Isotopic Labeling Experiments Research Articles

Low-crystallinity porous Ni2P2O7 nanowires with excellent CO2 capture ability for highly-efficient CO2 reduction

In this study, we prepared the porous Ni2P2O7 nanowires with low-crystallinity (LC-NPO) and high-crystallinity Ni2P2O7 nanosheets (HC-NPO), and employed them for the first time as catalyst in the CO2 photoreduction reaction. The photocatalytic performance of them was tested under the UV–Vis light irradiation and compared with the TiO2 commercial powder (P25). The CO2 photoreduction experiments shown that the LC-NPO had the highest ability than the HCNPO and the P25. Specifically, with triethanolamine (TEOA) as the sacrificial agent, LC-NPO achieved CO and CH4 yields of approximately 56.06 and 12.13 μmol·g−1 after 4 h UV–Vis light irradiation, which were 4.67 and 5.00 times of P25, respectively. The experimental results, and Density Functional Theory calculations proved that the unique porous nanowire structure enables LC-NPO to have a larger specific surface area, higher quantity of oxygen defects, and superior CO2 capture ability compared to HC-NPO. These features maybe the primary reasons for the excellent CO2 photoreduction ability. Besides, the photo-electrochemical characterizations revealed that the LC-NPO had the much superior photoelectrochemical properties than the HC-NPO, which was another reason for enhanced photocatalytic ability. In-situ FTIR and 13C isotope labeling experiments were used to monitor the CO2 reduction process, and finally a potential enhanced mechanism had been discussed.

TBuOK facilitated addition of diarylmethanes to styrenes through a radical reaction mechanism

Herein, we described the alkali-facilitated bonding of diarylmethanes to alkenes through a radical pathway. Diaryl compound products can be obtained under mild conditions with good yields. The reaction employed a practical approach by utilizing only 10% potassium tert-butoxide to synthesize diaryl compounds through a radical mechanism. The radical nature of this reaction was confirmed through radical capture experiments and EPR. Furthermore, we substantiated this characteristic with experimental evidence such as kinetic study and isotope labeling experiments.

Facet engineering of α-MnO2 for directly electrocatalytic oxygen atom transfer from water toward epoxidation

Electrocatalytic epoxidation, utilizing sustainable water and electricity as clean oxygen source and chemical oxidant surrogates respectively, represents an attractive approach toward industrial applications. However, the low yield and selectivity of epoxides are still a formidable challenge in satisfying industrial demand. Herein, we demonstrated a crystal facet engineering strategy over α-MnO2 catalysts for enhancing electrocatalytic epoxidation. Compared with α-MnO2-110 and α-MnO2-100, the α-MnO2 exposed with (310) facet exhibited excellent activity. Furthermore, a 21-fold yield increase was obtained compared with the reported superior Mn-based electrocatalyst. These results revealed that crystal facet engineering could induce the formation of more oxygen vacancies and weaken the interaction of Mn-O band to regulate oxygen atom transfer capacity. In addition, the reactive intermediate (MnOOH) was observed in an organic electrolyte, which was confirmed by in situ Raman spectra and isotope labeling experiment.

Ring–Opening Mechanism of O‐heterocycles into α,ω‐Diols over Ni−La(OH)3: C−O Bond Hydrogenolysis of THFA to 1,5‐Pentanediol as a Case Study

AbstractThe elucidation of the ring–opening reaction mechanism is a critical step towards improving the catalytic performance for the conversion of biomass into value–added chemicals. Herein, we focused on the stepwise C−O bond hydrogenolysis mechanism of oxygen–containing heterocycles (O‐heterocycles) into α,ω‐diols, in particular THFA to 1,5‐PeD, over selective Ni−La(OH)3 in hydrogen–donor isopropanol. A mechanistic study was carried out on a structurally well–defined Ni−La(OH)3, where the mechanism was elucidated using a combination of kinetic and 13C NMR isotope labeling experiments. It was suggested that both Ni nanoparticles and La(OH)3 support play a critical role in the reaction mechanism, where basic hydroxide species of the support initially deprotonate the CH2OH and −OH modified furan, tetrahydrofuran and tetrahydropyran rings, adsorbing them chemically on the catalyst surface. This step is followed by a direct hydride attack on the second carbon atom of these rings, which is proposed to be the key step for ring cleavage, as indicated by the increased deuteration at this position. In addition to the direct hydrogenolysis using gaseous H2, it is assumed that a catalytic transfer hydrogenolysis reaction (CTH) reaction of THFA is proposed to proceed via the dehydrogenation of isopropanol over isolated Ni species, forming hydrogen species that can be adsorbed on the Ni surface or desorb as H2 molecules.

Stabilizing Lattice Oxygen through Mn Doping in NiCo2O4-δ Spinel Electrocatalysts for Efficient and Durable Acid Oxygen Evolution.

Design the electrocatalysts without noble metal is still a challenge for oxygen evolution reaction (OER) in acid media. Herein, we reported the manganese (Mn) doping method to decrease the concentration of oxygen vacancy (VO) and form the Mn-O structure adjacent octahedral sites in spinel NiCo2O4-δ (NiMn1.5Co3O4-δ), which highly enhanced the activity and stability of spinel NiCo2O4-δ with a low overpotential (η) of 280 mV at j=10 mA cm-2 and long-term stability of 80 h in acid media. The isotopic labelling experiment based on differential electrochemical mass spectrometry (DEMS) clearly demonstrated the lattice oxygen in NiMn1.5Co3O4-δ is more stable due to strong Mn-O bond and shows synergetic adsorbate evolution mechanism (SAEM) for acid OER. Density functional theory (DFT) calculations reveal highly increased oxygen vacancy formation energy (EVO) of NiCo2O4-δ after Mn doping. More importantly, the highly hydrogen bonding between Mn-O and *OOH adsorbed on adjacent Co octahedral sites promote the formation of *OO from *OOH due to the greatly enhanced charge density of O in Mn substituted sites.

Upcycling of polyethylene to gasoline through a self-supplied hydrogen strategy in a layered self-pillared zeolite

Conversion of plastic wastes to valuable carbon resources without using noble metal catalysts or external hydrogen remains a challenging task. Here we report a layered self-pillared zeolite that enables the conversion of polyethylene to gasoline with a remarkable selectivity of 99% and yields of >80% in 4 h at 240 °C. The liquid product is primarily composed of branched alkanes (selectivity of 72%), affording a high research octane number of 88.0 that is comparable to commercial gasoline (86.6). In situ inelastic neutron scattering, small-angle neutron scattering, solid-state nuclear magnetic resonance, X-ray absorption spectroscopy and isotope-labelling experiments reveal that the activation of polyethylene is promoted by the open framework tri-coordinated Al sites of the zeolite, followed by β-scission and isomerization on Brönsted acids sites, accompanied by hydride transfer over open framework tri-coordinated Al sites through a self-supplied hydrogen pathway to yield selectivity to branched alkanes. This study shows the potential of layered zeolite materials in enabling the upcycling of plastic wastes.

The beneficial rhizobacterium Bacillus velezensis SQR9 regulates plant nitrogen uptake via an endogenous signaling pathway.

Nitrogen fertilizer is widely used in agriculture to boost crop yields. Plant growth-promoting rhizobacteria (PGPRs) can increase plant nitrogen use efficiency through nitrogen fixation and organic nitrogen mineralization. However, it is not known whether they can activate plant nitrogen uptake. In this study, we investigated the effects of volatile compounds (VCs) emitted by the PGPR strain Bacillus velezensis SQR9 on plant nitrogen uptake. Strain SQR9 VCs promoted nitrogen accumulation in both rice and Arabidopsis. In addition, isotope labeling experiments showed that strain SQR9 VCs promoted the absorption of nitrate and ammonium. Several key nitrogen-uptake genes were up-regulated by strain SQR9 VCs, such as AtNRT2.1 in Arabidopsis and OsNAR2.1, OsNRT2.3a, and OsAMT1 family members in rice, and the deletion of these genes compromised the promoting effect of strain SQR9 VCs on plant nitrogen absorption. Furthermore, calcium and the transcription factor NIN-LIKE PROTEIN 7 play an important role in nitrate uptake promoted by strain SQR9 VCs. Taken together, our results indicate that PGPRs can promote nitrogen uptake through regulating plant endogenous signaling and nitrogen transport pathways.

Stabilizing Lattice Oxygen through Mn Doping in NiCo2O4−d Spinel Electrocatalysts for Efficient and Durable Acid Oxygen Evolution

Design the electrocatalysts without noble metal is still a challenge for oxygen evolution reaction (OER) in acid media. Herein, we reported the manganese doping method to decrease the concentration of oxygen vacancy (Vo) and form the Mn−O structure adjacent octahedral sites in spinel NiCo2O4−δ (NiMn1.5Co3O4−δ), which highly enhanced the activity and stability of spinel NiCo2O4−δ with a low overpotential (η) of 280 mV at j = 10 mA cm−2 and long‐term stability of 80 h in acid media. The isotopic labelling experiment based on differential electrochemical mass spectrometry (DEMS) clearly demonstrated the lattice oxygen in NiMn1.5Co3O4−δ is more stable due to strong Mn‐O bond and synergetic adsorbate evolution mechanism (SAEM) for acid OER. Density functional theory (DFT) calculations reveal highly increased oxygen vacancy formation energy (EVO) of NiCo2O4−δ after Mn doping. More importantly, the highly hydrogen bonding between Mn−O and *OOH adsorbed on adjacent Co octahedral sites promote the formation of *OO from *OOH due to the greatly enhanced charge density of O in Mn substituted sites.

Isolated Ni sites anchored on zeolites for direct synthesis of acetic acid from methane oxidative carbonylation

Direct conversion of methane to acetic acid with high activity and selectivity over non-noble metal-based catalysts under mild reaction conditions remains a grand challenge. Specifically, 1.2Ni-ZSM-5 with 88.9 % isolated Ni sites achieved a high acetic acid yield of 2920 μmol/gcat. and a remarkable selectivity up to 82.3 % from methane oxidative carbonylation. Combined with high angle annular dark field scanning transmission electron microscopy, X-ray photoelectron spectroscopy, and X-ray absorption spectroscopy analyses, it was revealed that the isolated Ni species with Ni-O6 moieties anchored in the micropores of ZSM-5 were suggested to be the active sites. The isotopic labeling experiments and monitoring of reaction intermediates demonstrated that CH3COOH was formed via the direct coupling of methyl radicals and carbon monoxide molecules. This work suggests that the well-designed Ni sites can promote the transformation of CH4 to C2+ oxygenates instead of traditional C1 oxygenates as main products.

Electrokinetic Analyses Uncover the Rate‐Determining Step of Biomass‐Derived Monosaccharide Electroreduction on Copper

AbstractElectrochemical biomass conversion holds promise to upcycle carbon sources and produce valuable products while reducing greenhouse gas emissions. To this end, deep insight into the interfacial mechanism is essential for the rational design of an efficient electrocatalytic route, which is still an area of active research and development. Herein, we report the reduction of dihydroxyacetone (DHA)—the simplest monosaccharide derived from glycerol feedstock—to acetol, the vital chemical intermediate in industries, with faradaic efficiency of 85±5 % on a polycrystalline Cu electrode. DHA reduction follows preceding dehydration by coordination with the carbonyl and hydroxyl groups and the subsequent hydrogenation. The electrokinetic profile indicates that the rate‐determining step (RDS) includes a proton‐coupled electron transfer (PCET) to the dehydrated intermediate, revealed by coverage‐dependent Tafel slope and isotopic labeling experiments. An approximate zero‐order dependence of H+ suggests that water acts as the proton donor for the interfacial PCET process. Leveraging these insights, we formulate microkinetic models to illustrate its origin that Eley–Rideal (E−R) dominates over Langmuir–Hinshelwood (L−H) in governing Cu‐mediated DHA reduction, offering rational guidance that increasing the concentration of the adsorbed reactant alone would be sufficient to promote the activity in designing practical catalysts.

COF/In2S3 S-Scheme Photocatalyst with Enhanced Light Absorption and H2O2-Production Activity and fs-TA Investigation.

Photocatalytic hydrogen peroxide (H2O2) synthesis from water and O2 is an economical, eco-friendly, and sustainable route for H2O2 production. However, single-component photocatalysts are subjected to limited light-harvesting range, fast carrier recombination, and weak redox power. To promote photogenerated carrier separation and enhance redox abilities, an organic/inorganic S-scheme photocatalyst is fabricated by in situ growing In2S3 nanosheets on a covalent organic framwork (COF) substrate for efficient H2O2 production in pure water. Interestingly, compared to unitary COF and In2S3, the COF/In2S3 S-scheme photocatalysts exhibit significantly larger light-harvesting range and stronger visible-light absorption. Partial density of state calculation, X-ray photoelectron spectroscopy, and femtosecond transient absorption spectroscopy reveal that the coordination between In2S3 and COF induces the formation of mid-gap hybrid energy levels, leading to smaller energy gaps and broadened absorption. Combining electron spin resonance spectroscopy, radical-trapping experiments, and isotope labeling experiments, three pathways for H2O2 formation are identified. Benefited from expanded light-absorption range, enhanced carrier separation, strong redox power, and multichannel H2O2 formation, the optimal composite shows an impressive H2O2-production rate of 5713.2µmol g-1 h-1 in pure water. This work exemplifies an effective strategy to ameliorate COF-based photocatalysts by building S-scheme heterojunctions and provides molecular-level insights into their impact on energy level modulation.

Electrokinetic Analyses Uncover the Rate-Determining Step of Biomass-Derived Monosaccharide Electroreduction on Copper.

Electrochemical biomass conversion holds promise to upcycle carbon sources and produce valuable products while reducing greenhouse gas emissions. To this end, deep insight into the interfacial mechanism is essential for the rational design of an efficient electrocatalytic route, which is still an area of active research and development. Herein, we report the reduction of dihydroxyacetone (DHA)-the simplest monosaccharide derived from glycerol feedstock-to acetol, the vital chemical intermediate in industries, with faradaic efficiency of 85±5 % on a polycrystalline Cu electrode. DHA reduction follows preceding dehydration by coordination with the carbonyl and hydroxyl groups and the subsequent hydrogenation. The electrokinetic profile indicates that the rate-determining step (RDS) includes a proton-coupled electron transfer (PCET) to the dehydrated intermediate, revealed by coverage-dependent Tafel slope and isotopic labeling experiments. An approximate zero-order dependence of H+ suggests that water acts as the proton donor for the interfacial PCET process. Leveraging these insights, we formulate microkinetic models to illustrate its origin that Eley-Rideal (E-R) dominates over Langmuir-Hinshelwood (L-H) in governing Cu-mediated DHA reduction, offering rational guidance that increasing the concentration of the adsorbed reactant alone would be sufficient to promote the activity in designing practical catalysts.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade. The absolute configuration of 4,5-diepi-isoishwarane was determined using a stereoselective deuteration approach, revealing an absolute configuration typically observed for a microbial type sesquiterpene.

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis.

Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.

Zwei Sesterterpen‐Syntasen aus Lentzea atacamensis demonstrieren die Rolle der Konformationsvariabilität in der Terpenbiosynthese

AbstractStudien an zwei Multiprodukt‐Sesterterpensynthasen aus Lentzea atacamensis führten zur Identifizierung der Synthasen für Lentzeadien (LaLDS) und Atacamatrien (LaATS). Das Hauptprodukt von LaLDS (Lentzeadien) ist eine neue Verbindung, während eines der Nebenprodukte (Lentzeatetraen) das Enantiomer von Brassitetraen B ist und ein anderes Nebenprodukt (Sestermobaraen F) von einer überraschend weit entfernt verwandten Sesterterpensynthase bekannt ist. LaATS stellt sechs neue Verbindungen her, darunter das Enantiomer des bekannten Sesterterpens Bm1. Bemerkenswert ist, dass die Produkte beider Enzyme nicht alle aus ein und derselben Ausgangskonformation von Geranylfarnesyldiphosphat erklärt werden können, was die Notwendigkeit einer Konformationsflexibilität des Substrats in den aktiven Zentren der Enzyme zeigt. Für Lentzeadien wurde eine faszinierende thermische [1,5]‐sigmatrope Umlagerung entdeckt, die an die Biosynthese von Vitamin D3 erinnert. Alle Enzymreaktionen und die [1,5]‐sigmatrope Umlagerung wurden durch Isotopenmarkierungsexperimente und DFT‐Berechnungen untersucht. Die Ergebnisse unterstreichen auch die Bedeutung von Konformationsänderungen während Terpencyclisierungen.

Antagonism effect of residual S triggers the dual-path mechanism for water oxidation

Transition metal chalcogenides (TMCs) are recognized as pre-catalysts, and their (oxy)hydroxides derived from electrochemical reconstruction are the active species in the water oxidation. However, understanding the role of the residual chalcogen in the reconstructed layer is lacking in detail, and the corresponding catalytic mechanism remains controversial. Here, taking Cu1−xCoxS as a platform, we explore the regulating effect and existence form of the residual S doped into the reconstructive layer for oxygen evolution reaction (OER), where a dual-path OER mechanism is proposed. First-principles calculations and operando 18O isotopic labeling experiments jointly reveal that the residual S in the reconstructive layer of Cu1−xCoxS can wisely balance the adsorbate evolution mechanism (AEM) and lattice oxygen oxidation mechanism (LOM) by activating lattice oxygen and optimizing the adsorption/desorption behaviors at metal active sites, rather than change the reaction mechanism from AEM to LOM. Following such a dual-path OER mechanism, Cu0.4Co0.6S-derived Cu0.4Co0.6OSH not only overcomes the restriction of linear scaling relationship in AEM, but also avoids the structural collapse caused by lattice oxygen migration in LOM, so as to greatly reduce the OER potential and improved stability.

Highly efficient and selective electrosynthesis of urea via co-reduction of carbon dioxide and nitrate over the TiNx catalyst

Green and sustainable electrosynthesis of urea at ambient conditions through co-reduction of CO2 and NO3− (CR-CO2/NO3−) is regarded as a new avenue for nitrogen fertilizer production, however, the lack of novel electrocatalysts capable of selective C-N coupling hinders its wide application. Herein we reported a TiNx catalyst (TiN/TiN0.3) prepared by facile electric explosion method for electrocatalytic urea synthesis via CR-CO2/NO3−. The derived catalyst with porous nanostructure and specific active phase (TiN0.3) is more conductive for efficient C-N coupling. Consequently, a faradaic efficiency (FE) of 43.1 ± 3.1 % and a urea formation rate as high as 1488 ± 95 μg h−1 mgcat−1 were reached over the TiNx electrode, outperforming most of the reported CR-CO2/NO3− systems toward urea synthesis. In-situ attenuated total reflection Fourier transformed infrared spectroscopy and isotope-labeling experiments demonstrated the details about electrochemical coupling processes. The simulation study suggested that the TiN0.3 (100) facet facilitates the electroreduction of CO2 and NO3−, and decreases the energy barrier for generation of the key intermediates (*CO/*NH2), thus leading to the selective formation of urea. This work accomplished highly efficient electrochemical CR-CO2/NO3− toward nitrogen fertilizer production over a novel TiNx electrocatalyst for the target process.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch

AbstractSesquiterpene synthases (STPSs) catalyze carbocation‐driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation‐reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (−)‐cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (−)‐cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo‐electron microscopy‐derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure‐guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311‐S419‐C458]/[M311‐V419‐A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Fe-N-C core–shell catalysts with single low-spin Fe(II)-N4 species for oxygen reduction reaction and high-performance proton exchange membrane fuel cells

Fe-N-doped carbon materials (Fe-N-C) are promising candidates for oxygen reduction reaction (ORR) relative to Pt-based catalysts in proton exchange membrane fuel cells (PEMFCs). However, the intrinsic contributions of Fe-N4 moiety with different chemical/spin states (e.g. D1, D2, D3) to ORR are unclear since various states coexist inevitably. In the present work, Fe-N-C core–shell nanocatalyst with single low-spin Fe(II)-N4 species (D1) is synthesized and identified with ex-situ ultralow temperature Mössbauer spectroscopy (T = 1.6 K) that could essentially differentiate various Fe-N4 states and invisible Fe-O species. By quantifying with CO-pulse chemisorption, site density and turnover frequency of Fe-N-C catalysts reach 2.4 × 1019 site g−1 and 23 e site−1 s−1 during the ORR, respectively. Half-wave potential (0.915 VRHE) of the Fe-N-C catalyst is more positive (approximately 54 mV) than that of Pt/C. Moreover, we observe that the performance of PEMFCs on Fe-N-C almost achieves the 2025 target of the US Department of Energy by demonstrating a current density of 1.037 A cm−2 combined with the peak power density of 0.685 W cm−2, suggesting the critical role of Fe(II)-N4 site (D1). After 500 h of running, PEMFCs still deliver a power density of 1.26 W cm−2 at 1.0 bar H2-O2. An unexpected rate-determining step is figured out by isotopic labelling experiment and theoretical calculation. This work not only offers valuable insights regarding the intrinsic contribution of Fe-N4 with a single spin state to alkaline/acidic ORR, but also provides great opportunities for developing high-performance stable PEMFCs.