ELISA to elucidate the potential role of ANXA1, COL4A3BP, JAG1, MALT1, LGALS3 and PCSK9 as blood-based biomarkers of the disease.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8


Methacrylate is an isostructural analogof pyruvate and an informative irreversible inhibitor of pfl. Herewe demonstrate the mechanism of inhibition of pfl by methacrylate.Treatment of activated pfl with methacrylate results in the conversionof the G734· to a new radical species, concomitantwith enzyme inhibition, centered at g = 2.0033. Spectralsimulations, reactions with methacrylate isotopologues, and DensityFunctional Theory (DFT) calculations support our assignment of theradical to a C2 tertiary methacryl radical. The reaction is specificfor C418, as evidenced by mass spectrometry. The methacrylradical decays over time, reforming G734·, and thedecay exhibits a H/D solvent isotope effect of 3.4, consistent withH-atom transfer from an ionizable donor, presumably the C419 sulfhydryl group. Acrylate also inhibits PFL irreversibly, and alkylatesC418, but we did not observe an acryl secondary radicalin H2O or in D2O within 10 s, consistent withour DFT calculations and the expected reactivity of a secondary versustertiary carbon-centered radical. Together, the results support uniqueroles of the two active site cysteines of PFL and a C419 S–H bond dissociation energy between that of a secondaryand tertiary C–H bond.Pyruvate Formate Lyase (PFL) catalyzes acetyl transferfrom pyruvateto coenzyme a by a mechanism involving multiple amino acid radicals.A post-translationally installed glycyl radical (G Radical transfer from G· to a pair of cysteinesin the active site of PFL is unique among known GREs, and other thiylradical enzymes such as ribonucleotide reductases, which only containone substrate activating cysteine.13During anaerobic glycolysis in many bacteria,archaea, and someeukaryotes, acetyl-coenzyme A (acetyl-CoA) is produced for substrate-levelphosphorylation and biosynthesis by pyruvate formate lyase (PFL),which catalyzes the cleavage of pyruvate to acetyl-CoA and formate.19 Absent radical reduction potential changes due to the protein environment,21 this difference in reactivity presents an intrinsic challenge tothe study of the role of thiyl radicals in GRE catalysis. Consistentwith this apparent difference in radical stability, no thiyl radicalhas been directly observed in the catalytic cycle of PFL, or any otherGRE, to our knowledge. The mechanism of PFL and the role of the twocysteines are currently inferred from X-ray crystallographic structures24 and the effect of mutagenesis,26 isotopic substitution,28 and mechanism-based inhibitors34 on enzyme activity and G· characteristics, focused on the PFLfrom Escherichia coli. A plausible mechanistic proposalis depicted in 734· ⇌C419 ⇌ C418 (E. coli numbering) by sequential proton-coupled electron transfer events.The C418· then attacks the C2 carbonyl of pyruvate,followed by radical rearrangement and homolysis of the C1–C2bond of pyruvate to generate an acetylated C418 and a CO2·– radical anion, that abstracts aH atom from C419. Equilibration of C419·back to G734· completes the ping phase of the ping-pongmechanism.27 In the pong phase, H atomabstraction from the CoA sulfhydryl by C419·, acetylexchange, and radical equilibration completes the catalytic cycle.While this mechanistic proposal is consistent with a majority of structuraland biochemical observations, the lack of direct observations of radicalchemistry with structural specificity leaves ambiguity regarding theunique mechanism and the role of the two cysteines.As a free amino acid, G· exhibits a reductionpotential 110mV lower than the corresponding C·.34 Itis particularly illuminating as it provides a compelling case forthe unique role of the two thiyl radicals during the catalytic cycle.Incubation of active PFL (aPFL) containing the essential G734· with 1 mM methacrylate results in complete and irreversibleinhibition over 30 min with no apparent effect on the G734·.34 The product of inhibition isa thioether bond between C418 and C3 of methacrylate, whichis reduced stereoselectively to a S-(2-carboxy-(2S)-propyl) adduct.34 This biochemicaldata and the X-ray structure support the mechanism depicted in 418 and C419. Density functional theory(DFT) calculations also support a preference for C418·reaction with methacrylate over C419· and a slow reductionof the tertiary C2 methacryl radical by the C419 sulfhydryl,relative to inactivation.35 Unfortunately,these predictions are not supported by direct experimental evidence,limiting the mechanistic insight on the unique role of C418 and C419.Methacrylate is a substrate analog of pyruvateand acts as an irreversiblemechanism-based inhibitor of PFL.E. coli aPFL. Higher concentrationsof the inhibitor and faster quenching of the reaction were criticalto evidencing the intermediate by X-band electron paramagnetic resonance(EPR) spectroscopy, revealing an inhibitor radical consistent witha tertiary C2 radical of methacrylate. Proteolytic digestion of inhibitedPFL and peptide analysis by liquid chromatography-tandem mass spectrometry(LC-MS/MS) resolves a C418-methacrylate adduct. Densityfunctional theory (DFT) calculations based on this model and priorstudies35 reproduce the spectroscopic propertiesof the C2 radical, and support the stereoselective nature of radicalreduction.34 Kinetic analysis of the C2radical decay demonstrates that the reduction of the inhibitor radicalis indeed rate determining, as predicted theoretically.35 Using a combination of the methacrylate analog,acrylate, solvent kinetic isotope effects (KIE), and DFT calculationswe estimate the BDE of the C419 thiol S–H bond,placing the first energetic and mechanistic constraint on the H atomtransfer chemistry of this residue. The data are consistent with aC418· as the radical that adds to C3, and C419 as the H atom donor to the C2 radical.Here, we report the characterizationof the methacryl radical generatedupon inhibition of E. coli, NEBuilder HiFi DNA Assembly Master Mix, and Phusionpolymerase were purchased from New England Biolabs. SapphireAmp FastPCR-hot-start Master Mix was purchased from Takara Bio. Chloramphenicoland agarose were purchased from Apex Bioresearch Products. LB Millerbroth, Terrific Broth (Modified), citrate synthase (porcine heart),malate dehydrogenase (porcine heart), bovine serum albumin, tris(hydroxymethyl)aminomethanebase (Tris), β-nicotinamide adenine dinucleotide (NAD+), methacrylic acid, sodium acrylate, S-(5′-adenosyl)-l-methionine iodide, KH2PO4, Triton X-100,glycerol, MgCl2·6H2O, oxamic acid, sodiumpyruvate, CoA, malic acid, iodoacetamide, trifluoroacetic acid, dithiothreitol(DTT), l-cysteine, (NH4)FeII(SO4)2, formic acid, 4-hydroxy-TEMPO, urea, iron ICP-MSstandards (TraceCERT), ferrozine (3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4″-disulfonicacid sodium salt), neocuprine ,ascorbic acid, KMnO4, ammonium acetate, phenylmethylsulfonylfluoride (PMSF), lysozyme from hen egg white, Ribonuclease A frombovine pancreas, Deoxyribonuclease I from bovine pancreas, and AmiconUltra centrifugal filter units were purchased from Millipore Sigma.We note that Millipore Sigma sodium methacrylate contained variableamounts of an EPR-active species we ascribed to a polymerization inhibitornitroxide radical. For this reason, methacrylic acid was used withan EPR-silent 4-methoxyphenol polymerization inhibitor. Isopropyl-β-d-1-thioglactopyranoside (IPTG) was purchased from Merck. 5-Deazariboflavinwas obtained from Santa Cruz Biotechnologies. Perdeuterated d5-methacrylacrylic acid was purchased from PolymerSource Inc. DpnI fast digest and Lys-C were purchased from ThermoScientific. Sequencing grade modified trypsin, and electrocompetentBL21(DE3)pLysS E. coli were purchased from PromegaCorporation. HiTrap desalting 5 mL columns, and Sephacryl S-100 HRwere purchased from Cytiva Life Sciences. Nickel nitrilotriaceticacid (Ni-NTA) agarose resin was purchased from Prometheus ProteinBiology Products. Milli-Q water (>17 MΩ) was used for preparingall solutions. The TEVSH plasmid was a gift from Dr. Helena Berglund(Addgene plasmid # 125194).36 The plasmidpCAL-n-EK encoding the pflA gene was a gift fromProf. Joan Broderick.37 The pflB gene (Uniprot ID P09373) was codon optimized and synthesized byIntegrated DNA Technologies.DNA oligos were purchased from IntegratedDNA Technologies. Electrocompetent DH5α and BL21(DE3) E. coli PFL, we cloned the pflB gene intoa modified pCm1 plasmid (Addgene #174361) using Gibson assembly byPCR.39 The pCm1 plasmid was modified to encodea 6 × polyhistidine tag (His-tag), followed by an SSG spacerand a TEV protease site N-terminal to the codified protein, and theprevious C-terminal tag from pCm1 was removed. We term this new backboneplasmid pCm8. The pflB gene was also cloned intopCm8 using Gibson assembly by PCR. A DNA fragment containing pflB gene was amplified by PCR using the primers:In order to produce and purify Forward: 5′–tcagggcatgtccgagcttaatgaaaagttagcc–3′Reverse: 5′–agcctaggttacatagattgagtgaaggtacgagtaataacgt–3′The primers used to amplify the vector backbone were:Forward:5′–tcaatctatgtaacctaggctgctaaacaaagccc–3′Reverse: 5′–tcggacatgccctgaaaatacaggt–3′E. coli DH5αcells and streaked into chloramphenicol LB-agar plates. Successfultransformants were identified using colony PCR with the SapphireAmpfast PCR-hot-start master mix, as directed by the manufacturer, andthe fidelity of the cloning was confirmed by Sanger sequencing throughUC Berkeley DNA Sequencing Facility using the T7 promoter/terminatorand pflB specific primers:DNA fragments were assembled using NEBuilder HiFi DNA AssemblyMaster Mix following the manufacturer instructions. Assembly reactionproducts were transformed into Forward: 5′–ggtccggctaccaacgctc–3′Reverse: 5′–ccaggtcaacagccaggtc–3′418S and C419S weperformed site-directed mutagenesis (SDM). For C418S weused the following primers:To produce PFL mutants CForward: 5′–acgatgactacgctattgctagctgcgtaagcc–3′Reverse: 5′–ggcttacgcagctagcaatagcgtagtcatcgt–3′419S we used the following primers:For CForward:5′–ctacgctattgcttgcagcgtaagcccgatgat–3′Reverse: 5′–atcatcgggcttacgctgcaagcaatagcgtag–3′E. coli DH5αelectrocompetent cells and streaked the cells on chloramphenicol LB-agarplates. We confirmed the mutagenesis fidelity by Sanger sequencingas described above.We used Phusion polymerase to perform SDM by PCR per the manufacturer’sinstructions and treated the PCR products with DpnI at 37 °Covernight and then transformed them into 36The expressionand purification of the TEV protease was performed as previously described.418S, and C419S PFL using the same protocol. BL21(DE3) cellstransformed with the corresponding plasmid were inoculated into a15 mL preculture of LB broth supplemented with 50 μg/mL of chloramphenicol.The preculture was grown overnight at 37 °C in a 50 mL centrifugetube shaking at 200 r.p.m. The following day, 15 mL of the overnightpreculture were inoculated into 1.5 L of terrific broth, supplementedwith 50 μg/mL of chloramphenicol, and grown at 37 °C, shakingat 200 r.p.m. Protein expression was induced by adding 0.25 mM IPTGwhen the OD600 reached 1.0, and then the culture was maintainedat 25 °C overnight. Cells were harvested by centrifugation at8,000 × g for 10 min, and the collected cellpaste, approximately 10–15 g wet cell paste per liter, wasflash frozen in liquid N2 and stored at −80 °Cuntil purification.We expressed wild-type (wt), Cg for 30 min,and the cell debris was discarded. To remove nucleic acids, 1.5% w/vstreptomycin sulfate was added dropwise while stirring and incubatedfor 10 min before being centrifuged at 30,000 × g for 30 min. The pellet was discarded, and the supernatant was filteredthrough a 0.65 μm filter. The clarified lysate was applied toa 20 mL Ni-NTA column equilibrated with wash buffer composed of 30mM imidazole, 50 mM KPi, 150 mM NaCl, 1 mM DTT, and 5% glycerol atpH 8.0. The resin was washed with 20 column volumes of wash bufferand eluted with elution buffer in which the imidazole concentrationwas raised to 400 mM. Fractions containing protein were pooled andconcentrated using Amicon Ultra-15 50 kDa molecular weight cutoff(MWCO) centrifugal filter units and then buffer exchanged to resuspensionbuffer using a HiTrap desalting 5 mL column. To remove the N-terminalHis-tag, PFL was incubated with TEV protease in a 1:100 ratio of proteaseto protein overnight and then the digestion product was injected backinto the Ni-NTA column to remove the cleaved N-terminal tag, His-taggedprotease, and undigested PFL. The Ni-NTA flow-through was collectedand purified, and the untagged protein was concentrated and desaltedas described above. The purified protein was stored in resuspensionbuffer supplemented with 20% w/v glycerol at −80 °C untilused.For PLF protein purification we resuspendedcell paste in resuspensionbuffer consisting of 50 mM potassium phosphate (KPi), 150 mM NaCl,1 mM DTT, and 5% glycerol, adjusted to pH 8.0, and lysed the cellsby French press in an Emulsiflex C3 homogenizer at 14,000 psi. Everyfollowing step was performed at 4 °C. The cell extract was clarifiedby centrifugation at 30,000 × 40 BL21(DE3)pLysS cells transformedwith pCAL-n-EK-pflA were inoculated into a 15 mLpreculture of LB broth supplemented with 100 μg/mL of carbenicillinand 35 μg/mL of chloramphenicol. The preculture was grown overnightat 37 °C shaking at 200 r.p.m. 3 mL of cells from the overnightpreculture were inoculated into 300 mL of LB broth supplemented with100 mM KPi buffer pH 7.0, 100 μg/mL of carbenicillin and 35μg/mL of chloramphenicol and grew at 37 °C with shakingat 200 r.p.m. PFL-AE expression was induced by adding 0.25 mM IPTG,0.2 mM l-cysteine, and 0.2 mM (NH4)FeII(SO4)2 at OD600 of 0.8. The culturewas maintained at 30 °C for 5 h, and then l-cysteineand (NH4)FeII(SO4)2 wereadded to a final concentration of 0.4 mM. The culture was moved toa 4 °C refrigerator and sparged with Argon overnight (15–16h).We modified a previously reported method to express andpurifyPFL-AE.2 and transferred to centrifugebottles with an O-ring seal. Cell paste was harvested by centrifugationat 8,000 × g for 5 min. All following steps were performed inthe glovebox. Cells were resuspended into anaerobic lysis buffer consistingof 50 mM Tris, 50 mM KCl, 10 mM MgCl2, 1% w/v Triton X-100,and 5% w/v glycerol at pH 7.5 in a proportion of 1 mL of buffer pergram of wet cell paste. The resuspended cell pellet slurry was supplementedwith 1 mM phenylmethylsulfonyl fluoride, 0.2 mg/mL lysozyme, 1 mMDTT and 10 μg/mL of RNase and DNase, and homogenized on ice,with gentle stirring, for 1 h. The cell slurry was then lysed by ultrasonicationwith a Qsonica q125 sonicator for 10 min, in cycles of 20 s on–30s off, on ice. The sample was clarified by centrifugation at 15,000× g for 15 min and filtered using a 0.65 μmfilter. The protein extract was then injected into a 70 mL SephacrylS-100 HR size exclusion column, equilibrated with buffer consistingof 50 mM Tris, 100 mM KCl, and 1 mM DTT at pH 7.5, and resolved ata flow rate of 0.5 mL/min. We collected the dark brown fractions andconcentrated them using Amicon Ultra-0.5 10 kDa MWCO centrifugal filterunits. The concentrated protein was reinjected in the same columnunder the same conditions to further improve protein purity. Protein-complexediron was quantified using the ferrozine assay,41 using iron ICP-MS standards (TraceCERT) as an iron standard.Protein concentrations were quantified using the Bradford assay, usingbovine serum albumin as a standard.42For PFL-AE purification, the culture flasks were moved intoa coyglovebox <20 ppm of O2) by mixing 50 μMPFL, 5 μM PFL-AE, 2 mM SAM, 20 mM oxamate, and 100 μM5-deazariboflavin in activation buffer containing 100 mM Tris, 100mM KCl, 10 mM DTT, and 20% w/v glycerol at pH 7.6 in a 4 mm O.D. EPRtube. We exposed the mixture to a 1 W 405 nm LED light (Thor Laboratories)for 1 h in a thermal bath at 30 °C. The extent of activationwas estimated by activity assays and EPR quantitation of the G·(see below).We activated PFL in an anaerobic VACAtmospheres glovebox , and 0 to 120 μM for CoA (10 mMpyruvate). In order to calculate specific activities  we normalized the activity to the measured G·concentration determined by EPR (see below) assuming 1 mol of G·per mole dimer of active PFL, and fitted the calculated initial velocitiesto a Michaelis–Menten model described by For enzyme inactivation kineticswe incubated samples of aPFL with200 mM methacrylate or acrylate between 0 and 10 min. We took samplealiquots during inactivation and immediately determined the remainingPFL activity as described above. Inhibition time points were performedin duplicate and the average is reported.2. To analyzeradical intermediates during aPFL inactivation we mixed 180 μLof 50 μM aPFL, in activation buffer, with 20 μL of 2 Mmethacrylate or acrylate in H2O pH adjusted to 7.0. Sampleswere incubated at room temperature from 10 s to 7 min. The reactionswere flash frozen in EPR tubes in liquid nitrogen-cooled isopentane(<−130 °C). EPR spectra of the samples were collectedusing a Bruker EMXplus EPR spectrometer at 100 K with a frequencyof 9.38–9.44 GHz, power of 20 μW, modulation amplitudeof 2 G, modulation frequency of 100 kHz, time constant of 0.01 ms,scan time of 20 s, and conversion time of 16 ms. All reported spectraare the average of 30 scans. Spin quantitation was computed from thedouble integral of the first harmonic signal and referenced to a 4-hydroxy-TEMPOconcentration standard. All EPR spectral simulations were performedusing EasySpin 6.0.0 software, and confidence intervals and standarddeviations of the fitting parameters are reported.44All EPR samples were preparedin a VAC Atmosphere glovebox with <2 ppm of O2O-based buffer was prepared by lyophilizing 100 mMKCl and 100 mM Tris at pH 7.2 and rehydrating the buffer in the samevolume of D2O. The D2O-exchanged solution wasmeasured to be pH* of 7.2, corresponding to a final pD of 7.6 accordingto 45For preparing solvent H/D isotope effect samples,a D2O samples, aPFLprotein samples were subject to three cycles of 5-fold concentration,followed by 5-fold dilution in D2O buffer using 50 kDaMWCO centrifugal filters.To ensure water content <5% in theD2O and in D2O were determinedfrom fitting of therelative G· and C2· contributions to the composite EPR spectraas a function of time in COPASI 4.40. The reaction was fit to an irreversibleG· → C2· (k1) and thenC2· → G·* (k2) modelwhere G·* corresponds to the inhibited PFL G·. The errorassociated with the fit is reported as the error for the extractedrate constants, and the solvent kinetic isotope effect is reportedas k2(H2O)/k2(D2O).Kinetic constants for the C2·formation and decay in HWe assessed the formation of covalent adducts of methacrylate andacrylate with PFL using tryptic digestion of the protein followedby liquid chromatography tandem mass spectrometry (LC-MS/MS). To digestPFL 100 μg of protein was denatured in 20 μL of 8 M ureain 100 mM ammonium bicarbonate, and 5 mM DTT was added and incubatedfor 30 min at 37 °C to reduce cysteines. Unreacted cysteineswere alkylated with 15 mM iodoacetamide for 30 min in the dark, andthe reaction was quenched by adding 20 mM DTT and incubating for 10min. PFL samples were digested with 0.8 μg Lys-C for 2 h. Next,the digested samples were diluted to <2 M urea using 100 mM ammoniumbicarbonate and 0.16 μg of Trypsin were added, and digestedat 37 °C overnight. Reactions were stopped by adding formic acidto a final concentration of 1% v/v.2O with 0.1% v/v formic acid (A) and the apolar mobilephase acetonitrile 0.1% v/v formic acid (B). A solution of 150 pg/μLLeu-enkephalin and 100 fmol/μL Glu-fibrinopeptide B preparedin 25:75 acetonitrile:H2O with 0.1% v/v formic acid wasused as a lock mass, using the average of 3 scans injected every 30s. Peptides were resolved in a linear gradient from 20 to 35% B over30 min with a flow rate of 0.2 mL/min at 60 °C. Eluent from theLC was injected directly into the qToF. The mass spectrometer settingswere as follow: source capillary voltage 2.8 kV, sampling cone voltage20 V, source offset 20 V, source temperature 120 °C, desolvationtemperature 350 °C, cone gas flow was 50 L/h and desolvationgas flow was 800 L/h. The spectrometer was set in MS/MS detectionmode in positive polarity in the resolution regime, detecting from50 to 2000 Da, with a scan time of 3 s. Peptides corresponding withthe mass of the expected tryptic peptide with the methacrylate, acrylate,or carbamidomethyl modification were fragmented using an energy rampfrom 20 to 35 V and a cone voltage of 20 V. The LC-MS/MS data wereanalyzed using Masslynx.The digested samples (2–5μg at 0.4–1 μg/μL) were injected into a WatersAcquity H-class Ultra High-Pressure Liquid Chromatography system coupledwith a Waters Xevo G2-XS quadrupole time-of-flight (qToF) mass spectrometer.The UPLC stationary phase was a Waters BEH C18 column of 50 mm ×2.1 mm × 1.7 μm and the mobile polar phase a solution ofH418–C419 dipeptide for EPR spectral parameter predictions.35 Rotamers were constructed in ChimeraX by iterative10° rotation of the methacrylate moiety about the C418-bonded C3–C2 bond, and angles are reported relative to theinitial pro-(R) H–C3–C2-C1dihedral angle of 38°. Single point energies and EPR parametersat each angle were computed for the anionic methacryl radical in Orca5.0.146 using DFT48 with the B3LYP49 unrestrained hybridfunctional and a 6-311++G basis set and a conductor-like polarizationcontinuum model50 dielectric and indexof refraction of chloroform to model the protein environment. Spincontamination was evaluated by the computed expectation value forS2, which did not exceed 0.02 above the ideal value of0.75 for a pure doublet spin system.We obtained coordinate filesfor the methacryl radical from a previous DFT geometry optimized structureusing a small model containing methacrylate and the C418–S· to C3, and we used a small model previouslygeometry optimized composed of a methyl thiyl radical and methacrylate/acrylate(step 1).35 For the second step, methacryl/acrylradical C2· reduction by C419–SH (step 2),a second methyl sulfide was included, again starting from a previouslygeometry optimized state.35 All transitionstates exhibited a single imaginary vibrational frequency, and energiesare reported as the sum of Gibbs free energy correction (G – Eel) and electronic energy(Eel). To normalize the energy of theoverall reaction, the transition state (TS) and product (P) of bothstep 1 and step 2 were evaluated relative to their respective reactant(R1 or R2), and then the energies of R2, TS2, and P2 were scaled toP1 (P1 = R2).Reaction trajectories formethacrylate and acrylate were evaluated in two steps by geometryoptimizing reactant and product states and then performing a transitionstate search. For reactions with methacrylate, the C4 methyl groupin the methacrylate structures was replaced by a H and geometry optimizedas above. The first step involved the radical Michael addition ofCG‡) for the deuterated isotopologue reactant and transition states,assuming the same geometry for the deuterated and protonated reactantand transition states, and calculating kH/kD using kH/D arethe rate of the corresponding chemical step with either H or D, R is the ideal gas constant, and T is thetemperature in Kelvin. A similar treatment was used to compute theratio of the rate for inhibitor radical reduction by C419 for acrylate (kacryl) versus methacrylate(kmethacryl).The kinetic isotope effect of methacryl radicalreduction in step2 was computed using transition state theory by recomputing the Gibbsfree energy of activation . The expression yields were10–20 mg of purified PFL protein per gram of wet cell paste.During the recombinant expression of PFL-AE we applied a continuoussparge of argon to the cell culture after induction to protect the[Fe4S4] cluster from oxidation. Following anaerobicpurification, PFL-AE was estimated to be 60% pure with an overallyield of 10 mg of protein per gram of wet cell paste . Reconstitutionof the [Fe4S4] cluster resulted in PFL-AE ironcontent of 2.0 ± 0.2 Fe/mol of PFL-AE, likely an underestimatebased on the protein impurities.To investigate the mechanism of PFL inhibition by methacrylate werecombinantly expressed PFL, including wild-type (wt), CE. coli aPFL G734· product .5 Under our optimalconditions, PFL-AE was capable of charging 0.5–1.0 G·per PFL dimer of the wt PFL substrate, characteristic of the “half-of-sites”reconstitution and reactivity of the E. coli PFL.28 The C418S and C419S PFLvariants exhibited similar G· reconstitution efficiency, basedon G· EPR signal quantitation . We simulated the G· spectrumwith an isotropic giso of 2.0037, consistentwith prior studies,51 and hyperfine coupling(HFC) to a single 1H with an isotropic hyperfine couplingconstant (AαCH) of 40.5 MHz. Coupling to additionalnuclei, suggested by prior experimental and theoretical studies,52 did not impactfully improve the simulation to the experimental data,and were not included in experimental simulations. Transfer of aPFLfrom H2O-based to D2O-based buffer results ina narrowing of the doublet signal to an apparent singlet, due to theH/D exchange of the G· and the smaller gyromagnetic ratio ofdeuterium relative to hydrogen, which is facilitated by the ionizableC419 sulfhydryl group .26 The narrowing ofthe G· spectrum was thus used to confirm H/D exchange of aPFLin subsequent experiments. A table summarizing these, and all subsequentEPR spectral fitting parameters and associated fitting standard deviationsare provided in the Supporting Information Table S1.Treatment of PFL with PFL-AE,in the presence of a photochemical reduction system, and illuminationby a 405 nm LED resulted in the accumulation of an asymmetric doubletEPR signal characteristic of the 27 When normalized to reconstituted active sites,estimated by EPR quantitation of G·, we observed a kcat of 225 ± 9 s–1 and Km of 0.78 ± 0.09 mM for pyruvate and 12± 2 μM for CoA at 20 °C, consistent with prior reportsof E. coli PFL kinetic constants .27 The C418S and C419S variants exhibited activitiesbelow the detection limit of the coupled optical assay, which we estimateto be <0.01 s–1. Methacrylate is a mechanism-basedsuicide inhibitor of aPFL with a reported KI of 0.4 mM and kinact of 0.14 min–1.34 Indeed, we observetime-dependent inhibition of 50 μM aPFL upon exposure to activesite-saturating (200 mM) concentrations of methacrylate, but withan apparent kinact of 1 min–1 . Inclusion of an additional 1H did not statisticallyimprove the simulation fit, as judged by the residuals and the HFCconfidence intervals of the additional nuclei. The spectrum is consistentwith a C2· methacryl radical, which has been reported previously,54 but where the radical has minimal electron density overlap withthe second methylene 1H of C3. No change in the G·signal was observed at any time for C418S or C419S .Based on our abilityto temporally resolve the inhibition process, we sought to trap aradical intermediate in the inhibition of aPFL. Freeze-quenching theinhibition reaction at 10 s revealed a convoluted multiline EPR spectrum2A compos2O buffer  and C418–C419 (m/z =1150.6) containing peptides respectively, allowing for the unambiguousdetermination of post-translational modifications at each residue. Followingcomplete inhibition by methacrylate over 10 min, a new peptide isresolved at 14.8 min retention time with y9 unchanged,but y10 observed at an m/z of 1179.6, consistent with a methacrylate adduct to C418. No adduct is formed with unactivated PFL, confirming the radicalnature of the inhibition mechanism, nor in G·-reconstituted C418S or C419S, suggesting both residues participatein the inactivation process. We also performed the peptide analysison samples acid-quenched at 10 s, 30 s, and 1 min to terminate radicalchemistry at the time of observation of the EPR signal, yielding thesame y10 ion with the methacrylate adduct with variableintensity .Whileour EPR data support a methacryl C2· intermediate, theydo not inform on the site of reactivity with aPFL. We turned to peptideanalysis of tryptic digestions of PFL by LC-MS/MS. The parental ionof the peptide containing C418 through C3 as a C418–C419 dipeptide.35 The dihedral anglebetween the pro-(R) methylene H–C3–C2–C1was systematically rotated about the C3–C2 bond in 10°increments from −180° to +180° relative the initialangle of 38°, and the EPR giso andHFCs were calculated . Two dihedralangle zones with no van der Waals clashes were consistent with theexperimental HFCs, −35° and +145°, which show minimalsingly occupied molecular orbital (SOMO) overlap between with the pro-(R) methylene 1H . The−35° rotamer yields giso of2.0037 with ACH3 of 56 MHz and ACH2 of 65 and3 MHz, whereas the +145° rotamer yields giso of 2.0029 with ACH3 of 61 MHz and ACH2 of 68 and 2.6 MHz, both in agreement with the experimental spectrum(Supporting Information Table S2). Thevan der Waals clashes in the other two rotamers may be alleviatedby structural dynamics or relaxation, but were not investigated further.The methacrylate product observed by LC-MS/MS and EPR isconsistentwith a tertiary C2· methacryl radical, yet the lack of a fifthhyperfine interaction suggests a specific poise of the radical inthe protein active site. To investigate the conformational dependenceof the HFC to a putative C2· we performed DFT calculations withan unrestricted hybrid B3LYP functional to simulate the EPR parametersof the radical. As a starting structure, we used a geometry optimizedC2· bound to CSupporting Information Table S3). Using the EPRspectral parameters obtained from Supporting Information Figure S9). The extractedrates were 2.0 ± 0.2 min–1 for C2· formationand inhibition, and C2· decay at 1.7 ± 0.1 min–1. No other radical species were observed, and the reaction proceededwith <30% total radical loss.The methacryl radicalis unstable and decays with concomitant reformation of the G·over the course of minutes at 20 °C. We investigated the timecourse of C2· formation and decay during the methacrylate inactivationreaction by freeze-quenching reactions of aPFL from 10 s to 7 min3 using E2O buffer (–1), but a solvent KIE was observed for the C2· decay and G·reformation (0.50 ± 0.04 min–1) of 3.4 ±0.4, supporting a mechanism of radical reduction by H atom transferfrom an ionizable amino acid.To examine the mechanism of C2· reduction,we repeated theEPR analysis of the methacrylate inhibition process of aPFL in DO buffer 4. We obs2·– in thenative ping phase, or reactivity differences due to BDEs. To shedlight on these two potential contributions, we investigated the reactivityof aPFL toward acrylate, which we anticipated to form a much lessstable secondary C2· due to steric effects.57The relatively slow reactivity of the methacrylC2· radicalstands in stark juxtaposition to the native reactivity of aPFL, whereno radical intermediates have been identified. This could be due tosteric constraints of the covalently attached C2·, as opposedto the dissociated COkinact value of>15 min–1 relative to that of methacrylate . However, we were not able to capture an EPR spectrum of the acrylateC2· in H2O, D2O, or in D2O at4 °C at the fastest hand freeze-quenched time accessible of 10s and step 2 (C2· reduction by C419–SH) reactants,transition states, and products are shown in the Supporting Information Figure S11. The thiyl radical additionof C418–S· to C3 of either acrylate or methacrylateexhibited low barriers, with ΔG‡ of 9.4 and 4.6 kJ/mol, respectively, but were both driven to theC2· product with an overall exergonic ΔG of −16.7 kJ/mol and −22.1 kJ/mol, respectively. Asignificant difference was observed between the inhibitors in step2, where the predicted ΔG‡ were +33.1 kJ/mol for acrylate and +39.6 kJ/mol for methacrylate.The difference in activation barriers between the two substrates predictsa difference in reactivity of the C2· of acrylate relative tomethacrylate (kacryl/kmethacryl) of 14. For methacrylate, a KIE due to H/D exchangeof the C419–SH(D) is calculated to be 3.2. The formationof the product of step 2, namely a C419–S·,is endergonic for methacrylate by +7.5 kJ/mol, whereas this reactionis exergonic by −8.8 kJ/mol for acrylate, suggesting the C419 thiyl radical reactivity is between that the secondaryC2· of acrylate and methacrylate.To gain insight into the relative energetics ofthe radical chemistrybetween acrylate and methacrylate, we again turned to DFT. We performedgeometry optimizations and transition state searches in a truncatedmodel from a prior study for the reaction with both acrylate and methacrylateand a thiyl radical at C58 For PFL,this has led to some debate about the role of the two active sitecysteines during catalysis. Early studies on the mechanism of PFLsupported a “thiohemiketal” mechanism of catalysis inwhich the ping phase was radical-based and the pong phase involvednucleophilic displacement.25 Peptide mappingof aPFL reacted with 14C2-labeled pyruvate revealed radioactivityassociated with C419, which was proposed as the site ofradical reactivity and acetylation.30 Therole of C418 was suggested by the demonstration that reconstitutedC419S PFL was charged with an acetyl unit at C418 from acetyl-CoA.25 The ping and pongphases appeared to be connected by an acyl shift, which was evidencedby reactions of the (C419-)acetylated aPFL with hypophosphite,a formate analog, forming a C418-ligated acetylphosphonate.30 Conversely, the X-ray crystallographic structureof the unactivated PFL bound to pyruvate23 and peptide mapping studies with 14C2-labeled methacrylate,34 supported C418 as the site of radical-basedsubstrate activation and acetylation  or 2-(S) stereoisomer products, following reductionby H atom transfer. An inspection of the two DFT structures and theX-ray structure of the pyruvate-bound PFL , and the enantioselectivity of thereduction process,34 strongly favors the−35° orientation, forming the 2-(S) enantiomer,where the vinyl group of methacrylate takes the position of the carbonylin the pyruvate substrate.By examining the inhibitionof aPFL by methacrylate at significantlyhigher concentrations than those used previously, we have captureda radical intermediate by EPR spectroscopy that is kinetically consistentwith inhibition. Based on the radical intermediate EPR signature whengenerated in H2·– in the native reaction. We measured a solvent KIE of 3 for the C2·reduction in H2O versus in D2O, which confirmsthat the inhibitor radical is reduced by an ionizable residue capableof H atom transfer. The EPR spectrum of aPFL in D2O demonstratesthat the C419 sulfhydryl H/D exchanges with the solvent,and is the nearest redox-active residue to the substrate in the X-raystructure.23 The observed KIE is in agreementwith the DFT calculated KIE of 3.2, consistent with the proposed mechanismof C2· reduction by C419.The C2· radical quenching isinformative regarding the secondhalf of the ping phase of the reaction mechanism of PFL, where C2·serves as a surrogate for the CO35 The accumulation of C2·suggests that the tertiary radical is more stable than the correspondingC419 thiyl radical, but C2· reduction is ultimatelydriven forward by radical equilibration back to G734·,precluding the direct observation of a transient C419 thiylradical. Using acrylate as a methacrylate analog with a destabilizedsecondary C2·, we show that this radical is unstable, yet stillspecific for C418. Comparing the product and reactant statesof the acrylate reaction by DFT, we estimate that the C419 thiol S–H BDE is higher than a tertiary radical and nearlythermoneutral with a carbon secondary radical. Estimates of the bonddissociation energy of the corresponding isobutanoate C2 range from347 to 355 kJ/mol,60 and the shift from a tertiaryto secondary C2 increases the reactivity by +18 kJ/mol.60 Our DFT computed ΔG valuefor C2 reduction of the acryl radical is −8.8 kJ/mol placingthe corresponding C419 thiol between 356 and 364 kJ/mol,which is in agreement with prior experimental or theoretical studieson the free cysteine amino acid from 353 to 365 kJ/mol.62 These estimates neglect the roleof the protein environment, which may significantly alter the radicalreduction potentials of the inhibitor or the active site cysteines.The reported formate C–H BDE varies widely, but has been estimatedto be 360–380 kJ/mol, making the corresponding step 2 in thenative mechanism mildly exergonic.66Our observation ofa long-lived C2· and DFT analysis is consistentwith previous theoretical predictions of a rate determining C2·reduction.kcat > 100s–1), including the class II ribonucleotide reductase,68 PFL,27 and 1,2-eliminases . Interestingly, GREs proposed to activate primary carbon centers,including X-succinate synthases 75 and C–Plyase76 exhibit lower turnover rates (kcat < 1 s–1). Our resultsmay provide a thermodynamic explanation for the kinetic privilegeof the thiyl radical enzymes activating secondary, rather than primarycarbon centers, although a primary carbon radical was not investigatedin this study. This reactivity may also inform mechanistic investigation,where new tertiary centers may stabilize substrate analog radicalssufficiently long enough to be observed and characterized.Many thiyl radical enzymesactivate secondary carbon centers withhigh turnover frequencies (


The optical bandgap, magnetic parameters, and electrochemical capacitive behaviors of the prepared sample were compared with pristine electrospun MgFe2O4 and NiFe2O4 based on structural and morphological properties. XRD analysis affirmed the cubic spinel structure of samples and their crystallite size is evaluated to be less than 25 nm using the Williamson–Hall equation. FESEM images demonstrated interesting nanobelts, nanotubes, and caterpillar-like fibers for electrospun MgFe2O4, NiFe2O4, and Mg0.5Ni0.5Fe2O4, respectively. Diffuse reflectance spectroscopy revealed that Mg0.5Ni0.5Fe2O4 porous nanofibers possess the band gap (1.85 eV) between the calculated value for MgFe2O4 nanobelts and NiFe2O4 nanotubes due to alloying effects. The VSM analysis revealed that the saturation magnetization and coercivity of MgFe2O4 nanobelts were enhanced by Ni2+ incorporation. The electrochemical properties of samples coated on nickel foam (NF) were tested by CV, GCD, and EIS analysis in a 3 M KOH electrolyte. The Mg0.5Ni0.5Fe2O4@Ni electrode disclosed the highest specific capacitance of 647 F g−1 at 1 A g−1 owing to the synergistic effects of multiple valence states, exceptional porous morphology, and lowest charge transfer resistance. The Mg0.5Ni0.5Fe2O4 porous fibers showed superior capacitance retention of 91% after 3000 cycles at 10 A g−1 and notable Coulombic efficiency of 97%. Moreover, the Mg0.5Ni0.5Fe2O4//Activated carbon asymmetric supercapacitor divulged a good energy density of 83 W h Kg−1 at a power density of 700 W Kg−1.Ni-incorporated MgFe One-dimensional (1D) nanofibers providing numerous active sites for ion absorption are potentially one of the best options as an energy storage electrode owing to their magnificent morphologies2. Electrospinning is an efficient, cost-effective, controllable in diameter, convenient, and rapid method to fabricate different kinds of one-dimensional nanostructures such as nanofibers, nanobelts, and nanotubes with distinguished performances like excellent cycling stability, notable capacity, and suitable ionic conductivity. Various criteria, such as solution parameters , flow rate, applied voltage, heating rate, and temperature, have a significant influence on the morphology of electrospun nanostructures3. Metal spinel ferrites, generally known as MFe2O4 , which M and Fe ions may locate at both tetrahedral and octahedral positions in a cubic close packing of oxygen, attracted widespread attention in the past few years due to their simplicity of synthesis, high electrical conductivity, low electrical losses and inherent toxicity, physical and chemical stabilities, spontaneous magnetic and electrochemical nature resulting applications in different fields of technology5. Previously, the supercapacitive behavior of C/CuFe2O46, Fe2O3@SnO27, and ZnOFe2O48 nanofibers have been studied extensively.The ever-increasing worldwide demand for energy is urging the development of energy storage devices and materials with impressive specific capacitance and brilliant cycle stability, such as supercapacitors2O4), possessing a band gap of 2.18 eV, is a well-known n-type semiconducting material mainly used as a microwave absorber and lithium-ion batteries due to moderate saturation magnetization and high chemical stability11. Nickel ferrite (NiFe2O4) is an n-type semiconductor, having low coercivity and high electrical resistivity12. Both MgFe2O4 and NiFe2O4 possess cubic inverse spinel structures. In inverse spinel structure, a divalent cation  occupies half of the octahedral B-sites coordination and a trivalent cation (Fe3+) locates at tetrahedral A-sites as well as half of the octahedral B-sites17.Magnesium ferrite  nanoparticles using aloe vera extract solution. The results revealed that Ni0.25Mg0.75Fe2O4 electrodes show a high specific capacitance with cycling stability of 88.79% after a 1000 cycle21. Also, enhancing the surface area of the active materials by the fabrication of diverse morphology using various techniques is the most effective strategy to increase the performance of supercapacitors21. The physical properties of diverse morphology of MgFe2O4 and NiFe2O4 prepared by various methods such as hollow cubes27, core–shell28, rose-like nanoflower18, mesh-like structure19, thin film29, beehive-like nanosheets20, and nanoparticle21 have been reported earlier. One-dimensional nanomaterials, prepared via facile and low-cost electrospinning techniques, revealed superior electrochemical performance in supercapacitors owing to their unique morphology and fascinating properties, representing the great potential for boosting the performance of supercapacitors30. However, to the best of our knowledge, no reports are available focusing on the supercapacitive behavior of one-dimensional (1D) MgFe2O4 nanobelts, NiFe2O4 nanotubes, and ternary spinel Mg0.5Ni0.5Fe2O4 nanofibers. In this research, we have investigated the magnetic, optical, and electrochemical properties of these materials fabricated by the sol–gel electrospinning method. The results suggested that Mg0.5Ni0.5Fe2O4 caterpillar-like nanofibers can be used as a promising electrode for supercapacitor applications.NiFe3)2.6H2O, > 99%, Merck), nickel nitrate (Ni(NO3)2.6H2O, > 99%, Merck), iron nitrate (Fe(NO3)3.9H2O, > 99%, Merck), N, N-Dimethylformamide (DMF), Polyvinylpyrrolidone  and ethanol , carbon black (Alfa Aesar), polyvinylidene difluoride , N-methyl-2-pyrrolidone , and potassium hydroxide  were analytical grade and used without further purification. Deionized (DI) water (> 18.4 MΩ cm−1) from a Merck Millipore water purification system was used throughout the experiment. A flexible interconnected porous nickel foam with 1.5 mm thickness and a purity > 99% was supplied by Latech Scientific Supply Pte. Ltd. (Singapore).Magnesium nitrate (Mg(NO2O4 (MFO), NiFe2O4 (NFO), and Mg0.5Ni0.5Fe2O4 (MNFO) nanofibers were fabricated using the sol–gel electrospinning technique. Magnesium ferrite was synthesized by adding 0.2564 g of Mg(NO3)2.6H2O and 0.4039 g Fe(NO3)3.9H2O to 10 mL ethanol/DMF (50:50 w/w) mixed solvents. After that, 1 g of PVP was added dropwise into the above solution and stirred for 12 h. The formed homogeneous sol was loaded into a syringe equipped with a 25 G blunt stainless needle and placed in a syringe pump at a distance of 12 cm from flat aluminum foil as a collector. The syringe pump and collector were connected to a high-voltage power supply, applying a voltage of 17.5 kV. The electrospinning process was done at the rate of 0.5 mL h−1 under the controlled condition at room temperature with a relative humidity of 45%. The previous method was repeated to prepare Mg0.5Ni0.5Fe2O4 ferrite, where Mg(NO3)2.6H2O (1.282 g), Ni(NO3)2.6H2O (0.1454 g), and Fe(NO3)3.9H2O (0.4039 g) were used as precursors. All of the electrospun samples were dried at 100 °C for 12 h and then calcined at 600 °C in the air for 2 h with a heating rate of 2 °C/min. The schematic representation of Mg0.5Ni0.5Fe2O4 nanofibers preparation is shown in Fig. MgFe2). Afterward, the electrodes were dried at 80 °C for 3 h. The loaded mass on Ni foam was about 1 mg.The synthesized samples as active material (80 wt.%), carbon black (10 wt.%), and PVDF (10 wt.%), were mixed and a slurry was prepared by the addition of N-methyl-2-pyrrolidone (NMP) as a solvent. Then, the prepared slurry was coated onto the nickel foam  and Brunauer–Emmett–Teller (BET) techniques were used to measure the pore size distribution and surface area of the samples, respectively. The optical features of samples were explored using the Scinco-S4100 device. Magnetic measurements were done using MDK  device. A three-electrode system, consisting of a prepared electrode, Ag/AgCl, and platinum wire as working, reference, and counter electrodes in a 3 M KOH electrolyte, was used to test the electrochemical efficiency of the synthesized specimens. Using a ZAHNER-ZENNIUM device, cyclic Voltammetry, galvanostatic charge–discharge, and electrochemical impedance spectroscopy (EIS) measurements were conducted over a frequency range of 0.01–105 Hz at an AC amplitude of 10 mV. The Zview software was used to fit the EIS plot.Structural properties of prepared nanomaterials were investigated through X-ray diffraction analysis using the X'Pert Pro Philips device. The chemical bonds of nanofibers were studied by Fourier transform infrared analysis using the Alpha—Bruker device. The MIRA3TESCAN-XMU instrument was used to perform field emission scanning electron microscopy and energy dispersive spectroscopy to investigate nanofibers' morphological characteristics and elemental composition, respectively. N2O4, NiFe2O4, and Mg0.5Ni0.5Fe2O4 nanofibers in the range of 10–80° are demonstrated in Fig. 2O4 (JCPDS Card No. 36-0398) and NiFe2O4 (JCPDS Card No. 10-0325)31. No additional peaks related to any secondary phase were detected in the XRD pattern of synthesized nanofibers, indicating the high-purity phase in samples. On the XRD patterns, Rietveld fitting is conducted using MAUD software to further check the phase purity of the samples. The fitted XRD patterns are shown in Fig. 2+ incorporation in magnesium ferrite not only did not disturb the spinel structure but also the sharp diffraction peaks of the Mg0.5Ni0.5Fe2O4 nanofibers suggest the enhanced crystallinity compared to MgFe2O4 and NiFe2O4 samples. The lattice constant (a) was determined from the interplanar spacing (d) for the (311) plane, using the equation for a cubic lattice structure: a = d(h2 + k2 + l2)1/2, where h, k, and l are the Miller indices. The unit cell volume of the samples is denoted in Table 2+ was incorporated in magnesium ferrite, the calculated lattice parameter decreased from 8.38 to 8.33 Å due to the substitution of the smaller Ni2+ ions  for the larger Mg2+ ions , which is in line with the results reported for Mg0.5Ni0.5Fe2O4 nanoparticles by a hydrothermal route21.The X-ray diffraction patterns of synthesized MgFe32. The average crystallite size (D) and lattice strain (ε) of prepared samples are obtained from the intercept and slope of the linear fit of the Williamson–Hall (W–H) plot  occupy octahedral B-sites and trivalent ions (Fe3+) are equally distributed among tetrahedral A-sites and octahedral B-sites34. The bands at 438 and 465 cm−1 correspond to the intrinsic stretching vibration of the octahedral sites of Mg-O and Ni–O (νo); also the band at 580 cm−1 represents the intrinsic stretching vibration of the tetrahedral site of Fe–O (νt), respectively, confirming the formation of the spinel structure of MgFe2O4 and NiFe2O435. In the MNFO sample, the metal–oxygen absorption bands slightly shifted with the Ni2+ incorporation. This may be due to Mg, Ni, and Fe cations redistribution on both sites. The broad band around 3419 and the less intense band around 1637 cm−1 indicated the characteristic vibrational modes of O–H groups and vibrations of the absorbed water molecules, respectively36. The bands observed at 2859 and 2937 cm−1 disclosed the asymmetric and symmetric stretching vibrations of methylene (–CH2) groups, respectively12. The band at 1380 cm−1 was ascribed to the carboxylate group37. The bands in the range of 1000 to 1250 cm−1 correspond to nitrate ion traces as well as C–O bonding38. After calcination, they become weaker. Another band located around 2359 cm−1 was reasonably attributed to the anti-symmetrical vibration of carbon dioxide absorbed from the environment, observed only in the as-spun MgFe2O4 sample39.Figure 2O4, NiFe2O4, and Mg0.5Ni0.5Fe2O4 samples calcined at 600 °C are represented at three different magnifications in Fig. 2O4 sample demonstrates long and continuous solid inner belt-like morphology with rectangular cross-sections that possess an average diameter of 65 nm , Planck's constant, the light frequency, and the constant parameters-containing characteristics of the bands, respectively. Also, n = 1/2 is considered to determine the direct optical band gap (Eg) of MgFe2O4 and NiFe2O4, and the plot of (αhν)2 versus hν is shown in Fig. 44. The band gap of MFO nanobelts, NFO nanotubes, and MNFO caterpillar-like nanofibers was found to be 1.90, 1.80, and 1.85 eV, respectively. It is seen that the band gap of MNFO nanofibers is narrower than the MFO nanobelts sample because adding nickel in the MNFO preparation process induces inner states in the band gap, providing additional levels between the conduction and the valence bands44. The calculated band gap value of MNFO nanocomposite lies between the values obtained for the band gap of magnesium ferrite and nickel ferrite due to alloying effect and indicated suitable substitution of Ni ions on Mg sites in MgFe2O4. The band gap narrowing of magnesium ferrite nanoparticles by Ni substitution was reported by other researchers48.In order to investigate the optical properties of prepared nanofibers, the UV–vis-DRS spectra were recorded in the region of 300–900 nm Fig. a. Kubelkentclass1pt{minima13. From these hysteresis curves, the value of saturation magnetization (Ms), remnant magnetization (Mr), coercivity (Hc), and the magnetic moment  and spin canting at the surface of nanoparticles50. It is known that the crystallinity, surface imperfection, chemical composition, and cation distribution variation on octahedral and tetrahedral sites have a huge impact on the saturation magnetization of spinel ferrite nanostructures13. The increase in the Ms value of the MFO by Ni incorporation can be attributed to the larger magnetic moments of Ni2+  was utilized to measure the magnetization (M) of prepared nanomaterials as a function of the applied magnetic field (H). The hysteresis loops of prepared samples measured at room temperature are demonstrated in Fig. entclass1pt{minimaentclass1pt{minima2O4, NiFe2O4, and Mg0.5Ni0.5Fe2O4 electrodes was tested using a three-electrode system in 3 M KOH. The Cyclic Voltammetry (CV) of prepared samples at various scan rates of 10, 30, 50, and 80 mV s−1 with a potential window of 0–0.5 V is shown in Fig. 56. The energy storage mechanism of prepared samples is suggested by the following reactions58:The electrochemical performance of MgFe−1, revealing excellent electrochemical reversibility and prominent high-rate performance. However, the shifts of redox peaks towards lower/higher potentials may be attributed to the polarization effect. The specific capacitance from the CV profile was calculated according to the following equation:sp, −1), the integrated area under the CV plot, the mass of active material (g), the scan rate (V s−1), and the potential window (V), respectively59. The specific capacitance values of MgFe2O4, NiFe2O4, and Mg0.5Ni0.5Fe2O4 at the scan rate of 10 mV s−1 were 98, 385, and 965 F g−1, respectively, as shown in Fig. sp exhibited a higher specific capacitance at lower scan rates. This implies that the electrolyte ions had sufficient time to penetrate and access all the inner microstructures of the electrode material for charge storage60.The shape of the cyclic voltammogram of samples remains unchanged as the sweep rate increases from 10 to 80 mV s−1 demonstrated in Fig. sp, I, ∆t, m, and ∆V denote the current (A), the time of a full discharge (s), the mass of the active material (g), and the potential window (V)59. The specific capacitance of prepared samples at various current densities of 1, 3, 5, 7, and 10 A g−1 is exhibited in Fig. 2O4, NiFe2O4, and Mg0.5Ni0.5Fe2O4 at the current density of 1 A g−1 were obtained 97, 240, and 647 F g−1, respectively, which declined to 75, 150, and 325 F g−1 at the current density of 10 A g−1 due to reduced accessibility of active sites in high diffusion rate.The galvanostatic charge–discharge (GCD) profiles of prepared nanomaterials were recorded at a current density of 1, 3, 5, 7, and 10 A g2+ ions enhanced the electrochemical activities by enriching the redox states, and the whole process prominently improved the electrochemical chemical performance of the material63. (ii) The unique morphology of porous nanofibers, including high surface area and grain boundaries, played a major role in the electrochemical activities and capacitive energy storage of prepared materials65. The electrochemical behavior of nickel or magnesium-based ferrite has also been reported in the literature and some of them are listed in Table In both CV and GCD tests, the significantly high specific capacitance value of the MNFO samples is attributed to two main convincing reasons: (i) By adding nickel into the magnesium ferrite, the Ni−1, respectively. The Coulombic efficiency is also estimated according to the below equation:73. The Coulombic efficiency of prepared samples at various current densities of 1, 3, 5, 7, and 10 A g−1 is exhibited in Fig. 2O4 nanobelts, NiFe2O4 nanotubes, and Mg0.5Ni0.5Fe2O4 nanofibers demonstrated high Coulombic efficiency of 92, 95, and 97%, respectively, at the current density of 10 A g−1. Herein, the superior Coulombic efficiency of MNFO may be attributed to the appropriate formation of 1D surface area and designed architecture with high surface area, which cause the unique reversibility of the charge–discharge process of the prepared sample74.Long-term cycling stability as a criterion is also studied see Fig. a. The prmentclasspt{minimaThe electrochemical impedance spectroscopy (EIS) measurements of prepared nanomaterials were carried out in the frequency range of 0.01 Hz to 100 kHz.ct), solution resistance , Warburg resistance (Zw), and the constant phase element (CPE) are displayed in Fig. 61. The RS value of electrodes is very low, allowing the electrolyte to access the electrodes surface efficiently75. The Rct of MFO, NFO, and MNFO was calculated to be 6.64, 4.79, and 3.16 Ω, respectively. The lowest Rct value of nickel-substituted magnesium ferrite revealed that Ni incorporation in magnesium ferrite structure facilitates the charge transfer efficiency at the interface of electrode and electrolyte, affirming the higher specific capacitance of this electrode75. Also, steeper slopes of the plot in the low-frequency regions imply that the Mg0.5Ni0.5Fe2O4 sample has lower Warburg resistance (greater ionic conductivity) than other samples. Therefore, nickel incorporation in MgFe2O4 provided better ionic and electronic conductivity in the Mg0.5Ni0.5Fe2O4 sample.The Nyquist plot and the equivalent circuit diagram consist of charge transfer resistance  and power density (P) of MNFO//AC were calculated using Eqs.  as positive and negative electrodes, respectively, in a 3 M KOH electrolyte. At first, the electrochemical properties of AC electrode were investigated by the standard three-electrode system. Figure lts Fig. c. The ening Eqs.  and 10)−1 and cosp (F g−1) is the specific capacitance, ∆V (V) is the working voltage window, and ∆t (s) is the discharge time of the MNFO//AC cell. The results revealed a high energy density of 83 W h Kg−1 and a power density of 700 W Kg−1 at a current density of 1 A g−1.Where, C0.5Ni0.5Fe2O4 spinel ferrite nanofibers compared to pure MgFe2O4 nanobelts and NiFe2O4 nanotubes prepared by electrospinning technique. The XRD, FTIR, FESEM, EDS, DRS, and VSM studies are also done to show the maximum functionality of samples. XRD and FTIR results showed the well-crystallized cubic spinel phase and metal–oxygen bonds of the samples on the octahedral and tetrahedral sites, respectively. The optical band gap of Mg0.5Ni0.5Fe2O4 was narrower than MgFe2O4 nanobelts. The enhancement of saturation magnetization and coercivity of MgFe2O4 nanobelts via Ni2+ ions substation was confirmed using the VSM test. The electrochemical study revealed that although the specific capacitance obtained for the pristine magnesium ferrite nanobelts was small, the incorporation of nickel into its structure caused the formation of a novel ternary ferrite with a significant capacitance. The highest specific capacitance of 647 F g−1 for Mg0.5Ni0.5Fe2O4 with outstanding cycling stability of 91% after 3000 cycles at 10 A g−1 was achieved which is far greater than pristine MgFe2O4 and NiFe2O4. Furthermore, the Mg0.5Ni0.5Fe2O4//Activated carbon asymmetric supercapacitor cell could be cycled reversibly in the high-voltage range of 0 to 1.5 V and divulged intriguing performances with an energy density of 83 W h Kg−1 at a power density of 700 W Kg−1. Mg0.5Ni0.5Fe2O4 electrode with safe and suitable electrochemical performance is promising for practical application in energy storage devices and might play an important role in renewable energy, potentially reducing pollution and decreasing the consumption of hydrocarbon fuels. We hope that this work can open up new possibilities for exploring novel ternary ferrite spinels as electrode materials for application in the energy storage field.In this work, an attempt has been made to achieve the superior electrocapacitive performance from the novel and well-designed ternary Mg