Isotopic Labeling Experiments Research Articles

Oxidative Cleavage of Aromatic C-O Linkages by Oxoammonium Salts.

Oxidative cleavage of aromatic C(sp2)-O bond is important to the conversion of biomass and plastic wastes into value-added chemicals. Here we put forward the oxidative cleavage of para-C-O bonds in phenolic compounds in use of oxoammonium salts as oxidant and water as the oxygen source. The mechanism is that oxoammonium cation activates water to form hydroxy-oxoammonium adduct and thus realizes the ipso-substitution of 4-alkoxyphenol, which is proved by substituent effect, isotope labelling experiments, and kinetic analysis. Furthermore, this protocol is successfully applied into the depolymerization of both lignin model compounds with α-O-5 and 4-O-5 linkages and polyphenylene oxide (PPO).

Copper–nickel–MOF/nickel foam catalysts grown in situ for efficient electrochemical nitrate reduction to ammonia

Reducing nitrate (NO3−) in an aqueous solution to ammonia under ambient conditions can provide a green and sustainable NH3‐synthesis technology and mitigate global energy and pollution issues. In this work, a CuNi0.75–1,3,5‐benzenetricarboxylic acid/nickel foam (CuNi0.75–MOF/NF) catalyst grown in situ was prepared via a one‐pot method as an efficient cathode material for electrocatalytic nitrate reduction reaction (NO3RR). The CuNi0.75–MOF/NF catalyst exhibited excellent electrocatalytic NO3RR performance at −1.0 V versus a reversible hydrogen electrode, achieving an outstanding faradaic efficiency of 95.88 % and an NH3 yield of 51.78 mg h−1 cm−2. The 15N isotope labeling experiments confirmed that the sole source of N in the electrocatalytic NO3RR was the NO3− in the electrolyte. The reaction pathway for the electrocatalytic NO3RR was derived by in situ Fourier transform infrared spectroscopy and in situ differential electrochemical mass spectrometry. Density functional theory calculations revealed that the Ni element in the CuNi0.75–MOF/NF catalyst had excellent O–H activation ability and strong *H adsorption capacity. These *H species were transferred from the Ni sites to the *NO adsorption intermediates located on the Cu sites, providing a continuous supply of *H to Cu, thereby promoting the formation of *NOH intermediates and enhancing the hydrogenation process of the electrocatalytic NO3RR.

Corrosion behavior of Laves precipitates in FeCrAl-1Nb alloy at 290 °C and 7 MPa: The size effect and critical role of pH

The corrosion behavior of Laves precipitates in FeCrAl-1Nb alloy was investigated in four different deoxygenated solutions at 290 °C and 7 MPa. The Laves precipitates exhibit potential size effect during corrosion in a weak alkaline solution: the microscale precipitates undergo oxidation with amorphization and element dissolution, and these phenomena are not observed in nanoscale precipitates. The corrosion of nanoscale precipitates is less insensitive to slight changes in pH; however, micro-galvanic corrosion was observed between some nanoscale precipitates and the ferrite phase. Deuterium isotope labeling experiments observed hydrogen enrichment in some oxidized precipitates.

Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure.

While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in "reverse-labeling" mode: cells are initially grown in 13C-enriched media, with specific 12C-labeled amino acids added when protein expression is induced. 1 Because FTIR measurements require only around 20 μL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or β-sheet structures.

Tropical Leaf Wax n‐Alkane and n‐Alkanoic Acid Reflect δD of Precipitation During Early Stages of Leaf Growth: Insights From an Isotope Labeling Experiment

AbstractThe hydrogen isotopic compositions of leaf‐wax n‐alkanes (δDalk) and n‐alkanoic acids (δDacid) reflect ambient climatic conditions (including precipitation δD values, δDprecip). However, the understanding of climatic conditions of exactly which period (i.e., early or entire period of the leaf's lifespan) these biomarkers preserve is still evolving. Previous studies on the δDalk and δDacid values, done only in extra‐tropical regions, mostly indicate that δDalk values are biased toward the early growing season whereas δDacid values are not biased toward any season. To decipher the seasonal bias in δDalk and δDacid records from tropics, we conducted a long‐duration experiment wherein deciduous and evergreen species were grown using normal water (δD = −2‰) during early stages of the leaf growth and later using isotopically labeled water (δD = 1,000‰). Our experiment revealed (a) in deciduous and evergreen species, δDalk and δDacid values reflect δDprecip during early stages of the leaf growth, (b) synchronous synthesis of n‐alkanes and n‐alkanoic acids, and (c) in deciduous species, minor incorporation of the previous year's photosynthates in leaf wax pool of the current year's mature leaves. Our study suggests that δDalk and δDacid records in the tropics are biased toward the climatic conditions prevailing during early stages of leaf growth. This bias should be considered while comparing the δDprecip values generated from leaf wax proxy records and isotope‐enabled atmospheric circulation models.

Building organic-inorganic heterojunction NiCo2O4/h-BN toward artificial photosynthesis—boron as hole

To convert CO2 and water into organic chemicals via photoelectrocatalytic (PEC) reduction of CO2 under mild conditions is considered to be one of promising methods to address a series of problems like climate change and energy crisis caused by over-emission of CO2. Herein, the organic-inorganic heterojunctions were designed and fabricated by using organic h-BN and inorganic NiCo2O4 as precursors. The optimal NiCo2O4@h-BN-6 heterojunction exhibited impressive performance in CO2 reduction, yielding C2+ chemicals with 100% selectivity in a rate of 28.5μMcm−2 h−1. This phenomenon is attributed to the structural feature of three active sites of nitrogen in a unique hexacycle like benzene, which benefit the carbon-carbon coupling among the intermediates. The first observed *N-H species suggest that the N sites can adsorb protons and electrons and convert them into highly active hydrogen atoms. The verification experiments demonstrate that the material will oxidize intermediates to acetic acid where boron atoms are presented as holes in semiconductor. The isotopic labeling experiments of 13CO2 and H218O verified that the products were derived from CO2 and water. The key active intermediates such as *CHO, *=C=O, and *COCHO in the synthetic process of C2+ products had been confirmed by operando FTIR spectra and DFT calculations.

LuCl3/B(C6F5)3 Cocatalyzed Reductive Deoxygenation of Ketones, Aldehydes, Alcohols, and Ethers to Alkanes with Pinacolborane.

This report describes the LuCl3/B(C6F5)3 cocatalyzed reductive deoxygenation of 67 ketones, aldehydes, alcohols, and ethers to alkanes under mild conditions. The strategy tolerates reactive amino, hydroxyl, nitro, halogen, vinyl, and ester functional groups, and the results demonstrate rare chemoselective deoxygenation of α,β-unsaturated ketones. Isotopic labeling experiments, control experiments, and derivatization studies are used to elucidate the reaction mechanism.

Topological Synthesis of 2D High-Entropy Multimetallic (Oxy)hydroxide for Enhanced Lattice Oxygen Oxidation Mechanism.

Owing to sluggish reaction kinetics and high potential, oxygen evolution reaction (OER) electrocatalysts face a trade-off between activity and stability. Herein, an innovative topological strategy is presented for preparing 2D multimetallic (oxy)hydroxide, including ternary CoFeZn, quaternary CoFeMnZn, and high-entropy CoFeMnCuZn. The key to the synthesis lies in using Ca-rich brownmillerite oxide as a precursor, which possesses inherent structural flexibility enabling tailored elemental adjustments and topologically transforms from a point-shared structure of metal-oxygen octahedrons into an edge-shared structure under alkaline conditions. The presence of Zn in the catalysts causes a shift in the center of the O2p band toward the Fermi level, resulting in more Co4+ species, which drive holes into oxygen ligands to promote intramolecular oxygen coupling. The triggered lattice oxidation mechanism is identified by detecting peroxo-like (O2 2-) negative species using tetramethylammonium chemical probe, along with 18O isotope labeling experiments. As a result, the catalyst demonstrates an overpotential of 267mV at 10 mA cm-2, ranking it among the top-performing non-Ni-based catalysts. Importantly, the catalysts also show high Fe-leaching resistance during OER compared to conventional NiFe and CoFe hydroxides/(oxy)hydroxides. The assembled zinc-air battery enables stable operation for over 225 h at a low charging voltage of 1.93V.

Single-step conversion of methane-steam to methanol on single-atom Cu1/γ-Al2O3 catalyst prepared via electrostatic anchoring

Direct conversion of methane to methanol in heterogeneous catalysis is of paramount importance but remains a challenging issue in improving catalytic activity and selectivity. Here, we report that Cu single-atom sites anchored on γ-Al2O3 oxides (Cu1/γ-Al2O3) using surface electrostatic adsorption, are highly active (methanol yield of ∼47 mmol/molCu/h) and stable in continuous conversion of methane-steam to methanol at 200 °C, comparable to most of the state-of-the-art Cu-zeolite catalysts. The combined DFT calculation and isotope labeling experiments reveal the methane-to-methanol reaction pathway on Cu single-atom sites and methanol formation is the rate limiting step. The further theoretical study indicates that single Cu atom as active site can preferentially activate methane C-H bond instead of methanol and has larger advantage for highly-selective methanol yield compared with [Cu(II)-OH]+ and [CuOCu]2+ active sites. Our molecular-level findings on Cu1/γ-Al2O3 single-atom catalyst pave the way to prepare better non-zeolite catalysts for methane to methanol.

CDS2 expression regulates de novo phosphatidic acid synthesis.

CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.

Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways

Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.

Nitrate Enhanced Sulfamethoxazole Degradation by 222 nm Far-UVC Irradiation: Role of Reactive Nitrogen Species.

The application of 222 nm far-UVC irradiation for degrading organic micropollutants in water shows promise. Nitrate (NO3-), found in nearly all water bodies, can significantly impact the performance of 222 nm far-UVC-driven systems. This work was the first to investigate the effect of NO3- on sulfamethoxazole (SMX) photodegradation at 222 nm, finding that NO3- significantly enhances SMX degradation in different dissociated forms. Besides the hydroxyl radical (•OH), reactive nitrogen species (RNS) also played important roles in SMX degradation. With increasing NO3- concentration, the RNS contribution to SMX degradation decreased from 25.7 to 8.6% at pH 3 but increased from 1.5 to 24.7% at pH 7, since the deprotonated SMX with electron-rich groups reacted more easily with RNS. The transformation mechanisms of SMX involving isomerization, bond cleavage, hydroxylation, nitrosation, and nitration processes were proposed. 15N isotope labeling experiments showed that the RNS-induced nitrated products even became the major products of SMX in the 222 nm far-UVC/NO3- system at pH 7 and exhibited a higher toxicity than SMX itself. Further research is necessary to avoid or eliminate these toxic byproducts. This study provides valuable insights for guiding the utilization of 222 nm far-UVC for treating antibiotics in NO3--containing water.

Surface-controlled oxygen vacancy-rich Bi/BiVO4 Schottky junction for highly selective and active photocatalytic nitrogen fixation

The modulation of active site density and localized electron concentration represents a crucial step in enhancing nitrogen activation, thereby significantly augmenting the efficiency of photocatalytic nitrogen fixation. In this paper, Bi/BiVO4 Schottky junctions enriched with oxygen vacancies (Bi/Vo-BVO) were fabricated by in situ reduction. Compared to BiVO4, Bi/Vo-BVO composites exhibited 8.4-fold enhancement in ammonia synthesis yield, reaching a maximum rate of 167.74 μmol·g−1·h−1. In situ FTIR spectroscopy and isotopic labeling experiments definitively demonstrated that the nitrogen constituent of NH4+ is derived from N2. N2-TPD and DFT calculation collectively suggest that the surface of Bi/Vo-BVO, enriched with ligand-unsaturated V4+ species, furnishes a substantial array of chemisorption sites for N2 molecules, attributed to the abundance of oxygen vacancies. In situ XPS coupled with DFT corroborate that the photoexcited electrons within the BiVO4 valence band accumulate in the conduction band, thereby directly facilitating the activation of N2 adsorbed at the V4+ active sites; The built-in electric field established at the interface between Bi and BiVO4 substantially facilitates the enrichment of Bi-emitted thermal electrons within oxygen vacancies on BiVO4 surface. These electrons subsequently migrate towards and interact with the adsorbed N2 via the V4+ active sites. The synergistic photothermal electrons activation mechanism of N2 not only enhances the efficiency of light energy utilization but maximizes the catalytic efficacy of the photocatalyst through the full exploitation of its intrinsic catalytic potential.

Nickel-Catalyzed C-C Activation of Vinylcyclobutane with Visible Light: Scope, Mechanism, and Application to Chemically Recyclable Polyolefins.

N-heterocyclic carbene (NHC)-supported nickel complexes were investigated for the oxidative ring-opening of vinylcyclobutane (VCB) and photocatalytic activity. Addition of VCB to in situ generated [(NHC)Ni(0)] compounds furnished (NHC)Ni(VCB)2 that underwent oxidative addition and conversion to the corresponding Ni(II) alkyl, allyl-metallacycles. The (NHC)Ni(C6H10) metallacycles were isolated, characterized, and exhibited high thermal and chemical stability. Irradiation with visible light at ambient temperature produced a mixture of ethylene and 4-vinylcyclohexene and 1,5-cyclooctadiene, cycloaddition dimers of butadiene, arising from formal retro-[2 + 2] cycloaddition. A mixture of hexadiene products arising from β-H elimination from the metallacycle was also observed. Free ethylene also underwent a secondary reaction to form cyclopropane products through formal [2 + 1] cycloaddition. A series of sterically and electronically modified NHC ligands was evaluated to establish the structure-activity relationship governing the rate of photocatalytic conversion of VCB and the resulting product distribution. Isotopic labeling experiments, resting state analysis, and independent synthesis of a range of nickel bis(olefin) complexes provided insight into the mechanism of the reaction and origins of the organic product mixture. (NHC)Ni-catalysis was also applied toward the retro-[2 + 2] depolymerization of (1,n'-divinyl)-oligocyclobutane to butadiene dimers.

A Chemoselective Enrichment Strategy for In-Depth Coverage of the Methyllysine Proteome.

Proteomics is a powerful method to comprehensively understand cellular posttranslational modifications (PTMs). Owing to low abundance, tryptic peptides with PTMs are usually enriched for enhanced coverage by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Affinity chromatography for phosphoproteomes by metal-oxide and pan-specific antibodies for lysine acetylome allow identification of tens of thousands of modification sites. Lysine methylation is a significant PTM; however, only hundreds of methylation sites were identified by available approaches. Herein we report an aryl diazonium based chemoselective strategy that enables enrichment of monomethyllysine (Kme1) peptides through covalent bonds with extraordinary sensitivity. We identified more than 10000 Kme1 peptides from diverse cell lines and mouse tissues, which implied a wide lysine methylation impact on cellular processes. Furthermore, we found a significant amount of methyl marks that were not S-adenosyl methionine (SAM)-dependent by isotope labeling experiments.

A Chemoselective Enrichment Strategy for In‐Depth Coverage of the Methyllysine Proteome

AbstractProteomics is a powerful method to comprehensively understand cellular posttranslational modifications (PTMs). Owing to low abundance, tryptic peptides with PTMs are usually enriched for enhanced coverage by liquid chromatography‐mass spectrometry/mass spectrometry (LC–MS/MS). Affinity chromatography for phosphoproteomes by metal‐oxide and pan‐specific antibodies for lysine acetylome allow identification of tens of thousands of modification sites. Lysine methylation is a significant PTM; however, only hundreds of methylation sites were identified by available approaches. Herein we report an aryl diazonium based chemoselective strategy that enables enrichment of monomethyllysine (Kme1) peptides through covalent bonds with extraordinary sensitivity. We identified more than 10000 Kme1 peptides from diverse cell lines and mouse tissues, which implied a wide lysine methylation impact on cellular processes. Furthermore, we found a significant amount of methyl marks that were not S‐adenosyl methionine (SAM)‐dependent by isotope labeling experiments.

Picolinic acid-mediated Mn(II) activated periodate for ultrafast and selective degradation of emerging contaminants: Key role of high-valent Mn-oxo species

The utilization of periodate (PI, IO4-) in metal-based advanced oxidation processes (AOPs) for the elimination of emerging contaminants (ECs) have garnered significant attention. However, the commonly used homogeneous metal catalyst Mn(II) performs inadequately in activating PI. Herein, we exploited a novel AOP technology by employing the complex of Mn(II) with the biodegradable picolinic acid (PICA) to activate PI for the degradation of electron-rich pollutants. The performance of the Mn(II)-PICA complex surpassed that of ligand-free Mn(II) and other Mn(II) complexes with common aminopolycarboxylate ligands. Through scavenger, sulfoxide-probe transformation, and 18O isotope-labeling experiments, we confirmed that the dominant reactive oxidant generated in the Mn(II)-PICA/PI system was high-valent manganese-oxo species (Mn(V)=O). Due to its reliance on Mn(V)=O, the Mn(II)-PICA/PI process exhibited remarkable selectivity and strong anti-interference during EC oxidation in complex water matrices. Nine structurally diverse pollutants were selected for evaluation, and their lnkobs values in the Mn(II)-PICA/PI system correlated well with their electrophilic/nucleophilic indexes, EHOMO, and vertical IP (R2 = 0.79–0.94). Additionally, IO4- was converted into non-toxic iodate (IO3-) without producing undesired iodine species such as HOI, I2, and I3-. This study provides a novel protocol for metal-based AOPs using PI in combination with chelating agents and high-valent metal-oxo species formation during water purification.

Pyrolytic conversion of glucose into hydroxymethylfurfural and furfural: Benchmark quantum-chemical calculations.

Quantum chemical methods have been intensively applied to study the pyrolytic conversion of glucose into hydroxymethylfurfural (HMF) and furfural (FF). Herein, we collect the most relevant mechanistic proposals from the recent literature and organize them into a single reaction network. All the transition structures (TSs) and intermediates are characterized using highly accurate ab initio methods and the possible reaction pathways are assessed in terms of the Gibbs energies of the TSs and intermediates with respect to β-glucopyranose, selecting a 2D ideal-gas standard state at 773 K to represent the pyrolysis conditions. Several pathways can lead to the formation of both HMF and FF passing through rate-determining TSs that have ΔG‡ values of ~49-50 kcal/mol. Both water-assisted mechanisms and nonspecific environmental effects have a minor impact on the Gibbs energy profiles. We find that the HMF → FF + CH2O fragmentation has a small ΔrxnG value and an accessible ΔG‡ barrier. Our computational results, which are in consonance with the kinetic parameters derived from lumped models, the results of isotopic labeling experiments and the reported HMF/FF molecular ratios, could be useful for modeling studies including on nonequilibrium kinetic effects that may render more information about product yields and the relevance of the various pathways.

Novel Methods for the Formation of Free Carbyne Radicals in Solution.

Carbyne free radicals RC⋮ (RC) are usually associated with high-energy processes, thus research on their preparation, chemical reactivity, and prevalence under mild conditions is scarce. Recently, it was reported that metallo-complexes containing CR ligands may undergo spontaneous degradation in aqueous solutions to produce free RC radicals. These highly reactive species may react with each other to form the corresponding alkyne and other products. The reaction of 1,1,1 halo-carbo-hydrates with Cr(II) ions also forms RC radicals under mild conditions. Herein, we report two additional synthetic routes that produce free RC radicals under mild conditions. First, the reaction between metallic zinc and acetic anhydride produces 2-butyne and several other C2, C3, and C4 compounds. Isotopic-labeling experiments indicate that the formation of 2-butyne results from an inter-molecular reaction in which two RC moieties from two acetic anhydride molecules combine in solution. In addition, the degradation of the tri-molybdenum complex [Mo3(H3CC≡CCH3)(OAc)(H2O)2Br7], in which a 2-butyne ligand is coordinated to the Mo3 framework in a μ3-η2 (⊥) binding mode in aqueous solution produces several C2, C3, C4 and C5 molecules. This indicates the formation of free CH3C radicals by a homolytic cleavage of the carbon-carbon triple bond.

Insights into the multiple active sites in Bi-Co bimetallic oxide for a deeper understanding of nitrate electroreduction to ammonia

The electrochemical nitrate reduction reaction (NO3RR) is a promising approach for nitrate removal and NH3 production at ambient conditions. The development of various types of efficient electrocatalysts, especially bimetallic oxides with the advantages of superior durability and excellent catalytic activity, have become a research focus. However, the analysis of active sites for bimetallic oxide catalysts either emphasizes the role of bimetallic interactions or focuses on the role of oxygen vacancies (OVs), resulting in a lack of thoroughness. Herein, a Bi-Co bimetallic oxide (BCO) was constructed for NO3RR, illustrating 1.5∼7.8 folds higher NH3 yield rate than those of commercial single metal oxide (Bi2O3, Co3O4). The NO3RR pathway by BCO was explored according to H* capture test, DEMS, and isotope labeling experiment. An in-situ Raman test showed the structure evolution of BCO during NO3RR, and therefore the catalytic sites of BCO were further identified according to physical characteristics before and after NO3RR. Results suggest that Bi0, OVs and amorphous Co oxide as the real active sites play a vital role in nitrate electroreduction. Furthermore, research on 25 articles also indicates the presence of multiple catalytic sites in bimetallic oxides for NO3RR. This study provides a new and comprehensive insight into the active sites identification for the type of bimetallic oxide NO3RR electrocatalysts.