The N2 reduction drives biological nitrogen fixation and the global nitrogen cycle. Besides the conversion of N2, nitrogenases catalyze a whole range of other reductions, including the reduction of the small gaseous substrates carbon monoxide (CO) and carbon dioxide (CO2) to hydrocarbons. However, it remains an open question whether these ‘side reactivities’ play a role under environmental conditions. Nonetheless, these reactivities and particularly the formation of hydrocarbons have spurred the interest in nitrogenases for biotechnological applications. There are three different isozymes of nitrogenase: the molybdenum and the alternative vanadium and iron‐only nitrogenase. The isozymes differ in their metal content, structure, and substrate‐dependent activity, despite their homology. This minireview focuses on the conversion of CO and CO2 to methane and higher hydrocarbons and aims to specify the differences in activity between the three nitrogenase isozymes.Nitrogenases are the only known family of enzymes that catalyze the reduction of molecular nitrogen (N Nitrogenases reduce inert dinitrogen to bioavailable ammonia and thus ‘feed’ the Earth's biosphere. To catalyze this challenging reaction, nitrogenases are equipped with a complex reduction machinery that also permits the remarkable conversion of carbon monoxide and carbon dioxide. In particular, the formation of methane and hydrocarbons opens up the prospect of future biotechnological processes for carbon capture and utilization by nitrogenases. The majority of bioavailable N is derived from the atmosphere through the reduction of N2 to ammonia (NH3) in a process known as nitrogen fixation. For millions of years, nitrogen fixation was unique to diazotrophic microorganisms.Bioavailable nitrogen (N) is essential for all life on Earth to build central metabolites such as nucleotides and amino acids.th century, the industrial Haber–Bosch process was invented to reduce N2 to NH3, to produce explosives and fertilizers.2 fixation. Remarkably, evolution produced a sophisticated enzymatic reduction machinery that is capable of reducing N2 under physiological conditions.2 by consuming ATP and low potential electrons with the following canonical molybdenum (Mo) nitrogenase stoichiometry . Six Fe atoms form a trigonal prism around an interstitial μ6‐C atom that is capped with three μ2‐S2− ligands , the belt sulfides  and the opposite side is connected to the octahedrally coordinated Mo atom. The Mo atom is further coordinated by the bidentate ligand R‐homocitrate and H442 of NifD. The V‐cluster has the composition [VFe7S8C(CO3)‐(R)‐homocitrate] with the S3A belt sulfide being replaced by a CO3−‐ion, and V in place of Mo ‐homocitrate] with Fe at the Mo position.The three different active site cofactors  are the most complex metallocofactors observed in nature and constitute the most apparent difference between the three isozymes. The M‐cluster has the chemical composition of [MoFes Figure c.13]  7S9C‐, the spontaneous release of H2 gas can occur, leading from the nE state to the n‐2E state (2≤n≤4). After four electrons and protons are added to reach the 4E state, an exchange of the two bound hydrides by N2 occurs via a coupled reductive elimination (re) of the hydrides under release of H2 and oxidative addition (oa) of N2.4E(2N2H) state, subsequent electron flow leads to the full reduction of N2 to two equivalents of NH3 without the release of H2. This renders the 4E state the critical intermediate for N2 reduction, called Janus‐Intermediate.The Lowe–Thorneley scheme has been proposed on the basis of kinetic and spectroscopic investigations of the Mo nitrogenase. According to this scheme, the electron flux subsequently introduces additional electrons to the M‐cluster, starting from the most oxidized e Figure b.[4a] 2 fixation stoichiometry shows an increased ATP consumption and H2 production compared to the Mo nitrogenase. 1≤4 hydrocarbons, the activity of the Mo nitrogenase towards CO reduction was revisited by Ribbe and co‐workers. as electron source.4. The catalytic relevance of the isolated lo‐CO state was further demonstrated by turning over 13CO labeled lo‐CO VFe protein in the presence of 12CO, to release the partially labeled 13CH212CH2 product.Due to the CO reduction rates by the V nitrogenase, the isolation of the V nitrogenase corresponding to lo‐CO and hi‐CO states is hampered under turnover conditions, A. vinelandii with the VnfH of Methanosarcina acetivorans.For the first time, the hi‐CO EPR signal of VnfDGK was observed after reducing CO under a decreased electron flux, by exchanging the VnfH of 3−‐ligand  was already in the range of other bio‐ethylene formation methods without any optimization.Beyond the The CO‐reducing and C−C coupling activity of the V nitrogenase renders this enzyme system as a potential biological alternative to the industrial Fischer–Tropsch process for fuel formation.2.32 formation catalyzed by the Fe‐only nitrogenase is progressively inhibited by increasing CO concentrations until it reaches an inhibition of 35 % at a CO partial pressure of 0.4 atm.4 with a maximum specific activity at a CO partial pressure of ∼0.05 atm. The CH4 formation activity of the Fe‐only nitrogenase decreased to 10 % of the maximum value, when the CO partial pressure was further increased. Therefore, the CH4 formation alone cannot fully account for the observed inhibition of H2 formation at elevated CO concentrations and either CO inhibits the electron flux of the Fe‐only nitrogenase or an undetected product is formed.The H2H4) and the Fe‐only nitrogenase (only CH4) is so far unexplained, as no structural or spectroscopic characterization of the Fe‐only nitrogenase under CO has been carried out. However, a similar product shift was observed for the CO reduction by the isolated V‐ and L‐cluster under [Eu(II)‐DTPA].9S9C]. The L‐cluster closely resembles the proposed structure of the Fe‐cluster except that (R)‐homocitrate is missing. The preferred reduction of CO to CH4 by the L‐cluster indicates that the preference for CH4 formation of the Fe‐only nitrogenase could originate from the intrinsic cofactor properties rather than the protein environment. Further investigations are needed to elucidate the CO conversion by the Fe‐only nitrogenase.The differences in the product profile between the V nitrogenase (mainly C2.44×[nmol FeFe protein]−1×min−1) to the V nitrogenase (7.5 nmol C2H4×[nmol FeFe protein]−1×min−1).The specific activity for CO reduction is increasing from the Mo nitrogenase (traces) through the Fe‐only nitrogenase (4.5 nmol CH2 as electron sources.[V) and an M‐cluster containing VFe protein (VnfDGKM).V was 640 times smaller as the one observed for VnfDGK.M increased by 100‐fold compared to NifDK.M.The isolated M‐cluster and V‐cluster are able to produce hydrocarbons from CO at similar rates by using the strong reductants [Eu(II)‐DTPA] or SmI sources.67, 68Despite the differences in reactivity, the Mo and V nitrogenase show similar interactions with CO, forming a lo‐ and hi‐CO state, and for both nitrogenases the states are interconvertible. The same behavior and mechanism seem to apply to the Fe‐only nitrogenase. On the basis of the lo‐ and hi‐CO structures of the V nitrogenase, a mechanistic scheme was proposed Scheme .[64b]2E state, CO binds to the t‐site and replaces the hydride at the μ‐site ((2)–(4)). The reaction can then proceed either by CO insertion of the hydride in (4) leading to the formyl adduct (7) followed by the reduction of CO to hydrocarbons in a cascade of reductions and a CO addition ((8)–(11)) or by hydride protonation forming the lo‐CO state (5). The lo‐CO state (5) can bind an additional CO molecule to form the hi‐CO state (6). This mechanism explains why the hi‐CO state cannot couple both bound CO molecules, as the terminal CO at the t‐site of (6) has to be exchanged with a hydride to form intermediate (4) before the CO reduction to hydrocarbons is initiated. The preference of C2H4 formation by the V nitrogenase is explained by the preferential β‐H elimination in (11) over the methyl protonation in intermediate (8). On the other hand, the Fe‐only nitrogenase produces mainly CH4, which could be explained by a facilitated protolysis of intermediate (8) or by the occurrence of a σ‐bond metathesis between intermediate (8) and a generated H2 molecule.After reaching the To summarize, the activation of CO by the Mo and V nitrogenases seems to depend on the same CO binding mode to the M‐ and V‐cluster. Nonetheless, the CO activity and product profile between the three nitrogenase isoenzymes differs significantly. The activity differences appear to mainly originate from the different protein scaffolds  as the introduction of the M‐cluster into the V nitrogenase and the reverse exchange, the introduction of the V‐cluster into the Mo nitrogenase, suggest. However, the exact influence of the protein scaffold on the activity could not be clarified, yet.32 is the main source of carbon in the atmosphere. The anthropogenic CO2 emissions of the last century increased the abundance of CO2 in the earth's atmosphere by 40 %, compared to the pre‐industrial era.2 concentration is the main contributor to global warming.2 renders it a cheap and accessible source of C1 building blocks. Hence, carbon capture to reduce the CO2 concentration and conversion of CO2 into value‐added products are pressing challenges for chemistry and biology. Recently, nitrogenase proteins were discovered to be capable of activating and reducing CO2 to hydrocarbons. Potentially, this reactivity offers new solutions to the great challenge of carbon capture and utilization.The greenhouse gas CO3.1et al. first reported the reduction of CO2 to CO by the Mo nitrogenase.in vitro assay, but was not suitable for the detection of alternative CO2 reduction products. The formation of H2 during Mo nitrogenase turnover at elevated CO2 concentrations (0.45 atm) is inhibited by 16 %.2 reduction. HCOO− was later identified by 1H and 13C NMR spectroscopy as the main product of the CO2 reduction by the WT Mo nitrogenase and its mutants. HCOO− is formed at a rate of 9.8 nmol×[nmol MoFe protein]−1×min−1. The formation of CO is much slower and proceeds at a rate of 0.03 nmol×[nmol MoFe protein]−1×min−1, although it was discovered first. This product profile was corroborated by quantum mechanical calculations. The reaction pathway of direct hydride transfer from the 2E state to CO2, releasing HCOO−, is favored over the alternative pathway of CO2 activation at the M‐cluster with the reduction of the Fe‐CO2 intermediate leading to CO. The product profile of the CO2 reduction by the Mo nitrogenase could be extended to CH4 by introducing two mutations to the NifD subunit (V70A and H195Q).4 likely proceeds over an intermediate CO in a non‐dissociative mechanism. The addition of the CO binder deoxyhemoglobin reduced the amount of formed CH4 by 25 % in the CO2 reduction assays. The coupling of two or more CO2 molecules to longer hydrocarbon chains was not detected. Instead, the Mo nitrogenase could couple C2H2 and CO2 to C3H6.Seelfeldt 2 reduction behavior of the MoFe protein was electrochemically investigated by immobilizing the MoFe protein in a polymer film on an electrode and cobaltocene was used as the electron mediator.2 reduction by the MoFe protein showed similar product distributions with 8 % and 9 % of electron flux directed towards the formation of HCOO−, respectively. The CO2 reduction activity of the Mo nitrogenase double mutant (V75A and H201Q) was further demonstrated in vivo using the anoxygenic phototroph Rhodopseudomonas palustris.in vivo formation of CH4 under photochemotrophic growth is dependent on light for the ATP generation and requires an electron source like acetate (H3CCOO−) or thiosulfate (S2O32−). The yield of CH4 was further increased by directing metabolic electrons to the nitrogenase proteins. This was partially achieved, either, by preventing further bacterial growth via resuspension of the cells in a nitrogen‐depleted medium or by deletion of the Calvin‐Benson‐Bassham (CBB) cycle from the organism, which is an alternative electron accepting pathway for anoxygenic phototrophs.Moreover, the COfate S2O32−. The y2 mainly to HCOO− and a little bit of CO. Nonetheless, Mo nitrogenase mutants can also reduce CO2 via the intermediate CO to CH4.In short, the Mo nitrogenase converts CO3.22 to CO at similar rates as the Mo nitrogenase.et al. detected the formation of CD4 and the C−C coupling products C2D4 and C2D6 in sub‐stoichiometric amounts by changing the medium from H2O to D2O. In contrast to the V nitrogenase, no C−C coupling products were observed for the Mo nitrogenase. The carbon source for the formed hydrocarbons was traced to CO2 by 13C‐labeling. Moreover, it was shown that the formation of C2D4 and C2D6 proceeds independently from the produced CO, while CD4 partially originated from CO. The product distribution of the CO2 reduction reaction is modulated, when the reductant [Eu(II)‐DTPA] is used as the electron source.4 and the C−C coupling products occur in H2O. Furthermore, the product profile is extended from C2H4 and C2H6 to C3H6, C3H8, C4H8 and C4H10. The hydrocarbon formation activity increases dramatically in D2O for the V nitrogenase under [Eu(II)‐DTPA] and CO2, with the exception of CD4, which was not detected under these conditions. Both findings, the longer hydrocarbon chains and the increased product formation indicate that D2O facilitates C−C coupling by the V nitrogenase. The ability of the VFe protein to C−C couple CO2 to C2H4 and C3H6 was also demonstrated in the electrochemically driven CO2 reduction by the VFe protein, using 1,1’‐dicarboxycobaltocenium as the electron mediator.4 from H2CO3 inside R. palustris. Increased amounts of CH4 were observed in strains expressing a double mutant of the VFe protein (V57A and H180Q in VnfD).The V nitrogenase can reduce CO2 to CO. Furthermore, hydrocarbon chains can be produced, either, by replacing H with D in the buffers or by using the strong reductant [Eu(II)‐DTPA].To summarize, also the V‐nitrogenase reduces CO3.32 by the Fe‐only nitrogenase has been characterized in AnfH/ATP‐dependent in vitro assays and electrochemically, using a polymer‐coated electrode containing the FeFe protein and relying on cobaltocene as electron mediator.2 to HCOO−, respectively. For the AnfH/ATP‐driven in vitro assays, the formation of trace amounts of CH4 were reported, but the possible formation of CO was not mentioned.The reduction of CO2 reduction activity by the Fe‐only nitrogenase was also confirmed in non‐growing R. palustris. In contrast to the Mo and the V nitrogenase, strains expressing the wild type Fe‐only nitrogenase formed already elevated amounts of CH4.4 in R. palustris.The CO2. Remarkably, the same portion of electrons (31 %) are used either for the reduction of N2 or for the reduction of CO2 under the according atmosphere.In a nutshell, the WT Fe‐only nitrogenase has the highest inherent activity for the reduction of CO3.42 to CO.in vitro CO2 reduction assays that used dithionite to reduce the enzymes [Fe4S4] cluster to the [Fe4S4]1+ state. The produced amounts of CO were increased for VnfH compared to NifH, which is consistent with the lower reduction potential of VnfH compared to NifH. The activity was further improved by adding ATP to the Fe proteins, as the nucleotide‐bound states of both proteins exhibit lower reduction potentials. Turnover numbers of up to ∼8 were achieved for the CO2 reduction to CO by using the reductant [Eu(II)‐DTPA] in repeated additions. [Eu(II)‐DTPA] reduces the [Fe4S4] cluster to the all‐ferrous state ([Fe4S4]0). The Fe proteins can also catalyze the reverse reaction, the oxidation of CO to CO2, when the reduced [Fe4S4]+ cluster was reoxidized to the [Fe4S4]2+ state by an excess of indigo disulfonate. This suggests a potential involvement of all available [Fe4S4] cluster redox states of the Fe proteins  in the CO2 reduction and establishes the Fe protein as an enzymatic mimic of the nickel‐iron CO dehydrogenase, the only other enzyme known to interconvert CO2 and CO. Moreover, the CO2 reduction to CO was demonstrated in vivo in A. vinelandii strains expressing only the Fe protein of the Mo or V nitrogenase. After 14 h under 40 % CO2, the maximum turnover numbers of 140 and 110 were reported for VnfH and NifH, respectively. These turnover numbers indicate the formation of the all‐ferrous state in vivo.In 2017, Yu and co‐workers demonstrated that the Fe proteins of the Mo and V nitrogenase (NifH and VnfH) are capable of reducing CO2 by Fe proteins was further investigated for the NifH analogue of Methanosarcina acetivorans (NifHMa). This organism is naturally adapted to the CO2 reduction and occurs in habitats with high CO2 concentrations.Ma reduces CO2 to CO under [Eu(II)‐DTPA]. However, the amount of formed CO by NifHMa decreased for concentrations above 20 mM [Eu(II)‐DTPA]. Instead of forming CO, electrons were redirected into the formation of CH4, C2H4, C2H6, C3H6 and C3H8. In contrast, the activity of NifH from A. vinelandii (NifHAv) for the reduction of CO2 to CO was enhanced by increasing the concentration of [Eu(II)‐DTPA] from 10 mM to 100 mM, no hydrocarbons were detected. NifHAv and NifHMa were also tested for their capability to reduce CO. While NifHAv shows no catalytic activity, NifHMa formed C1≤4 hydrocarbons with a similar product distribution as observed for the CO2 reduction. Also the hydrocarbon formation from CO increased with higher [Eu(II)‐DTPA] concentrations. The differences in the CO2 reduction between NifHAv and NifHMa likely originate from their different affinities towards CO. Under dithionite NifHMa can reduce CO to hydrocarbons at low yields, which emphasizes a potential physiological relevance of this reaction.The reduction of COrans NifHMa. ThisMa ([Fe4S4]1+ state) was crystallized in the presence of the alternative CO2 source bicarbonate. A 2.4 Å crystal structure was obtained, leading to mechanistic insights into the CO2 reduction by the Fe proteins  is highly conserved among Fe proteins. While the R100 residues in the dithionite reduced structure of NifHAv are facing away from the [Fe4S4] cluster  of the [Fe4S4] cluster, with the R100B interacting with the bound CO2 via a hydrogen bond. On the basis of DFT calculations a mechanism was proposed. CO2 is guided by R100B to the Fe(3) of the [Fe4S4] cluster and subsequently activated. The carboxylate intermediate is then protonated by R100B or by the buffer. Subsequent electron uptake and donation of the [Fe4S4] cluster to the carboxy‐group results in the formation of H2O and a CO bound intermediate. The CO is then released and the [Fe4S4]1+ cluster reduced back to its [Fe4S4]0 state by [Eu(II)‐DTPA] to close the catalytic cycle.Dithionite reduced NifHMa 4].2 and CO reduction activity of biogenic and synthetic [Fe4S4] clusters renders them an interesting research field for the development of new homogenous CO2 reduction catalysts.Moreover, it was shown that the CO42, DMSO reductases use excess electrons to reduce DMSO, and nitrogenases use electrons for N2 and H+ reduction and the formation of NH3 and H2.[2 formation by the Mo nitrogenase is increased in vivo upon interruption of the CBB cycle by deletion of RuBisCO.2 by the alternative nitrogenases potentially contributes to this mechanism of redox cofactor recycling.Organisms that grow anaerobically encounter the problem of accumulating abundant amounts of reducing equivalents in ferredoxins and flavodoxins, if their carbon source is more reduced than their biomass. The enrichment of reduced redox cofactors ultimately prevents growth, if the organism cannot transfer the electrons to an electron acceptor. Three viable mechanisms have been identified in purple non‐sulfur bacteria to independently support anaerobic phototrophic growth from such carbon sources. In the CBB cycle electrons are accepted by CO3 and H2.76, 842 and H+ reduction.Rhodobacter capsulatus for CO concentrations of up to 3.5 %. However, the deletion of CowN did not affect the cell growth sustained by the Fe‐only nitrogenase under CO. Interestingly, AnfA, the transcriptional activator of the Fe‐only nitrogenase, represses the expression of CowN, indicating that the CO protection is not required for the Fe‐only nitrogenase. Therefore, the promiscuous activity for CO reduction by the V and Fe‐only nitrogenase could indicate a function of these nitrogenases as detoxifying enzymes. The amount of CH4 formed from CO2 by the Fe‐only nitrogenase in R. palustris was also sufficient to support the growth of a co‐cultivated methanotroph Methylomonas sp. LW13.[4 formed by nitrogenases implies that the CO and CO2 reduction might play a role in the symbiosis of microbial communities, and not only presents a detoxifying function. Our understanding regarding the biological role of the nitrogenases‘ ‘side reactivities’ is very fragmented and calls for further investigation.As discussed above, the Mo nitrogenase is strongly inhibited by CO, even under environmental concentrations. To protect the Mo nitrogenase from CO, some diazotrophs express the protein CowN. CowN interacts with the Mo nitrogenase and prevents its inhibition by CO, without decreasing the nitrogenase activity for Nsp. LW13.8, 9b 5in vivo reduction of N2, CO and CO2 by the WT V and Fe‐only nitrogenases implies a potential evolutionary link between the nitrogen and carbon cycle on earth.2 reduction. Repurposing the nitrogenase machinery for the reduction of CO and CO2 to form C−C bonds is an exciting path to biotechnological processes and the synthesis of value‐added products. Recently, V nitrogenase expressing A. vinelandii cells were explored for the production of C2H4 from CO in 2 l bioreactors.2H4 productivity could not compete with industrial methods. Still, this process can be improved by changing the reaction conditions or by protein engineering. The effect of protein engineering was highlighted by introducing double mutations to the MoFe (V75 A and H201Q) and VFe (V57A and H180Q) proteins, increasing the CO2 reduction and CH4 formation activity.2 and is capable to produce CH4 inside cells. This activity renders the Fe‐only nitrogenase an ideal basis for the development of reduction catalysts for the utilization of the ubiquitous greenhouse gas CO2.The The authors declare no conflict of interest.Niels N. Oehlmann obtained his BS and MS degrees in Chemistry at the Goethe‐Universität Frankfurt. In 2020, he joined the research group of Dr. Johannes G. Rebelein at the Max Planck Institute for Terrestrial Microbiology for his Ph.D. studies. His research focuses on the conversion of carbon containing substrates by nitrogenases. Currently, he is a Kekulé fellow of the Fonds der Chemischen Industrie.Johannes G. Rebelein received his BS and MS degrees in Biotechnology from the TU Braunschweig. In 2016, he received his PhD degree from the University of California in Irvine. During his postdoctoral stay at the University of Basel, he worked as an EMBO long‐term fellow on the construction of artificial metalloenzymes. He has been leading an Emmy‐Noether research group since 2020, at the Max Planck Institute for Terrestrial Microbiology.


Substantial electron transfer from the electride support to copper nanoparticles via electronic metal–support interactions results in the accumulation of excess electrons at the surface of copper nanoparticles. Moreover, the surface‐accumulated excess electrons prohibit the oxidation of copper nanoparticle, thereby maintaining the metallic surface in a negatively charged state and activating both (aza)indoles and disulfides under mild conditions in the absence of any further additives. This study defines the role of excess electrons on the nanoparticle‐based heterogeneous catalyst that can be rationalized in versatile systems.Engineering active sites of metal nanoparticle‐based heterogeneous catalysts is one of the most prerequisite approaches for the efficient production of chemicals, but the limited active sites and undesired oxidation on the metal nanoparticles still remain as key challenges. Here, it is reported that the negatively charged surface of copper nanoparticles on the 2D [Ca 2N]+∙e− electride are responsible for the catalytic reaction that has not been accomplished with typical copper nanoparticles.A combination of copper nanoparticle and exotic electride support provides novel heterogenous catalysts for selective sulfenylation of (aza)indoles with diaryl disulfides under mild conditions in the absence of any additives. Surface‐accumulated excess electrons of the negatively charged copper nanoparticles anchored on dicalcium nitride, [Ca It has been well established that the electronic and geometric structures of metal NPs are the key component to design a catalyst and control its catalytic performance, thus manipulating the size, shape, and composition of metal NPs is the primary approach for developing an efficient catalyst. Furthermore, a profound impact on the catalytic behavior of metal NPs is made upon the metal–support interaction (MSI), which includes the phenomena such as charge transfer and the interfacial perimeter. Indeed, the redistribution of electrons within both NPs and supports can induce the change in oxidation states of metal atoms of NPs and metal ions of supports. However, this phenomenon is typically limited to a few atomic layers of the interfacial region. Considering that the difference in work function (ϕ) values between the metal NP and the support determines the direction of the charge transfer and its magnitude, an appropriate selection of a support material with extremely low ϕ can further drive the charge transfer to metal NPs, ultimately activating the metal atoms to have an intriguing electronic state and thus realizing numerous active sites over the whole surface area of NPs.Heterogeneous catalysis plays a critical role in the chemical and energy industries with the undisputable advantages such as the sustainability and facile product separation.−3 Am[−] When excess electrons are transferred to the metal NPs, which is a good conductor, excess electrons are not bound to a particular metal ion but rather free to move around. These free electrons will be rearranged and accumulated near the metal atoms at the surface of NPs. Indeed, this phenomenon can be found in metal clusters such as Na and Ag clusters. More critically, in the metal NPs with surface‐accumulated excess electrons, the intriguing negatively charged surface state can offer numerous sites for reactant activation to generate the intermediates in the targeted chemical reaction; therefore, facilitating the massive charge transfer from a support material to metal NP is a straightforward approach for an efficient metal NPs‐based heterogeneous catalyst. Recently, it is found that the bare copper NPs (Cu NPs) grown on the [Gd2C]2+·2e− electride with the ϕ of ≈2.8 eV show the negatively charged surface state due to the surface‐accumulated excess electrons and the ultrastrong oxidation resistance in air, keeping the metallic surface state during a long period over several months. Thus, in order to prove the potential of the electride as a support material and demonstrate the metal NPs with numerous active sites on excess electrons accumulated surface, we design the bare Cu NPs supported on [Ca2N]+·e− electride (Cu NPs/[Ca2N]+·e−) for an efficient heterogeneous catalyst.A strong electronic interaction between an electroactive support material with high‐density mobile electrons and metal NP enables a substantial charge transfer, leading to the negatively charged surface state of metal NPs according to the Gauss's law.−14 W2N]+·e− electride has a low ϕ value of ≈2.6–3.5 eV depending on the surface orientation. Moreover, benefited from the fully delocalized nature of anionic electrons at interlayer space, the electronic mobility of ≈160 cm2 V−1 s at 300 K was obtained, while the [Gd2C]2+·2e− electride with strongly localized anionic electrons showed the value of ≈20 cm2 V−1 s at 300 K. Owing to the low ϕ originating from the high electron density with ≈2.3 × 1022 cm−3 and delocalized nature of anionic electrons, the 2D [Ca2N]+∙e− electride spontaneously transfers its highly mobile electrons to the Cu NPs (≈4.5 eV). In contrast to the low selectivity of conventional Cu‐based heterogeneous catalysts due to various Cu oxidation states originated from the formation of Cu oxides,[−] the Cu NPs on the [Ca2N]+·e− electride can improve the oxidation state‐ and site‐dependent catalytic activity because the system retains the metallic surface with the transferred excess electrons that can provide numerous active sites regardless of morphology and facet. Here, we show an efficient catalytic sulfenylation of indole compounds as a model substitution reaction utilizing Cu NPs/[Ca2N]+·e− catalyst. Among the indole derivatives, 3‐sulfenylindoles are currently attracting considerable attention due to their therapeutic value in the treatment of HIV, cancer, obesity, heart disease, and allergies.[−] While several strategies have been explored for the 3‐sulfenylation of indoles with thiols, disulfides, sulfenyl halides, N‐thioimides, sulfonium salts, quinone mono‐O,S‐acetals, and arylsulfonyl chlorides, the required excess sulfenylating agents or additives, limited substrate scope, and undesired byproducts have been unsettled obstacles.[−] The present sulfenylation reaction using Cu NPs/[Ca2N]+·e− catalyst is successfully accomplished with various functional groups on both reactants, with a broad substrate scope. The performed high‐yield regioselective synthesis of 3‐sulfenylindole compounds is ascribed to the surface‐accumulated excess electron on the Cu NPs, suggesting that the catalytic activity of Cu NPs can be effectively enhanced by inducing a negatively charged surface state through a strong electronic MSI.It is noted that the [Caentation.17 Mor22.1Figureϕ for enhancing charge transfer to the Cu NPs by comparing the energy diagrams with typical heterogeneous catalysts. The zinc dust and reduced titania  natural formation of copper oxide indoles with disulfides  under the optimized solvent and reaction temperature conditions  and [Ca2N]+·e− electride (entries 1 and 2). It is noticeable that sole Cu NPs and [Ca2N]+·e− electride showed no reaction, whereas the reaction of Cu NPs/[Ca2N]+·e− produced the desired 3‐phenylthioindole (3aa) in 93% yield (entry 3), indicating that the catalytic activity was verified in the present heterogenous system of the Cu NPs grown on the [Ca2N]+·e− electride support. Furthermore, we also examined the catalytic performance of the separated Cu NPs from the electride . To validate the efficiency of the electride support in the sulfenylation reaction, alternative electron donating supports, such as sodium ascorbate and Zn dust, were examined with Cu NPs and CuI compound (entries 6–8). These reactions showed no catalytic activity, emphasizing the excellent charge transfer ability of the [Ca2N]+·e− electride that activates the Cu NPs as a catalyst. Furthermore, other tested Cu compounds, such as CuI and CuBr2 showed no reaction (entries 9 and 10), but their combined systems with the [Ca2N]+·e− electride produced the product 3aa in 91% yield (entries 11 and 12). These results indicate that Cu+ and Cu2+ cations of the CuI and CuBr2 compounds can be reduced by the excess electrons from the electride, probably forming negatively charged Cu clusters and acting as active sites for the catalytic sulfenylation.2.32N]+·e− electride and compare its electronic and surface state with those of the commercial Cu NPs, the separated Cu NPs from the electride, and Cu NPs supported by Zn dust. Figure2N]+·e− electride by solid‐state reaction , where Cu NPs with a 30 ± 7 nm diameter form on the surface of [Ca2N]+·e− electride. MSI‐induced charge transfer significantly changed the ϕ value of the Cu NPs. The ϕ histograms obtained with Kelvin probe force microscopy (KPFM) show an average value of ≈3.9 eV for the Cu NPs, which is lower than the ϕ value of Cu metal (4.5 eV), suggesting that the electride with a ϕ of ≈2.9 eV provides excess electrons to the grown Cu NPs on the surface  of Cu metal, while a slight positive shift for the Cu NPs/Zn dust is observed. For a comparison, XPS spectrum of Cu foil is provided in Figure  In addition, the Cu compounds combined with the [Ca2N]+·e− electride, which showed an efficient catalytic sulfenylation  measurements Figure , it is rn Figure 41 In 3M45M45 Auger spectrum  image, which shows the face‐centered cubic (fcc) structure at the surface with an interplanar distance of 0.18 nm, which corresponds to the fcc Cu {100} plane indoles with disulfide , with the 3‐position blocked, and observed that the sulfenylation did not occur at the 2‐position , p‐methoxy (2c), and o‐amino (2d) groups as well as electron‐withdrawing substituents, such as p‐chloro (2e), m‐trifluoromethyl (2g), and o‐bromo (2f) furnished the corresponding 3‐arylthioindoles (3). Importantly, the reactions were highly regioselective with respect to the 3‐sulfenyl products. The reactions of substituted indoles  afforded the corresponding 3‐arylthioindole products (3) in good yields. Notably, the mild reaction conditions tolerated various functional groups, such as —Cl, —Br, —CF3, —NO2, and —NH2, in both 1 and 2. The synthetic utility of the transformation was examined in a gram‐scale synthesis, where the product 3aa was prepared on a 5 mmol scale, and the yield was similar to that obtained on the 0.2 mmol scale reaction. Upon investigation of the indole framework scope, we extended the transformation to 7‐azaindole (8) (Table 8 with various diaryl disulfides (2) containing both electron‐donating and withdrawing groups effectively proceeded under the standard conditions to generate the corresponding 3‐arylthioazaindoles (9) in high yields.Furthermore, we investigated the substrate scope of the sulfenylation of indole derivatives Table2. The 32N]+·e− electride support has been developed for the regioselective sulfenylations of (aza)indoles, producing various 3‐arylthio(aza)indoles in a high yield. While the catalytic activity of the commercial Cu NPs and negatively charged Cu NPs separated from the electride for the sulfenylation has been of scarce, the negatively charged surface of the Cu NPs supported by the [Ca2N]+·e− electride, which has excess charge of 1.6e− per surface Cu atom, shows the efficient catalytic reactions in the absence of any additives. In particular, the critical role of substantially high‐density surface excess electrons on the negatively charged Cu NPs to trigger the catalytic reactions was evidently demonstrated from the comparative experiments and mechanistic studies. We believe that our protocol represents a practical sulfenylation reaction and provides a milestone for the application of the unique electride support with excellent electron donating ability and negatively charged Cu NPs in synthetic chemistry. Because in principle the metal NPs can be charged in a negative state by the charge transfer via a physical contact with an electride support having a lower ϕ value than that of the metal NPs, this work will stimulate the further utilization of negatively charged metal NPs supported by the electride for multiple electrons involving catalytic reactions.A heterogeneous catalytic system of negatively charged Cu NPs grown on [Ca42N]+·e− electride. To synthesize a stoichiometric polycrystalline [Ca2N]+·e− electride, Ca3N2 powder and Ca shots in a 1:1 ratio was mixed, and then pressed them into a pellet form. The pelletized mixture was annealed at 800 °C for 48 h under vacuum (≈10−3 Pa) in a silica tube. After annealing, the sample was quenched into water. The synthesized sample was pulverized into a powder in an agate mortar and reannealed under the same conditions to improve homogeneity. To synthesized the Cu NPs on the [Ca2N]+·e− electride, polycrystalline [Ca2N]+·e− powder (5.31 mmol), and dried Cu(II) acetate (0.0786 mmol) were mixed with n‐heptane in an agate mortar in the glove box and transferred into a silica tube for heating at 150 °C under vacuum (≈10−3 Pa) for 24 h. The synthetic procedures of separated Cu NPs from the electride can be found in the previous report.All the synthesis processes were carried out in glove boxes filled with high‐purity argon gas (Ar 99.999%) to minimize the oxidation of raw materials and synthesized [Cas report.412N]+·e−  and dimethylformamide, DMF (1 mL). The reaction was allowed to proceed at 80 °C and the progress was monitored using thin layer chromatography or gas chromatography. After completion (~ 4 h), the reaction mixture was then diluted with EtOAc and washed with water. The organic layers were dried over MgSO4, concentrated in vacuo, and purified by silica gel flash column chromatography using a hexane‐ethyl acetate mixture as the eluent to give the corresponding 3‐arylthio(aza)indole product.An oven‐dried resealable test tube, equipped with a magnetic stir bar, was charged with (aza)indoles derivatives (0.2 mmol), and aryl disulfide derivatives (0.15 mmol). In a nitrogen‐filled glove box, the reaction mixture was charged with Cu NPs/[Ca2N]+·e− was analyzed using field‐emission SEM . STEM‐EELS measurements were performed using a Cs‐corrected TEM  operating at 200 kV. All samples were dispersed in n‐heptane, applied to a 300‐mesh gold holey carbon grid (TED PELLA INC.) in glovebox, and dried in a vacuum chamber. To prevent oxidation of the STEM sample, a single‐tilt vacuum transfer holder  was used. EELS measurements were performed in the STEM mode using the same microscope equipped with a Gatan imaging filter (GIF) detector (Gatan Quantum 965ER). XPS and AES measurements were performed to investigate chemical and electronic states of Cu NPs on the surface of [Ca2N] +∙e− electride. The photoemission results were obtained using a Scienta R4000 electron analyzer with an Al Kα X‐ray source (1486.7 eV) and a discharge lamp that emitted an excitation line of He Iα (21.2 eV). Core spectrum of Cu 2p and Auger spectrum of Cu L3M45M45 were measured for analysis, and all spectra were fitted by comparing the binding energies of the component peaks with CasaXPS. AES was modeled by a symmetric mixed Gaussian–Lorentzian function. Every peak was calibrated by the spectrum of hydrocarbon on the sample and reference Au attached next to the sample. The surface work function of the Cu NPs/[Ca2N]+·e− placed on an Au‐coated Si substrate was measured by an atomic force microscope  equipped with a sealed electrochemistry cell filled with argon gas. The KPFM experiments were conducted using Ti/Ir‐coated Si probe  with a force constant of 2.8 N m−1 and a resonance frequency of 75 kHz. The work function of the probe was calibrated using a highly ordered pyrolytic graphite (HOPG) as a reference with the work function of ≈4.6 eV. During the KPFM scanning process, the scan rate and set point were 0.8 Hz and 0.5 V, respectively. Moreover, an a.c. voltage Va.c.  =  1 V was applied to the tip, and the tip was lifted up 30 nm from the sample surface.The morphology of Cu NPs/[Ca ≈4.6 eV.45 DurAll statistical analyses of the STEM‐EELS spectra, KPFM data, XPS, and AES spectra were performed using the Origin software.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5′ side of an AP site, thereby forming a single–strand break containing 3′–OH and 5′–dRP ends. In addition, Apn2 has 3′–phosphodiesterase activity (removing 3′–blocking groups) and 3′ → 5′ exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3′–5′–exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single–strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped–flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5′ overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate–limiting step in the enzymatic reaction. We determined an influence of the nature of the 3′–terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context–specific.In yeast  Maintenance of DNA integrity in cells of living organisms is performed by repair enzymes which detect and remove damaged nucleotides, thus leading to the restoration of the original DNA sequence . One wayThe process of DNA damage removal and AP site processing has been studied for many years, and until recently, it has been thought that the main biological function of AP endonucleases is the hydrolysis of the phosphodiester bond on the 5′ side of an AP site, followed by the formation of a 2′–deoxyribophosphate residue at the 5′ end and a 3′–hydroxyl group ,9,10. NoIt should be noted that in mammals, the main AP endonuclease APE1 participates in the processes of BER and NIR . AdditioSaccharomyces cerevisiae, two AP endonucleases have been identified too: Apn1 and Apn2  and 50 mM [K+]. There is also a fairly wide range of optimum pH levels in the buffer: 7.25 to 8.5. The results are in good agreement with the literature data on chimeric protein (GST)–Apn2 [Optimal conditions for the 3′–5′–exonuclease reaction of Apn2 were found by means of a model DNA duplex called ST)–Apn2 .To investigate the influence of the nature of the cleaved off 3′–terminal nucleotide on the course of the 3′–5′–exonuclease reaction, substrates with various terminal complementary and noncomplementary pairs were tested A. KinetiFAMExo–C/G, FAMExo–G/C, and FAMExo–T/A, but only ~20 min for substrate FAMExo–A/T, and 10 min for substrates containing noncanonical terminal pairs C/A, C/T, and C/C  took 1 h for fully complementary substrates  and C/C B. Intere and C/C A was fouThe obtained data suggest that the 3′–5′–exonuclease reaction proceeded in a context–specific manner. Thus, in the presence of 3′–terminal adenosine, the 3′–5′–exonuclease reaction proceeded much faster as compared to substrates with other complementary terminal nucleotides. Stability of the terminal pair also had a major impact on the course of the 3′–5′–exonuclease reaction. During the digestion of DNA substrates containing a noncomplementary terminal pair, the reaction rate was comparable to that of a substrate containing a 3′–terminal adenine.To make sure that Apn2 preferentially cleaves off adenosine, a similar experiment was conducted with substrates containing a triad of a nucleotide at the 3′ end . As one Ks and kcat are listed in kobs is the observed rate constant for the formation of S–1, e0 denotes initial concentration of Apn2, Ks represents the binding constant, and kcat means the catalytic rate constant of hydrolytic removal of a 3′–terminal nucleotide.The kinetic curves of accumulation and consumption of a product corresponding to the hydrolytic removal of the first nucleotide were fitted to a sum of exponentials; the dependence of the observed rate constant for the formation of product S–1 on the enzyme concentration is given in FAMExo–A/T), the catalytic rate constant (kcat) was approximately two times greater than that for other substrates with a terminal complementary pair and approximately two times less than the catalytic rate constant for substrates with a noncomplementary terminal pair. Constants of formation of the enzyme–substrate complex (Ks) are approximately the same if we take into account the margin of error. This finding suggests that the nature of the terminal nucleotide does not affect the stage of substrate binding. The value of kcat for substrate FAMExo–AAA/TTT significantly exceeds that of all other constants characterizing the catalytic stage of the process. This result confirms our supposition that the presence of 3′–terminal adenine increases the rate of the 3′–5′–exonuclease reaction.One can see that for the substrate containing the 3′–terminal adenosine  in the presence of various 2′–deoxyribonucleoside monophosphates. For this purpose, the enzyme was incubated for 5 min with a considerable excess of a dNMP (5 mM), after which the enzyme was mixed with a substrate , which could characterize changes of enzyme conformation in the course of interaction with DNA. Changes in fluorescence intensity of Trp residues during the binding and hydrolysis of substrate kobs. The regular residuals of fit curves are presented in kobs on substrate concentration proved to be linear  to calculate observed rate constant e linear B, which py) made it possible to register conformational changes in DNA substrates in the course of interaction with the enzyme. It has been shown earlier for human APE1 and Nfo [j/T  are suitable for 3′–5′ exonuclease activity. A comparison of kinetic data obtained by fluorescence and PAAG electrophoretic analyses for human APE1 and 5′–32P–labeled substrates Exo–aPuj/T revealed that the phase of the increase in aPu fluorescence intensity coincides with the removal of the aPu nucleotide from the jth position of the hydrolyzed oligonucleotide [py residue either at the first and second positions from the 3′ end of the hydrolyzable strand or in the complementary strand opposite the first nucleotide that can be cleaved off.Usage of the substrates containing fluorescent analogs of nitrogenous bases such as 2–aminopurine (aPu) or pyrrolocytosine ; accordingly, for this substrate, the interaction with the enzyme probably did not cause a substantial change in the environment of aPu. It should be noted that no changes in aPu fluorescence intensity were detectable at time points up to 10 s, suggesting that the use of this fluorescent analog does not permit the registration of conformational transitions in DNA at the stage of enzyme–substrate complex formation. The increase in fluorescence intensity of aPu for substrate Exo–aPu1 at time points > 50 s represents detachment of aPu from the 3′ end of the oligonucleotide as suggested by PAAG electrophoresis experiments  and catalytic step rate constant kcat (0.005 ± 0.001 s−1) were computed. The constants are in good agreement with the data from PAAG electrophoresis  were used: acrylamide, N,N′–methylene bisacrylamide, 2–amino–2–(hydroxymethyl)–1,3–propanediol (Tris), tetramethylethylenediamine (TEMED), urea, boric acid, dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) and its sodium salt, Coomassie Brilliant Blue R–250, ammonium peroxodisulfate, 4–(2–hydroxyethyl)–1–piperazineethanesulfonic acid (HEPES), sodium dodecyl sulfate (SDS), magnesium chloride, potassium chloride, and glycerol. All solutions were prepared with double–distilled water.py), or 6–carboxyfluorescein (FAM)   as part Escherichia coli Arctic DE3 cells transformed with the pET28c plasmid carrying the S. cerevisiae AP endonuclease gene by the following procedure. E. coli Arctic DE3 cells were cultivated at 37 °C in the 2YT medium (1 L) containing 20 µg/mL gentamicin, 10 µg/mL tetracycline, and 50 µg/mL kanamycin to an optical density of 0.6–0.7 at a wavelength of 600 nm. After that, the temperature was lowered to 16 °C, and transcription was induced by the addition of isopropyl–β–d–thiogalactopyranoside to a concentration of 0.2 mM. The cell culture was incubated for 24 h. The cells were then pelleted by centrifugation for 20 min at 5000× g at 4 °C, and a suspension of the cells was prepared in 30 mL of a buffer composed of 20 mM HEPES–NaOH pH 7.8, 40 mM NaCl, and a mixture of protease inhibitors . The resuspended cells were lysed using a French press. All subsequent procedures were carried out at 4 °C. The resultant cell lysate was centrifuged . Then, a solution of NaCl and imidazole was added to the supernatant to concentrations of 500 and 20 mM, respectively. The resulting solution was mixed with 1 mL of the Ni SepharoseTM High Performance resin  and stirred for 1 h. The enzyme was eluted with 5 mL of a buffer consisting of 20 mM HEPES–NaOH pH 7.8, 500 mM NaCl, and 600 mM imidazole. The obtained enzyme–containing fraction was diluted to a final NaCl concentration of 40 mM and applied to a HiTrap–Heparin™ column  at a flow rate of 0.4 mL/min. The chromatography was performed in a buffer containing 20 mM HEPES–NaOH and a linear 40 → 1000 mM gradient of NaCl; optical density of the solution was recorded at 280 nm. The purity of the Apn2 protein was determined by gel electrophoresis , 1 mM EDTA, 3 mM MgClAnalysis of the hydrolysis of model DNA substrates was performed via the following procedure. To 10 μL of a buffer containing a substrate, 10 μL of the enzyme in a buffer was added at 37 °C. The concentration ratio of the enzyme to substrate and time were varied depending on the type of experiment. To obtain kinetic dependences of the degree of substrate cleavage on time, to 50 µL of a buffer containing 2 µM substrate, 50 µL of the enzyme was added at concentrations of 1, 1.6, 2, 3, 4, or 5 µM in the same buffer. The reaction was stopped by adding 20 μL of a solution containing 7 M urea and 25 mM EDTA.2 concentration, a buffer was utilized in which the Mg2+ concentration was varied from 0 to 10 mM. To determine the optimal KCl concentration, a buffer was used in which the KCl concentration was varied from 0 to 300 mM. To find the optimal pH of the solution, a buffer was employed in which pH was varied from 6.5 to 8.5.To find the optimal MgClTo determine the effect of 2′–deoxyribonucleoside monophosphates on the Apn2 enzymatic activity, 10 mM dNMP was added to 40 µL of a buffer containing 2 µM enzyme and incubated for 5 min. Next, a 10 µL aliquot was taken and added to 10 µL of 2 µM substrate in the same buffer; the reaction time was 10 min.Separation of the products by electrophoresis was carried out in a 15% PAAG under denaturing conditions (7 M urea) in a vertical thermostatted Protean II xi unit  with voltage 200–300 V at 50 °C. The gel was visualized by means of an E–Box CX.5 TS gel documentation system .The degree of substrate transformation was determined in the Gel–Pro Analyzer 4.0 software . The degree of transformation was computed as the ratio of the sum of peak areas of cleavage products to the sum of the peak areas of the products and of a starting oligodeoxyribonucleotide. Expected error in determination of the degree of transformation, as a rule, did not exceed 20%.py were used, as was amino acid residue tryptophan contained in the protein. Registration of the data was conducted under conditions close to one turnover of the enzyme, i.e., at enzyme and substrate concentrations of the same order of magnitude. These experiments were conducted essentially as described previously [Kinetic curves of fluorescence intensity were recorded on an SX.20 stopped–flow spectrometer . The instrument’s dead time is 1.0 ms. As fluorophores, aPu and Ceviously ,39,40,41The enzyme Apn2 (500 µL) and duplex DNA (500 µL) in a buffer at various concentrations were placed into syringes, then the solutions were rapidly mixed. The reaction was carried out at 37 °C.To calculate rate constants of conformational transitions, we employed the OriginLab 15.0 software , which helped to compute parameters of the catalytic process (kinetic constants) by the nonlinear regression method.ikobs, the kinetic curves were fitted to the equationF(t) is observed fluorescence intensity at time t, F0 denotes a background signal level, F1 means the maximum amplitude of fluorescence intensity change at t → ∞, and kobs is the observed rate constant. Under quasi–equilibrium conditions, kobs is expressed ask1 and k−1 are rate constants for the formation and dissociation of the enzyme–substrate complex.To calculate the observed rate constants characterizing a change in fluorescence intensity, namely, py residues in DNA. It is shown for the first time that Apn2 and DNA undergo conformational transformations during their interaction. According to these experimental data, a kinetic scheme was derived that describes the mechanism behind the interaction of Apn2 with DNA substrates, and we determined the constant for the enzyme–substrate complex formation and rate constants of the reactions included in the kinetic scheme.In this study, pre–steady–state kinetics of conformational transformations of the enzyme Apn2 and model DNA substrates were investigated by the stopped–flow method with registration of changes in fluorescence intensity of this protein’s tryptophan residues as well as in the fluorescence of aPu or Cn) takes place at the first stage; this process was registered by means of the changing fluorescence of Trp and Cpy in substrates Exo–oppositeCPy and Exo–2CPy. The change of fluorescence intensity in substrate Exo–oppositeCPy between 10 s and 100 s indicates the process of conformational rearrangements, probably induced by the formation of a catalytically competent enzyme–substrate complex. By means of changes in fluorescence intensity of Cpy and aPu located at the 3′ end of the hydrolyzable strand (substrates Exo–1CPy and Exo–aPu1) at time points > 100 s, the catalytic stage of the process was detected. For the substrate containing Cpy at the second position of the 3′ terminus of the hydrolyzable strand (Exo–2CPy), an additional stage of fluorescence intensity alternation was documented, most likely explained by the translocation of the enzyme and by the assembly of a new catalytically competent enzyme–substrate complex.During the interaction of Apn2 with a DNA substrate, the formation of an enzyme–substrate complex (E•DNAE. coli Nfo [An influence of the nature of the cleaved off 3′–terminal nucleotide on the course of the 3′–5′–exonuclease reaction catalyzed by Apn2 is demonstrated. It is shown that the efficiency of the enzymatic reaction is context–specific, while the preferred nucleotide to be removed is 2′–deoxyadenosine, whereas the presence of a noncomplementary pair at the 3′ end raises the rate of degradation of the substrate. It should be noted that the Apn2–mediated mechanism of DNA degradation at single–strand breaks can be associated with the action of Apn1 and probably other BER proteins such as PCNA, as demonstrated for human APE1/APE2 . Therefocoli Nfo . It can py fluorescence in DNA made it possible to register conformational changes in Apn2 and its DNA substrates during the 3′–5′–exonuclease reaction and to assign them to certain stages of the enzymatic process.Thus, this study on pre–steady–state kinetics of tryptophan residues’ fluorescence in the enzyme and of aPu and C


Here, a non‐noble bimetallic catalyst of CoFe/Fe3O4 nanoparticles is successfully designed with a core–shell structure that is well dispersed on the defect‐rich carbon substrate for the hydrogenation of CO2 under mild conditions. The catalysts exhibit a high CO2 conversion activity with the rate of 30% and CO selectivity of 99%, and extremely robust stability without performance decay over 90 h in the reverse water gas shift reaction process. Notably, it is found that the reversible exsolution/dissolution of cobalt in the Fe3O4 shell will lead to a dynamic and reversible deactivation/regeneration of the catalysts, accompanying by shell thickness breathing during the repeated cycles, via atomic structure study of the catalysts at different reaction stages. Combined with density functional theory calculations, the catalytic activity reversible regeneration mechanism is proposed. This work reveals the structure–property relationship for rational structure design of the advanced non‐noble metallic catalyst materials with much improved performance.Non‐noble metal catalysts now play a key role in promoting efficiently and economically catalytic reduction of CO 3O4 core–shell structures, used for CO2 hydrogenation with promoted performance by rational structure design. Such phenomenon results from the oxide shell “breathing” induced by cobalt reversible exsolution/dissolution in the shell during the cycling reaction.A reversible deactivation/regeneration of the catalytic activity is found in non‐noble bimetallic CoFe/Fe Developing and applying sustainable green energy in human ordinary life is urgent as an effective strategy to address the energy and environmental concerns. Catalysis plays a critical role in conversion waste gas to renewable energies, especially for the carbon dioxide (CO2) hydrogenation. The catalytic conversion of CO2, known as the reverse water gas shift (RWGS) reaction, paves the way toward the formation of value‐added chemicals , and meanwhile, it can effectively ameliorate worldwide energy crisis and environmental concerns caused by massive CO2 emissions. Unfortunately, direct reduction of CO2 molecule is extremely difficult due to its thermodynamical stability and chemical inertness. Besides, it generally suffers from multiply sub‐reactions and creates diverse byproducts in the hydrogenation process. Therefore, new catalysts with high activity, selectivity, stability, and low cost have been an urgent need in the industrially catalytic conversion of CO2.The over consumption of fossil fuels has brought serious energy depletion and environmental issues, that is, climate change, pollution, ocean acidification, etc.,1 whic Au, Pd, Ru, etc. have been extensively studied due to their high activity. However, most noble metals have strong adsorption to CO* intermediate, which might lead to the catalysts sintering, coking and poisoning. Additionally, the high cost and low availability greatly limit their applications in industry catalysis. Therefore, more attentions turned toward non‐noble metal catalyst categories, including Co, Fe, and Cu. Their catalytic performances, that is, activity and selectivity, can be optimized to similar level as noble metals via rational structure and component design. For instance, creating surface defects, strong metal−support interactions (SMSI), improving particle dispersion, etc. Among them, alloying is one of the most common and effective strategies to control the principles of economy and more importantly, to construct multicomponent systems with tunable physical and chemical properties. A wide range of diverse bimetallic alloy catalysts including Ni–Co alloy, Ni–Au, Fe–Cu, Co–Fe, have been successfully prepared and examined for CO2 conversion in RWGS reaction process by far.Metal‐based catalysts have been widely applied in various catalytic applications, resulting in both highly scientific interest and potentially industrial practices. Especially, noble metals, such as Pt,4 Au,[2 hydrogenation with high activity and CO selectivity. Groot et al. found the bimetallic CoFe catalysts with Co and Fe segregating to form Janus‐like nanoparticles, displayed higher activity compared to a single element for Fisher–Tropsch (FT) reaction. Apart from the tunable properties by multi‐component system, carbon materials are often involved to effectively support and uniformly disperse catalyst nanoparticles due to their plentiful properties, including high temperatures stability (even above 750 °C under inert atmosphere), strong reducibility to metallic phases, controllable adjustment , low price, environmental friendliness, etc. Meanwhile, carbon matrix can certainly inhibit the non‐synchronization of catalytic reaction process in heterogeneous catalysis by providing more active sites. Especially, defect‐rich carbon materials display excellent CO2 adsorption and activation ability via inducing Lewis base sites on the surface. Lee et al. used CoFe2O4 derivatization to obtain bimetallic alloy carbides ((Fe1−xCox)5C2) supported on the CNTs, and the heterogeneous catalysts provided higher CO2 hydrogenation activity than the phase‐separated Janus‐like structure of CoFe nanoparticles. However, multi‐component systems might undergo out‐of‐step reactions of the metallic elements with different oxidation states, which increases the difficulty in the identification of active sites, element synergistic effects, and catalytic reaction mechanisms. Thus, atomic surface structure change and its corresponding effect on the catalytic properties of bimetallic catalyst, what is more, the bimetallic‐carbon support interaction are still unclear up to now. Deeply understanding the bimetallic catalyst structural and chemical evolution that governs the catalytic performance is the key to develop new catalysts and optimize advance catalyst with high activity.CoFe‐based alloy architectures were developed for COot et al.16 fou3O4 nanoparticles with core–shell structure, loaded on defect‐rich amorphous carbon (α‐C) for catalytic CO2 hydrogenation. This nanoarchitecture exhibits enhanced activity, selectivity and stability of the RWGS reaction. Catalytic properties of different bimetallic core–shell structures, with alloy core of ≈10 nm and oxide shell of ≈2 nm, can be effectively controllable by tuning only the Co/Fe atomic ratios while the size of nanoparticles remains almost unaltered. Defect‐rich α‐C matrix boosts its catalytic performance by providing more active sites to absorb H2 and CO2 molecules, and simultaneously, assisting the uniform dispersion of catalyst nanoparticles. Bimetallic Co–Fe catalyst with atomic ratio of 1:1 displays the best activity, the CO2 conversion is 30%, and CO selectivity is over or nearly 99% at 450 °C. Moreover, it also has stable durability without conversion rate loss after being cycled by 90 h. Interestingly, we found that the reversible dissolution of Co element in iron oxide shell would result in the degradation of CO2 conversion, accompanying with a phase transformation from Fe3O4 to CoFe2O4. Density functional theory (DFT) calculations were as well performed to help to understand the catalytic properties of the bimetallic core–shell structures. This work provides a fundamental interpretation on the structure–activity relationship for multicomponent system of advanced non‐noble metal catalysts in great potential energy and environmental applications.Herein, we reported an unique nanoarchitecture of bimetallic CoFe/Fe22.13O4 core‐shell structures formed on the defect‐rich α‐C substrate by a specific thermal reduction reaction of CoFe2O4 nanoparticles loaded on layered g‐C3N4 nanosheets in the mixed reaction gas  at 450 °C  and (200) of Co3Fe7 alloy, and the diffraction peaks at 35.2° and 62.4° belong to the (311) and (440) of CoFe2O4, respectively. It implies that CoFe2O4 is partially reduced in H2 and then oxidized in air. While increasing the loading mass to 15 wt%, only a single phase of Co3Fe7 can be detected by XRD patterns. By tuning the atomic ratios of Co and Fe in precursors, a phase transformation from Co3Fe7 to CoFe occurs with same loading amount, as shown in Figure 3N4 nanosheets  is ≈15 nm  in 4 Figure . The corTo further understand the chemical composition of the core–shell structure, we carried out electron energy loss spectrum (EELS) mapping on the dotted square in Figure 2.23O4 for RWGS reaction at atmospheric pressure has been comprehensively investigated to evaluate the catalytic activity for CO2 reduction. Figure 2 conversion and CO selectivity of the bimetallic catalysts with different atomic ratios of Co and Fe. Surprisingly, as the atomic ratio is 1:1, it shows appealing catalytic activity with CO2 conversion of 30% and CO selectivity of ≈99% compared to those with other ratios, and even among the widely explored catalysts , which is mainly contributed by Fe3O4. The lower chemical state of the sample before oxidation is further confirmed in Fe k‐edge XANES  and X‐ray absorption fine structure (XAFS), when Co is migrated into versus out of the shell, as shown in n Figure 20 theS Figure . The cor2.4Figure d‐spacing  samples are still α‐C and CoFe alloy, and the diffraction peaks of 27.0° and 44.4°, can be attributed to (200) of α‐C and (110) of CoFe, respectively  was successfully modified on TiO2 and MXene supports  among different supports, thereinto, the activity of α‐C supported bimetallic catalyst is 2.2 times higher than that on MXene and 1.4 times higher than that on TiO2   caused by oxygen adsorption has been found in the O 1s region. Simultaneously, CO2‐temperature programmend desorption (CO2‐TPD) was performed to illustrate the effect of reactive gas adsorption of different supports on catalytic activity, as shown in Figure α‐C/CoFe has much higher CO2 adsorption in the moderate region than the other supports, which is attributed to the abundant Lewis base sites provided by the defects. This once again confirmed that the defect‐rich α‐carbon substrate contributes to the adsorption of CO2. Moreover, the amount of bimetallic CoFe alloy increases gradually with the rise of reduction temperature  on the former surface than on the latter (−3.40 eV), the corresponding adsorption energy values are shown in Table 2O4 tends to bind H atoms stronger than that in Fe3O4. The H adsorption on CoFe2O4 is so strong that is will further cause difficulty of H desorption, and thus less active on CoFe2O4 for the considered reaction. That should mainly account for high catalytic activity of the bimetallic CoFe/Fe3O4 core–shell catalysts. In addition, the formation energy of oxygen vacancy on CoFe2O4 (4.67 eV) is about 0.5 eV less than on Fe3O4 (5.15 eV) because of weaker bond of Co—O than Fe—O. However, it is thermodynamically unfavorable (bonding energy of O2 is 3.0 eV per O atom) for the formation of oxygen vacancies to release O2 on both CoFe2O4 and Fe3O4,To check the influence of core–shell structure on its catalytic activities, DFT calculations were performed on the highly active CoFe/FeFigure 3O4 outer layer, compared to perfect Fe3O4 crystal (8.39 Å), we had compared the surface properties of Fe3O4 in Table 3O4 core–shell catalysts.2 adsorption on the catalyst surface, the sample was first treated in a pure CO2 environment, then driven out all CO2 in reactor by Ar, and then H2 began to be fed until the left CO2 had been completely reacted. Surprisingly, we detect the existence signal of CH4 but not CO , and it is easily desorbed. Thus, it should be that defect‐rich amorphous carbon substrate provides more active sites for excess CO2 adsorption and activation to the Lewis base sites, assisting to boost the catalytic performance. And the selectivity tends to CH4 due to the lack of hydrogen assisted in the CO2 activation process, resulting in the production of intermediates conducive to methanation. Controversially, if the sample was first treated in H2, following by removing all H2 in reactor via Ar, and then CO2 began to be fed up till H2 was completely depleted. A large amount of CO is identified, yet the yield of CH4 is 0  acetylacetonate (98%), iron (III) acetylacetonate (97%), triethylene glycol (99%), and ethyl acetate (99%) were purchased from J&K Scientific. Urea (98%) was purchased from Sinopharm Chemical Reagent Co., Ltd. All chemicals were used here without further purification.3N4 nanosheets were first prepared according to a typical two‐step thermal polymerization method by using urea as precursor. Secondly, CoxFe2−xO4 nanoparticles were synthesized through a simple wet‐chemistry procedures, and the tunability of composition can be achieved by change the molar ratios of cobalt and iron salt precursors, that is, 2:1, 1.5:1, and 1:1, details are in the Supporting Information. Then prepared g‐C3N4 nanosheets and CoxFe2−xO4 nanoparticles were respectively weighed and dispersed in water by ultrasonic to obtain a uniform dispersion. During ultrasonication, 15.3 mL of 1.5 mg mL−1 CoFe2O4 nanoparticle dispersion was added dropwise to 100 mL of 1.3 mg mL−1 g‐C3N4 nanosheet dispersion, continued to be ultrasonicated for 1 h, and freeze‐dried to obtain a CoFe2O4 content of 15 wt%. The g‐C3N4/CoFe2O4 nanocomposite are obtained. Different loading amount specimen could be obtained by changing the added volume of CoFe2O4 dispersion. Finally, a series of bimetallic CoFe samples were obtained by pretreatment in a reaction atmosphere. The sample obtained after pretreatment was marked as CoFe (X:Y), where X/Y represents the atomic ratio of Co/Fe. The different substrate synthesis strategy, for example, TiO2 and MXene, shared the same merit of experimental parameters with g‐C3N4.Porous g‐Crecursor.25 Secα radiation (λ = 1.5418 Å). The morphology images were collected by a field emission scanning electron microscope . The electronic structure was obtained by X‐ray photoelectron spectrometer . The high‐resolution transmission electron microscopy (HRTEM) images and atomic HAADF STEM images were characterized by utilizing Cs‐corrected transmission electron microscope , which was operated at 300 kV. The EDS and EELS measurements were characterized by related detectors configured in the STEM system. Soft and hard XAFS were recorded at Shanghai Synchrotron Radiation Laboratory (BL02B02). CO2‐TPD is performed via an integrated resonant micro‐cantilever and developed into micro‐electromechanical system‐based TGA .The phase structure was identified by powder X‐ray diffraction  with Cu Kd = 6 mm) under atmospheric pressure. Typically, 20 mg of catalyst was placed between two layers of quartz wool in the center of the reactor tube. Before the catalytic test, the catalyst was pretreated at 450 °C for 3 h with reaction gas  under atmospheric pressure. Subsequently, the performance at different temperatures (300–500 °C) was tested under the same feed gas conditions. The product was analyzed by an online gas chromatograph  equipped with TDX‐01 column, a thermal conductivity detector, and flame ionization detector. The CO2 conversion and CO selectivity were calculated by Equations  functional under the generalized gradient approximation, and the projected augmented wave (PAW) pseudopotentials. The GGA+U approach by Dudarev et al. was employed to treat the on site Coulomb interactions on the localized 3d electrons of Fe and Co, with the U values of 4.0 and 3.3 eV, respectively, taken from the ref. [k‐points sampling in all the calculations.Spin‐polarized DFT calculations were performed by using the Vienna Ab initio Simulation Package (VASP),27 witthe ref. . The ene3O4 was modeled by a 1 × 1 unit cell of Fe3O4 (100) of 17‐atom‐layer thickness supported on top of the 2 × 2 unit cell of CoFe (100) alloy surface with 6‐atom‐layer thickness. According to the measured distance of (110) on the CoFe/Fe3O4 surface of 2.14 Å, the lattice vector length of the slab model of CoFe (100)/Fe3O4 (100) was 6.06 Å. Here, the “6.06 Å” was corresponding to the lattice constant of 8.57 Å for Fe3O4 shell of cubic phase, which was slightly expanded compared to experimental value of 8.394 Å in perfect Fe3O4 crystal. As a result, the thickness of the 17‐atom‐layers Fe3O4 (100) had about 1.9 nm, which was comparable to that of the 1.95 nm in the CoFe/Fe3O4 system found in the experiments. In modeling the CoFe2O4 (100) surface, all the Fe atoms at the tetrahedron sites were replaced with Co atoms. The magnetic ordering in the CoFe alloy was set to ferromagnetic, and in Fe3O4 and CoFe2O4 the metal atoms at the octahedral sites were antiferromagnetically coupled to those at the tetrahedral sites, details are in the Supporting Information.The core–shell structure of CoFe/Fe3O4 (100) and CoFe2O4 (100), all atoms were allowed to relax until the residual forces on each atom are less than 0.03 eV Å−1. For CoFe/Fe3O4, the bottom four layers of the CoFe metal atoms were fixed to their bulk positions, and all other atoms were allowed to relax. Afterward, in the calculation of H atom adsorption and O vacancy formation, only the top four layers of the oxide surfaces were allowed for further relaxation. The surface adsorption energy of H atom, ΔEadsH, and oxygen vacancy formation energy, ΔEvacO, were respectively defined as:In the structure optimization of FeThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The process of phase evolution in the coating was observed in situ by HT-CLSM. The Mo2FeB2 phase with good thermodynamic stability can exist in the high-temperature liquid phase. It also has a phenomenon of connection and merging and turns into different morphology during the plasma cladding process. The hardness value of coating was much higher than the base metal, and the hardness value of Mo2FeB2 (785.5 HV) was higher than the eutectic matrix (693.2 HV). The wear mechanisms of the cladding under dry sliding were primarily caused by adhesive wear, accompanying slight oxidation wear. The Mo2FeB2 phase has an important effect on the wear resistance property.In this study, an Fe–Cr–Mo–Ni–B coating was prepared using plasma cladding on Cr5 steel substrate. The microstructure, phase evolution and tribological performance of the Fe–Cr–Mo–Ni–B coating were investigated. The microstructure is mainly composed of Mo Fe–Cr–B–C alloy was studied widely because the fracture toughness is improved owing to the entry of Cr into Fe2B [2FeB2 cermets, which were prepared by the two-step sintering technique. Mo2FeB2 showed outstanding wear resistances over wide temperature ranges under dry sliding [2FeB2 metal-based ceramics. The hardness and toughness of Mo2FeB2 metal-based ceramics were the highest when the C content was 0.5%, and the transverse rupture strength shows a maximum value of 1.9 GPa at a Mo/B atomic ratio of 0.9. The hardness decreased with the Mo/B atom ratio from 0.8 to 1.1, and the strength reached the highest when the Mo/B atom ratio was 0.9. The suitable amount of boron addition into the raw material was around the range of 1–2 wt.% [2FeB2 cermets are prepared by reaction sintering, and few studies of Mo2FeB2-reinforced iron base coatings were reported. Xu et al. [2FeB2 coating prepared by argon-arc cladding is better than Mo2FeB2 cermets prepared by vacuum sintering. Mo2FeB2 coating prepared by an arc welding method has good diffusion and metallurgical bonding with the base material, and there are no obvious pores. Although the microstructure with different component content was already investigated, the distribution and morphology of Mo2FeB2 particles prepared by cladding technology were complicated and variable, the distribution and form of particles especially are difficult to control, and good forming and performance cannot be guaranteed. Therefore, it is necessary to reveal the phase evolution and the growth mechanism of Mo2FeB2 particles. Moreover, the nucleation and growth of boride is related to quantity and distribution of the powders; this work invented a novel Fe–Cr–Mo–Ni–B powder prepared by vacuum gas atomization which reduces the inhomogeneity of the distribution.The development of wear-resistant materials is driven by complex and adverse working conditions. Various cladding techniques have been used to prepare wear-resistant coatings on the surface of workpieces to repair and strengthen the workpieces. Plasma cladding is one of the most promising techniques for surface modification ,2. Due tnto Fe2B . The weanto Fe2B . Binary nto Fe2B ,10. Zhannto Fe2B  reported sliding . Yu et a sliding ,14 inves1–2 wt.% . At presu et al.  reported2FeB2 particles and binary boride hard phase within the coating was elucidated. The hardness and wear mechanism of boride-reinforced Fe-based cladding were also investigated.In the present work, a novel Fe–Cr–Mo–Ni–B wear-resistant coating was fabricated on Cr5 steel by plasma cladding technology. The formation mechanism of the MoAnnealed Cr5 steel with a size of 200 mm × 100 mm × 15 mm was used as a substrate. The Fe–Cr–Mo–Ni–B alloy powders prepared by actual air atomization method were used as cladding powders. The morphology of cladding powders is shown in 3 and H2O with a ratio of 1:1:25) at room temperature for 90 s. The microstructure of the coating was observed by OMLPUS BX-6 metallographic microscope and the field emission scanning electron microscope  equipped with an energy dispersion spectroscope . A high-resolution X-ray diffraction  with a step size of 0.02° and 2θ range from 10° to 90° was used to identify the phase composition of the coating. The clad specimens were cut into cross-section and longitudinal section with a size of 10 mm × 20 mm by using wire electrical discharge machining, using a vernier caliper to measure the size. Then, these specimens were pre-treated using the standard metallographic techniques and etched using the corrosive solution . The Fe–Cr–Mo–Ni–B coating was machined into a cylindrical specimen with the size of Φ 5 mm × 3 mm. Before heating, the HT-CLSM chamber was filled with argon in order to avoid the oxidation phenomenon, and the specimen was prepared by mechanical polishing. The sample was heated following the heating cycles with a rapid heating rate of 500 °C/min from 100 °C to 1150 °C because the phase transition temperature ranged from 1012.9 °C to 1149 °C, according to the DSC curve of the Fe–Cr–Mo–Ni–B coating, as shown in The Rockwell hardness was tested by the Rockwell hardness tester  with a load of 150 kg. Different areas on the surface of the Fe–Cr–Mo–Ni–B coating were selected for 8 tests, and the average value after removing the maximum value and the minimum value were taken. The microhardness was measured by Vickers microhardness tester  with a load of 100 g and a loading duration of 15 s. A ball-on-disk wear tester  was used to evaluate the wear resistance of the cladding under dry condition at room temperature. The loads applied on the counterpart were 20, 30, 40 and 50 N. GCr15 balls with a diameter of 5 mm were selected as counterparts. The frequency of reciprocating wear, sliding speed and wear time were 6 Hz, 60 mm/s and 30 min, respectively, because after 30 min there was little change according to the results of the pre-experiment. The worn surfaces of clad samples were examined by Bruker direct reading spectrometer  to obtain wear size and calculate wear rate. Morphology of the worn surfaces was observed by scanning electron microscope.2FeB2 ternary boride. In point 2 and 3, the eutectic matrix structure mainly contains the Fe element and a small amount of the Cr, Mo and Ni elements. The XRD pattern of the Fe–Cr–Mo–Ni–B coating is shown in 2FeB2, Fe2B and MoB. The observed microstructure of the Fe–Cr–Mo–Ni–B coatings are shown in 2FeB2 phase distributed in the eutectic matrix. The grain size of the Mo2FeB2 phase is mostly between 5–30 μm. Due to the rapid heating and cooling rate of the plasma cladding process, the agglomerated, elongated and square Mo2FeB2 phase in the eutectic matrix is different from that in the cermets [2FeB2 increased significantly and irregular coarse Mo2FeB2 appeared. In this study, Fe–Cr–Mo–Ni–B powders were prepared by vacuum gas atomization. So, the excessive cooling rate leads to insufficient element diffusion and uneven distribution of component concentrations in the molten pool. The square Mo2FeB2 phase is formed because of the same growth rate in growing directions. The Cr element could significantly decrease the hard phase grain size, and the Mo2FeB2 phase may inhibit the growth of the hard phase particles in the length direction [2FeB2 phase changed. However, with the high cooling rate of the molten pool, the nucleation rate of the Mo2FeB2 phases increases. A large number of elongated and irregular coarse Mo2FeB2 particles are observed in the coating. During the rapid cooling, the Mo2FeB2 particles would grow to connect to each other and turn into irregular, coarse hard phase due to an uneven distribution of temperature in the molten pool. The same crystal structure strengthens the connection between the Fe2B and Mo2FeB2 phases. With the decrease in the Mo2FeB2 phase, the volume fraction of the eutectic structure which formed by Fe and B elements increases substantially. The volume fraction of the Mo2FeB2 phase and the eutectic boride is negatively correlated, and the microstructure is complicated and varied.The SEM image of the Fe–Cr–Mo–Ni–B coating and EDS sweeping analysis area are shown in  cermets . Accordi cermets ,19, withirection . The par2FeB2 phases and eutectic matrix. Due to the low solid solubility of B in austenite and ferrite, B is mainly used for the formation of boride phase rather than dissolution in the matrix. The addition of alloying elements will improve the solid solubility of B [2FeB2 increased significantly and an irregular Mo2FeB2 phase appeared. B and Mo are mainly consumed during the formation of the Mo2FeB2 phase. Fe is mainly distributed in the eutectic structure and matrix, while a small amount of Cr, Mo and Ni are distributed in the iron matrix. The Cr and Ni elements are evenly distributed in the Fe–Cr–Mo–Ni–B coating. Cr and Ni are considered to partially substitute Mo and Fe in the Mo2FeB2 phase to form complex borides [3B2 formed by the dissolution of Cr and other alloying elements into the Mo2FeB2 phase. Ni is often dissolved in the iron matrix as an austenite-forming element, which is conducive to ensuring the strength of the coating and improving the plasticity and toughness of the Fe–Cr–Mo–Ni–B coating. Due to the replacement and solid solution of Mo, Cr, Ni and other alloyed elements, the lattice distortion of the borides Mo2FeB2 and Fe2B is induced to form multiple composite borides M3B2 and M2B, and the mechanical properties are improved.The elemental distribution maps of the Fe–Cr–Mo–Ni–B coating are shown in ity of B . Therefo borides . The ter2B→L occurs at about 1079 °C, as shown in 2B phases connect to each other and turn into an eutectic network matrix, and the network breaks during heating . This solid solution of alloying elements improves austenite stability, so that a part of γ-Fe, rich in Cr, Mo and Ni, is retained without undergoing an allotropic transformation from γ-Fe to α-Fe [According to the binary phase diagram of Fe–B and Mo–B, the precipitation temperature of molybdenum and boron compounds is much higher than that of Fe–boron compounds, which could prove that the Moid phase . Hence,  to α-Fe . It is b2FeB2 hard phase of the Fe–Cr–Mo–Ni–B coating results in the fluctuation of the microhardness. The hardness is 785.5 HV, where the Mo2FeB2 hard phase is agglomerated, and the hardness of the eutectic matrix is 600.6 HV. The average Vickers hardness of the Fe–Cr–Mo–Ni–B coating is 693.2 HV. Therefore, the hardness improvement of the Fe–Cr–Mo–Ni–B coating mainly comes from the formation of the Mo2FeB2 phase. The boride hard phase can improve the hardness and wear resistance of the Fe–Cr–Mo–Ni–B coating, and the matrix can guarantee the toughness of the Fe–Cr–Mo–Ni–B coating, which effectively prevents the spalling of the Mo2FeB2 phase. Wear scar size, wear volumes and wear rate of the Fe–Cr–Mo–Ni–B coating under the four different loads are listed in F is the load in wear test and D represents the Sliding distance in the wear test. The friction coefficient and the microstructure of the worn surfaces of the cladding are shown in 2FeB2 phase on the surface of the Fe–Cr–Mo–Ni–B coating, the grinding ball impinged with the Mo2FeB2 particles at the initial stage of friction and wear. The curve of friction coefficient tends to flatten after a rapid increase. With the increase in the load, the average friction coefficient of the Fe–Cr–Mo–Ni–B coating decreases gradually due to the formation of an oxide layer and increase in the contact area between the grinding ball and the Fe–Cr–Mo–Ni–B coating. To better describe the wear mechanism, a schematic illustration of the wear process under different loads is shown in 2FeB2 phase protruding. The debris and the protruding Mo2FeB2 particles are relatively wear-resistant, resulting in great fluctuations in the friction coefficient.Where V is the wear volume, 3B2 and M2B, micro-cutting of the Fe–Cr–Mo–Ni–B coating is hindered. The hardness of GCr15 is higher than that of the coating, so the grinding ball would easily penetrate into the surface and produce wear volume loss. Plastic deformation and adhesion lead to the spalling of the eutectic matrix. With the increase in temperature in the friction process, slight oxidation was found on the worn surface. Under 40 N and 50 N, the spalling of eutectic matrix is more serious and some of the brittle M3B2 phase breaks under extrusion. The connection between the eutectic borides and M3B2 enhance the bonding force of the M3B2 phase. The M3B2 phase plays an important role in resistance to friction and wear. Therefore, there is plastic deformation and the formation of friction oxides on the worn surface. Additionally, the wear mechanism includes adhesive wear, slight oxidation wear and abrasive wear.Under 20 N and 30 N, the wear surface of the Fe–Cr–Mo–Ni–B coating shows shallow wear marks along the sliding direction and slight adhesive wear. Due to the existence of large areas of boride hard phases in the matrix, such as M2FeB2 boride particles and binary borides in Fe–Cr–Mo–Ni–B coating are studied, as well as the effect of borides on the hardness and wear resistance of the Fe–Cr–Mo–Ni–B coating. The main research conclusions are as follows:(1)2FeB2 hard phase connects with other phases and grows to a different morphology.The Fe–Cr–Mo–Ni–B coating was prepared by plasma cladding technology. The Mo(2)2FeB2 phase has good thermodynamic stability and stably exists in the high-temperature liquid phase. The growth and coalescence process of the eutectic matrix and Mo2FeB2 grains could occur at about 1150 °C.The microstructure of the Fe–Cr–Mo–Ni–B coating remains stable when the temperature is below 1050 °C. The Mo(3)2FeB2 phase plays an important role in the wear-resistance property.The hardness value of the Fe–Cr–Mo–Ni–B coating was much higher than that of the base metal. The wear mechanisms of the Fe–Cr–Mo–Ni–B coating under dry sliding were primarily caused by adhesive wear, accompanying abrasive wear and oxidation wear, and the MoIn this study, the formation mechanism, morphology, distribution position and size of various forms of boronized phases such as Mo


Herein, we show for the first time that oxalate oxidase (OXO) is an efficient in situ source of H2O2 for one of these biocatalysts, which is known as unspecific peroxygenase (UPO). OXO is reasonably robust, produces only CO2 as a by‐product and uses oxalate as a cheap sacrificial electron donor. UPO has significant potential as an industrial catalyst for selective C−H oxyfunctionalisations, as we confirm herein by testing a diverse drug panel using miniaturised high‐throughput assays and mass spectrometry. 33 out of 64 drugs were converted in 5 μL‐scale reactions by the UPO with OXO (conversion >70 % for 11 drugs). Furthermore, oxidation of the drug tolmetin was achieved on a 50 mg scale (TONUPO 25 664) with 84 % yield, which was further improved via enzyme immobilization. This one‐pot approach ensures adequate H2O2 levels, enabling rapid access to industrially relevant molecules that are difficult to obtain by other routes.H 2O2‐generation using oxalate oxidase in bioconversions catalysed by unspecific peroxygenase. This enzymatic tandem exhibits extraordinary potential for selective C−H oxyfunctionalisation reactions of complex drug scaffolds.High‐throughput μL‐scale screenings revealed optimal conditions for in situ H Oxidoreductases that use H2O2 as an electron acceptor include unspecific peroxygenases . These enzymes, which incorporate one oxygen atom from H2O2 into the reaction product, have attracted much attention over the past decade. UPOs catalyse C−H oxyfunctionalisations of a wide variety of industrially relevant molecules, offering advantages over their transition metal catalyst counterparts in terms of selectivity and sustainability.Enzymes are gaining increasing importance in industrial synthetic chemistry.2O2 in the reactions.2O2 lead to irreversible inactivation of the UPO by heme degradation  OXO (HvOXO) is very resistant to heat and proteases and can be expressed in yeast.2O2‐generation system. In the present study, HvOXO was produced by fermentation of Komagataella phaffii (Pichia pastoris) and purified using Ni‐sepharose resin . To assess the efficiency of the HvOXO‐oxalate system for H2O2‐generation, the previously studied Agrocybe aegerita UPO (AaeUPO) variant, known as PaDa‐I,Previous studies showed that Tm) was determined by the ThermoFluor method . Higher thermostability was similarly observed for PaDa‐I at pH 4.0 and 5.0, though PaDa‐I was more rapidly inactivated at pH 3.0 . Next, we studied the influence of pH on the kinetic parameters for HvOXO. kcat and Km values increased 3‐ and 244‐fold as the pH was raised from 3.0 to 5.0, respectively  as a model substrate, which gives the products (R)‐1‐phenylethanol (2) and acetophenone (3)2O2‐sources in previous studies (Table S1). In parallel, the drug tolmetin (4) was assayed as a PaDa‐I substrate to start investigating the potential of combining these enzymes for late‐stage functionalisation of bioactive compounds, since there is a considerable interest in using enzyme‐based C−H diversification strategies to increase the efficiency of drug discovery processes.4). The TONUPO for ethylbenzene (37 780) slightly improved over other oxidase‐based H2O2‐generation systems  conversion (from 1.6 mg/100 μL to 50 mg/7.5 mL). The resulting products 5, 6 and 7  and the combi‐CLEA (TONUPO 30 699), respectively. In the case of the combi‐CLEA reaction, enzymes were active for a longer time since mainly product 7 was observed after the same incubation time (96 h), and the negligible amounts of other minor products were not isolated. In the case of the reaction with soluble enzymes, the isolated yield for 5, 6 and 7 was 16, 24 and 44 %, respectively.These results encouraged us to scale up the tolmetin  was used as a model substrate to study the influence of various parameters on μL‐scale reactions containing PaDa‐I and HvOXO. First, increasing HvOXO concentrations were tested . At pH 3.0 and 4.0, an equimolar concentration of HvOXO and PaDa‐I was optimal. Contrarily, a 3‐fold higher HvOXO:PaDa‐I ratio was required at pH 5.0 to obtain the same conversion as that observed for the reactions containing 10 mM H2O2 instead of HvOXO and oxalate. This is due to the fact that HvOXO exhibits a high Km at pH 5.0  led to full conversions after 2 h . The same outcome was observed when H2O2 was replaced with HvOXO and oxalate, only with extended incubation times due to the slow release of H2O2. However, the HvOXO‐oxalate system is advantageous for reactions which contain higher tolmetin concentrations  resulted in around 2.6‐fold less product than when using HvOXO after 20.5 h. Only in the case of reactions with a single addition of H2O2, were conversions similar after 2 and 20.5 h due to PaDa‐I inactivation .Next, we used an equimolar concentration of HvOXO and PaDa‐I at pH 4.0 to further study the efficiency of their partnership. Reactions containing low tolmetin and H2O2 addition were not influenced by 25 % acetonitrile, which was the best cosolvent for PaDa‐I. Tolmetin and other drugs exhibit low solubility in glycerol, while acetone is very volatile. Thus, the best option for reactions with both PaDa‐I and HvOXO is 10 % acetonitrile, and 10 % tetrahydrofuran is a good alternative. Similarly, we tested various temperatures (25–50 °C) for the reactions containing these enzymes, which indicated that a temperature of 25 °C is optimal .The influence of twelve co‐solvents on tolmetin conversion were also investigated, since they are often needed for substrate solubilization. In reactions with 10 % co‐solvent and HvOXO, conversions were higher than 75 % in the presence of glycerol, acetonitrile, acetone or tetrahydrofuran , oxidation on the tetrahydrofuran ring likely produced compound 9 which was followed by ring opening to form a carboxylic acid product (10). PaDa‐I presumably accomplished the aromatic mono‐hydroxylation of methotrimeprazine (11) and triflupromazine (13), while this enzyme found two oxidation sites on the indole ring of fluvastatin (15). Phenylbutazone (17) was likely hydroxylated by PaDa‐I on the butyl side chain (18). In the clozapine (19) reaction, compounds 20 and 21 were tentatively assigned as the major reaction products. Using 5‐benzyloxygramine (22) as a PaDa‐I substrate, formation of a product with an aliphatic N‐oxide (23) likely occurred. In the ketoconazole (24) reaction, one of the main products was O‐dealkylated ketoconazole (25). In the case of raloxifene (26), our results suggested that PaDa‐I peroxidative activity (Scheme S1) may yield substrate radicals which were subjected to non‐enzymatic formation of a covalent raloxifene homodimer (27). Nevertheless, dimerization site on raloxifene was not unequivocally identified. These results confirm the extraordinary ability of PaDa‐I to enable various types of oxygenation reactions and one‐electron oxidations of non‐native substrates.After performing the reaction optimization, we used the best conditions to demonstrate the ability of PaDa‐I to catalyse late‐stage oxidations of a diverse drug panel using the HvOXO‐oxalate system in 5 μL reactions. 33 out of 64 drugs were used as a substrate by PaDa‐I, with a conversion higher than 70 % for 11 drugs . The corresponding UPLC‐QTOF/MS2O2‐source in UPOs‐catalysed oxyfunctionalisations using an inexpensive sacrificial electron donor. Furthermore, this work showed for the first time that it is possible to do high‐throughput screening using 5 μL‐scale UPO reactions by converting 33 drugs. One example reaction was successfully scaled up to 50 mg and featured a synthetically useful oxidation of tolyl methyl to carboxylic acid. Libraries of drug analogues resulting from miniaturised assays are useful assets for bioactivity screening and in the synthesis of human drug metabolites. For example, different non‐purified μL‐scale UPO reactions may be subjected to biophysical methods such as surface plasmon resonance (SPR)In summary, we have demonstrated here that HvOXO can play an important role as an HThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.