Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα′βγ)2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα′βγ)2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα′ possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined.Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and P Aromatic hydrocarbons are one of the most abundant classes of organic compounds in nature. They are primarily produced by plants as soluble secondary metabolic products or as components of the structural polymer lignin2Aromatoleum aromaticum strain EbN12 heterooctamer with a molecular mass of 482 kDa. It contains 2 Zn2+, although the activity of Apc is strongly inhibited by further added Zn2+ ions. Kinetic studies further indicated the requirement of the Apc core complex, Apcε, and ATP, but not of biotin, for catalytic activity. 2 ATP are hydrolyzed to 2 ADP and 2 Pi per one acetophenone carboxylated. In addition, uncoupled ATPase activity with either bicarbonate or acetophenone was detected in the absence of the second substrateEthylbenzene is aerobically or anaerobically catabolized via different pathways in various bacteria45A. aromaticum is encoded by the genes apcA-E (coding for Apcα′ (70 kDa), Apcγ (15 kDa), Apcα (80 kDa), Apcβ (75 kDa) and Apcε (32 kDa), respectively) which are located in an apparent operon together with the gene for the following enzyme of the metabolic pathway, benzoylacetate-CoA ligase. Apcα and Apcα′ are structurally related and have a sequence identity of 30%. Sequence comparison studies revealed phylogenetic relationships between Apc, acetone carboxylases (Acx) and ATP-dependent hydantoinases/oxoprolinases (Hyd) with sequence identities between 20 and 30%11hydA and hydB), an αβγ protomer for Acx  and an αα′βγε protomer for Apc .Apc of 2 heterohexamers. Upon carboxylation of acetone to acetoacetate by HCO3−, one ATP is either stepwise hydrolyzed to ADP and AMP plus 2 Pi by the enzymes from Xanthobacter autotrophicus or Rhodobacter capsulatus1314i without the transient formation of ADP by the enzyme from A. aromaticum2 heterotetramers that act on diverse substrates as cyclic amidohydrolases with concomitant hydrolysis of ATP to ADP and Pi16Acetone carboxylases (Acx) are organized as Acx(αβγ)For a more profound understanding of the complex carboxylation reactions, detailed structural data are indispensable. In this report, we describe the first X-ray structure of a member of the hydantoinase/ketone carboxylase family, the 482 kDa Apc core complex, at ca. 3 Å resolution. The structural details allow to outline a first proposal of the catalytic reaction cycle of Apc. Carboxylation of inert methylketones is a basic chemical process which makes the Apc reaction also attractive for biotechnological applications.2 heterooctamer which confirms the previously reported oligomeric state derived from a gel filtration–based molecular mass determination in solution2; 4% of the surface area2). Apcα′ is located at the periphery of the Apc core complex and only attached to Apcα. The small Apcγ partly envelops Apcβ and is itself embraced by the extended C-terminal arm of Apcβ  cosubstrates, forming phosphoenol-acetophenone and carboxyphosphate  into organic matter and extend organic molecules by forming new carbon-carbon bonds. Nature developed diverse types of carboxylases. They can be subgrouped into reductive and non-reductive enzymes, which are further subdivided into carboxylases dependent or independent on biotin2. Enolate intermediates are formed by ATP-independent and ATP-dependent tautomeric keto-enol shifts as catalyzed by RuBisCO3− in Apc is shared with biotin-dependent carboxylases. The generated carboxyphosphate might be channeled to the appropriate active site and decompose in-situ to the electrophilic CO2 which then reacts with the substrate either indirectly via the biotin cofactorCarboxylases are key enzymes in the biosphere because they catalyze the fixation of inorganic carbon (CO2 formed from carboxyphosphate with phosphoenol-acetophenone (2). The postulated movement of the exposed Apcα′ part into the wide hollow formed by Apcαβγ might be induced by the binding and activation of both substrates. However, due to steric hindrances the residual distance between the kinase sites of Apcα and Apcα′ in a modelled “tightened” complex is still predicted to be around 20 Å (from modelling results treating Apcα and Apcα′ as rigid-bodies). The apparent need of extensive conformational changes within the Apc complex during the catalytic cycle may implicate a role for the still elusive subunit Apcε. Apcε might be involved in the transitions between the “tightened” and “relaxed” forms of the Apc core complex and/or in the further bridging of the gap between the kinase sites. Consistent with kinetic and structural data we propose a first scenario for the mechanism of Apc  and carboxyphosphate (Apcα′) in the determined Apc core structure are ca. 50 Å apart from each other, which clearly precludes any direct interaction of COophenone . Therefoophenone . The looophenone  is also m of Apc : The reae hollow . Subseque hollow . As prop partner , or indulacetate 10. Finaletate  or methylene protons in case of longer substituents . We assume that this expensive activation strategy was specifically developed for carboxylating such chemically inert ketones. In other carboxylases substrate activation is achieved by biotin/ATP, by using electron-rich substrates such as phosphoenolpyruvateCompared to other carboxylases, Apc and Acx consume  two ATP equivalents per carboxylation event instead of one (as in biotin-dependent carboxylases) or none (as in RuBisCO). While activation of HCOA. aromaticum seems to be a special case of an organism that contains one member of all three branches of this enzyme family, namely ApcMicrobial genome analysis reveals the existence of a large new family of proteins containing highly related α and β subunits which are annotated as putative Hyd, Acx, and Apc complexes. hydAB. Gene clusters encoding acetone carboxylases exhibit the same order of the genes for α and β subunits (acxAB), but additionally contain a third gene for the γ subunit (acxC) downstream of acxB. Finally, gene clusters encoding acetophenone carboxylase or similar enzymes contain four or five genes in the order apcABCD(E), which code for the subunits Apcα′, Apcγ, Apcα and Apcβ of the core complex, followed by the gene encoding the additional subunit Apcε. Note that the fifth subunit is not present in every predicted operon for an acetophenone carboxylase-like enzyme, and that the sequences of these subunits are much more divergent than any of the other subunits. Close orthologues to Apcε from A. aromaticum are only found in the gene clusters of Nevskia ramosa (43% identity) and Rubrobacter xylanophilus (26% identity).Firstly, we investigated the organization of the encoding genes, which appears to be conserved even if more than one enzyme of the family is encoded in the genome of a bacterial strain . The geni appear to form separate subbranches121314Secondly, we constructed phylogenetic trees for the hydantoinase/ketone carboxylase family based on multiple sequence alignmentsThirdly, an approximate distinction between Apc, Acx and Hyd is possible by the different lengths of their subunits. In particular, Acxβ consists of ca. 720 amino acids, which is about 50 more than in Apcβ and about 150 more than in Hydβ orthologues. Most of the additional amino acids are located at the N- and C-terminal ends of the subunits. Acxα orthologues are ca. 30 amino acids longer than those of Apcα and more than 100 amino acids longer than Hydα orthologues. Finally, Acxγ orthologues contain ca. 50 more amino acids in their N-terminal regions, compared to Apcγ.Fourth, a comparative structure-based sequence analysis revealed several signature motifs for the hydantoinase/keton carboxylase family at the interface regions of subunits, at the metal binding site of subunit γ containing four conserved cysteines, at the ATP binding sites of subunits α (and α′) and at the glycine-rich segments characteristic for the new protein fold in subunits β. However, a specific identification of Apc, Acx and Hyd based on structural differences i.e. of the substrate binding sites reflected in a consensus sequence failed. In particular, members of the Apc and Hyd branches are phylogenetically highly diverse. In addition, only few enzymes are biochemically characterized yet (in the case of the Apc branch only one) and an assignment of the function of more distantly related species has to be regarded with caution.After annotating specific family members as Apc, Acx or Hyd based on items 1–3, preliminary structural models for the AcxABC and HydAB complexes can be calculated on the basis of the established structure of the Apc core complex and sequence identities between the subunits of 20–30% i.e. with the Swissprot serverXanthobacter Acx subbranch121314While the structure of the Apc core complex provides plausible insights into the functionality of the kinase-related partial reactions of the enzymes of the hydantoinase/ketone carboxylase family, the partial reactions related to carboxylation in Acx and Apc substantially differ as suggested by structural and biochemical dataA. aromaticum cells and purification of the Apc core complex were performed as described previously14C]-bicarbonate to [14C]-benzoylacetate−1 enzyme in 10 mM Tris buffer, pH 7.5, containing 0.1 M KCl and 5 mM acetophenone and used for crystallization.Cultivation and harvesting of ethylbenzene-degrading III edge with the peak wavelength of 1.008 Å were collected at the beamline PXII of the Swiss-Light-Source in Villigen (Switzerland) and processed with the program XDSP6522 and the cell axes are 240.1 Å and 336.6 Å, best compatible with an Apc(αα′βγ)2 heterooctamer in the asymmetric unit. The positions of three anomalously scattering Hg atoms in the asymmetric unit were detected with the programs SHELXC/D422 of Apcα′ substantially complicated the fitting of the polypeptide into the solvent-flattened electron density map, which was only feasibly because of its structural relationship to Apcα. The particularly low quality of the electron density for Apcα′ reflects its high mobility compared to Apcαβγ which might even be higher in solution because of the absence of an observed crystal lattice contact to Apcα′of a neighboring complex.The resulting model is reminiscent to that of many membrane proteins in exhibiting a high temperature factor of the coordinates (Baver = 109 Å2). Refinement was performed with REFMACfree factor was 20.2/24.0% in the resolution range 50.0–2.84 Å  using the Cartesian Honey Bee robot system. The optimized conditions are listed in 0–2.84 Å . The geoSequence similarities were analyzed by using BLAST , and phylogenetic trees were constructed using Clustal omega  and iToL .Atomic coordinates and structure factors for the crystal structures are deposited in the protein databank under the accession code 5L9W.How to cite this article: Weidenweber, S. et al. Structure of the acetophenone carboxylase core complex: prototype of a new class of ATP-dependent carboxylases/hydrolases. Sci. Rep.7, 39674; doi: 10.1038/srep39674 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The addition of NH3 also facilitated NO elimination at a fixed reaction temperature. Furthermore, of the products of the elimination process, zirconia (ZrO2) powder is a kind of biocompatible material, red phosphorus can be used to produce safety matches, organophosphorous pesticide and phosphor bronze, and the produced N2 might be collected and used as a protective gas or be converted into liquid nitrogen for other purposes.Zirconium phosphide (ZrP) powders were synthesized by elemental combination method via the direct reaction of zirconium powders with red phosphorus, and characterized by XRD, SEM, XPS, XRF, SAED and TEM measurements. The obtained ZrP powders were found to exhibit apparent activity in the ready eliminateion of nitric oxide (NO) via facile redox reactions, and the elimination dynamics was evaluated within the context of various important experimental parameters, such as reaction temperature and gas concentration. At a fixed amount of ZrP powders, an increasing amount of NO would be eliminated with increasing reaction temperature, and complete conversion of NO to N In the pursuit of a better living environment and with increasingly stringent environmental regulations, NOx emissions have become a research hotspot in the field of environmental science and engineering. NOx storage and reduction (NSR) plays an important role in controlling NOx emissions from automobile sources while permitting operation under predominantly lean-burn conditions6. So far, the most efficient technique to control NOx emissions from coal-fired power plants and automobiles is the selective catalytic reduction (SCR) of NO (4NO + 4NH3 + O2 = 4N2 + 6H2O)11 and NO2 (NO + 2NH3 + NO2 = 2N2 + 3H2O) with NH3 using various catalysts13. For SCR of NO by NH3, it has been found that NO and NH3 are introduced into the reaction vessel at a ratio of 1:1, and a certain amount of O2 is essential for the reaction. However, the excessive use of NH3 can cause air pollution and the corrosive nature of NH3 is also harmful to the experimental apparatus, which may cause secondary pollution. Therefore, it is necessary to reduce the use of NH3 for the elimination of NO. Also, the removal of NOx is generally carried out at high temperatures (>600 °C), where many catalysts may lose their activity18, leading to reduced elimination efficiency of NOx. Within this context, it is extremely urgent to find more suitable active species or to develop an efficient, green approach to the elimination of NOx at high temperatures.Nitrogen oxides  are known for their hardness and chemical inertness even at elevated temperatures21. In the past few years, substantial efforts have been devoted to the study of the catalytic properties of transition metal carbides and nitrides23. Notably, transition metal phosphides possess similar physical properties and even better catalytic activity and selectivity in many reactions such as hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), and have become a new research focus in the field of catalytic materials and water oxidation catalysis. For instance, Ni2P has been used as an excellent catalyst precursor for water oxidation catalysts24, and FeP as an efficient catalyst for HER26. Also, there are many studies on the catalytic performance of zirconium phosphate (ZrP2O7)31. In contrast, whereas ZrP has been synthesized by a variety of methods in the past decades, such as the reactions of zirconium metal with red phosphorus 33, zirconium or zirconium tetrachloride with Ca3P220, zirconium with PH333, or sodium co-reduction of ZrCl4 and PCl334, there are few reports on the chemical properties of ZrP and, up to date, there is no study of the activity of ZrP towards NO elimination.Transition metal phosphides  and the nitrogen atoms in the NO are positively charged (N2+), ZrP might be used to reduce NO by the reaction, ZrP + 2NO → ZrO2 + N2 + P. The results showed that the efficiency of NO elimination by ZrP increased with increasing temperature. In addition, it was found that when a small amount of NH3 was added to the reaction system, the following reaction could occur, 8ZrP + 22NO + 4NH3 → 8ZrO2 + 13N2 + 8P + 6H2O, leading to enhanced efficiency of NO elimination. The experimental results showed that at the ratio of the NH3 to NO concentration of 2:11, 0.5 g of ZrP powders was sufficient for the complete reduction of 500 ppm NO gas for up to 14 h at 750 °C. As for the products of the elimination reaction, ZrO2 powders can be used as a biocompatible material36 and catalysts/catalyst support, red phosphorus can be used to produce safety matches and organophosphorous pesticide, and the produced N2 may be collected and used as a protective gas or be converted into liquid nitrogen for other purposes. In this study, we not only identified an active species for NO elimination, but also reduced the amount of NH3 for NO reduction. The elimination process was of high efficiency and all reaction products could be used for other purposes.In this study, ZrP was synthesized by elemental combination method via the direct reaction of zirconium powders with red phosphorus in a quartz tube. Since the phosphorus atoms in the ZrP are negatively charged (PThe structures of the obtained ZrP were first characterized by X-ray diffraction (XRD) measurements. As shown in the Fig. The microstructures of ZrP were then investigated by SEM measurements. From the SEM images Fig. , it can 44.The microstructures of ZrP were also investigated by TEM measurements Fig. . From thIn view of the same diffractions of XRD for ZrP samples prepared at different temperatures Fig. , only thXRF analysis was then carried out to further analyze the composition of the ZrP samples, and the results are summarized in Table °C. In the course of experiment, a mixture of gases containing 500 ppm NO balanced with N2 was introduced into the reactor at the 174th min. It can be seen that at the controlled temperature of 650 °C, after the introduction of NO, the concentration of NO at the outlet increased rapidly and equal to the concentration of NO in the inlet, which means that there was no reaction between NO and ZrP. Yet, when the temperature was raised to 800 °C, no signal of NO was detected at the outlet for 1080 min (18 h), indicating that NO was completely reduced by ZrP in the reactor within this period. At lower temperatures (700 and 750 °C), the period of time where no NO was detected was shorter, suggesting that temperature was an important factor for the elimination of NO. Such a disparity of the reaction activity can also be manifested in XRD measurements of the solids after reaction. From Supporting Figure 2, indicating that part of the ZrP was oxidized into ZrO2 due to reduction of NO to N2. At the even higher temperature of 800 °C, no diffraction pattern of ZrP was detected and only ZrO2 diffraction patterns were observed, most likely due to complete consumption of ZrP in the reduction of NO, (ZrP + 2NO = ZrO2 + N2 + P).Figure 3 addition in NO elimination. As depicted in Fig. 3, 0.5 g of ZrP powders can reduce 500 ppm NO gas for 5 h at 750 °C. With the addition of NH3 into the reactor where the molar ratio of NH3:NO increased from 1:3 to 2:3, the time for the complete conversion of NO into N2 was increased from 6 h to 8 h. Yet, too much NH3 is not conductive to the elimination of NO. For instance at the mole ratio of NH3:NO = 3:3, the time for the complete conversion of NO by ZrP actually decreased to only 3 h, even shorter than that without any ammonia addition at the same condition (~5 h). The optimal NH3:NO ratio was identified at 2:11, where 0.5 g of ZrP powders were found to reduce 500 ppm NO gas for up to 14 h at 750 °C. This may be attributed to the following reaction, 8ZrP + 22NO + 4NH3 → 8ZrO2 + 13N2 + 8 P + 6H2O. From this reaction, we can see that at a fixed amount of ZrP, the addition of a small amount of NH3 could facilitate the reduction of NO. In fact, this elimination process not only reduces the use of NH3, but also greatly improves the elimination efficiency of NO as compared to SCR of NO by NH3.The tests were also carried out to elucidate the influence of NH2, some red brown powders were found adhering to the inner wall at the end of the quartz tube  and red phosphorus. In summary, a new method based on ZrP was developed for the reductive elimination of NO, where the reaction products might be collected and used for other applications.ZrP powders were prepared by thermal treatment of zirconium and red phosphorus in an argon atmosphere at controlled temperatures, which exhibited apparent activity in the reductive elimination of NO to N38.All chemicals were purchased from Alfa Aesar and used as received without further purification. To prepare ZrP, zirconium powders were placed in a quartz boat at one end of a quartz tube and the required quantity of 99.99% pure red phosphorus powders were placed at the other end under an atmosphere of purified argon. The temperature was slowly raised (2 °C/min) from room temperature to 800 °C~1000 °C and kept for 6 hThe phase structures and morphologies of the ZrP powders were characterized by X-ray powder diffraction , Scanning electron microscope , X-ray photoelectron spectroscopy  and X-ray fluorescence  measurements. The lattice fringes of the obtained samples and the corresponding selected-area electron diffraction (SAED) patterns were examined by using a high-resolution transmission electron microscope .39. 0.5 g of ZrP powders were sieved with a 40–60 mesh and placed on the quartz wool held in the reactor, and the reactor was heated by a vertical electrical furnace. The total flow rate was 198 mL·min−1 (room temperature), the mass of ZrP was 500 mg, and the corresponding gas hourly space velocity (GHSV) was 6 × 104 cm3·g−1·h−1, which was evaluated by the equation , and balance of N2. The concentration of NO was continuously detected by a gas chromatographic analyzer equipped with a flame photometric detector (Beijing Beifen-Ruili 3420 A). The NO conversion was calculated according to the equation .Supplementary information


We observe a clear electric field effect only when SrIrO3 is inserted between SrRuO3 and a gate dielectric. Our results establish that strong SOC of nonmagnetic materials such as SrIrO3 is essential in electrical tuning of these Hall effects and possibly other SOC-related phenomena.One of the key goals in spintronics is to tame the spin-orbit coupling (SOC) that links spin and motion of electrons, giving rise to intriguing magneto-transport properties in itinerant magnets. Prominent examples of such SOC-based phenomena are the anomalous and topological Hall effects. However, controlling them with electric fields has remained unachieved since an electric field tends to be screened in itinerant magnets. Here we demonstrate that both anomalous and topological Hall effects can be modulated by electric fields in oxide heterostructures consisting of ferromagnetic SrRuO Spintronic devices are typically controlled with magnetic fields or current injection but incorporating these mechanisms into high-density devices is difficult. Here the authors present an oxide device where the topological and anomalous Hall effects can instead be modulated using an electric field. Such properties have been attracting great interest as foundations for high-density and low-power-consumption spintronic devices because the properties coupled to the spin states enable spin manipulation without magnetic field variation or large current injection. Among the magneto-transport processes, of interest here is the intrinsic anomalous Hall effect2 (AHE), which is related to magnetization (M) in Hall resistivity (ρAHE = RSM). In some itinerant ferromagnets such as SrRuO34, the proportionality factor (RS) is governed by k-space monopoles, i.e., singularities originating from band-crossings gapped by SOC; not M but RS is potentially tuneable by electric field there. Another intriguing example is the topological Hall effect (THE) originating from scalar spin chirality concomitant with non-coplanar spin structures such as in a frustrated pyrochlore magnet5 and in metallic magnets characterized by a non-zero skyrmion number (Nsk)9. In the case of Nsk = 1, the topologically protected spin swirling texture is called magnetic skyrmion11. Despite mounting interest in electrical control of these Hall effects, however, their control in itinerant magnets has been elusive in conventional field-effect structures, that is, magnetic materials adjacent to gate dielectrics. Recent discovery of the THE in an oxide heterostructure composed of SrRuO3 and SrIrO312 brings an opportunity to develop electrical control because the strong SOC in SrIrO313 induces a SOC-related phenomenon in neighboring ferromagnetic SrRuO3. In addition to the intrinsic AHE inherent to SrRuO33, the combination of the strong SOC and the broken inversion symmetry gives rise to substantial Dzyaloshinskii–Moriya interaction (DMI)14 and the concomitant THE; the underlying spin texture has been deduced to be the interface-driven skyrmion12. In view of the fact that ultrathin films of itinerant magnets have been a playground for electrical manipulation of spin states19, an electric field applied to the ultrathin heterostructure may provoke effective manipulation of the spin state contending with screening effects by itinerant electrons of SrRuO3. Furthermore, we can expect that the strong SOC in SrIrO3 plays a positive role at the interface as well because both AHE and THE are magneto-transport phenomena driven by SOC.Profound implications for the electric field control of spin states have been revealed through magneto-transport properties arising from the spin-orbit coupling (SOC)3/SrIrO3/SrTiO3 from magneto-transport and magneto-optic Kerr effect (MOKE) measurements. The electrical modulation is effective only when SrIrO3 is inserted between SrRuO3 and a gate dielectric, i.e., SrTiO3. This indicates the essential role of the strong SOC in nonmagnetic materials for the electrical tuning of these Hall effects and possibly other SOC-related phenomena.Here, we show the clear modulation of both AHE and THE by an electric field in the heterostructure of SrRuO3 thin films were epitaxially grown on SrTiO3(001) substrates by pulsed laser deposition in three stacking orders with SrIrO3 (see Methods): a single layer of SrRuO3 (SRO5/Sub), a bilayer of SrRuO3 and 2-unit-cell SrIrO3 (SIO2/SRO5/Sub), and that with the inverted deposition order  and magnetization perpendicular to the film plane in the three samples. All of them show metallic conduction and ferromagnetic magnetization whose Curie temperatures (TC) are lower than the bulk value (160 K)3. These transport and magnetic properties are consistent with previously reported ultrathin SrRuO3 films20. Figure σΑΗΕ) as a function of magnetization with temperature as a control parameter. The σAHE of the three samples behave the same way; when the magnetization gets larger, the sign of σAHE is inverted from positive to negative. This sign inversion is consistent with the previous experiments12, manifesting the above-mentioned band anti-crossing singularities of SrRuO3. Conversely, such temperature dependence in AHE implies the potential for electrical control of AHE through the modification of the SOC-induced band.Five-unit-cell SrRuOρAHE + ρTHE) under applied electric fields are shown in Figs. B). The B dependence of ρAHE corresponds to that of magnetization normal to the film plane, whereas ρTHE shows up with the formation of the topological spin texture as exemplified by the peak at around 3 T in Fig. B-linear ordinary Hall term (RHB) from the Hall resistivity (ρyx). Both in SRO5/Sub . The sums of anomalous and topological Hall resistivities  to positive (200 V), while ρAHE is close to vanish under zero bias. This sign inversion indicates the sign reversal of the proportionality factor RS because MOKE measurements reveal that only a minor fraction of M, <10% of the magnetization, is electrically modulated . The insertion of the SrIrO3 layer makes a more important contribution to the AHE modulation than the EF shift in SrRuO3 does. The controllability may partly rely on the lower carrier density of inserted semimetallic SrIrO3, the order of 1019 cm−322.As shown in Fig. 3 −ρAHE (VG = −180 V)) in SRO5/SIO5/Sub with that in SRO5/SIO2/Sub, where we can clearly see the suppression of the modulation in the thicker SrIrO3 sample within all the measured temperatures. We attribute this suppression to the significant screening of the electric field within the thicker SrIrO3 layer.In order to clarify the effectiveness of the heterointerface close to the gate dielectric, we also fabricate the heterostructure with thicker (5 unit cells) SrIrOρTHE from magneto-transport measurement, ρAHE has to be subtracted from ρAHE + ρTHE. Since MOKE is proportional to magnetization as anticipated from its perturbative nature and also as experimentally verified in the previous report12, MOKE under applied electric field can be utilized as the reference of ρAHE, which is also proportional to magnetization at a constant temperature. The magnetic field dependences of Kerr rotation are shown by the broken lines in Fig. ρAHE at high magnetic field under each applied electric field; the normalized curves represent the B dependence of ρAHE. By subtracting them from ρAHE + ρTHE, we deduced ρTHE  gate voltage is applied, ρTHE gets smaller (larger) and the magnetic field region gets shrunk (enlarged). The variation in peak of ρTHE (ΔρTHE = ρTHE (VG = 200 V)−ρTHE (VG = −180 V)) normalized by zero-bias value (ΔρTHE/ρTHE (0 V)) is as large as 55%. The sign of THE remains the same during the sign inversion of AHE by electric field, supporting that the origins of AHE and THE are clearly distinct from each other as previously indicated12.THE is also modulated by electric field in the structure of SRO5/SIO2/Sub. In order to evaluate ρTHE is an outgrowth of ordinary Hall effect under fictitious magnetic field (beff) caused by the non-coplanar spin texture of skyrmion6. Since one skyrmion generates one flux quantum (Φ0 = h/e), ρTHE is described as a function of skyrmion density (nsk):P is spin polarization of conduction electrons in SrRuO3. Ordinary Hall coefficient RH is inversely proportional to carrier density. The change of P or RH is negligible because EF in SrRuO3 remains almost unchanged judging from the variation of ρxx as small as the order of 1% as discussed later. Therefore, the modulation of ρTHE indicates the change of nsk. The skyrmion size, estimated by VG = 200 V) to 12 nm (VG = −180 V) adopting P = −9.5% as a typical value of SrRuO323 and RH evaluated from the B-linear component of Hall resistivity under VG = 0 V at 30 K. Since rsk is proportional to the ratio of ferromagnetic interaction J to DMI D, or J/D6, the positive (negative) gate voltage should increase (decrease) J/D. The controlling knob of ρTHE is thought to be D rather than J; the variation of J is negligible because the applied electric field hardly varies TC, which can be evaluated by a kink structure in ρxx3  is not unity27. The emergent fictitious magnetic field can thus be generalized as ntopoNskΦ0, where ntopo is the density of the topological spin texture. Since Nsk is constant inherent in the topological spin structures and hence independent of the gate bias, the electric field modulation of ρTHE is attributed to the modulation of ntopo. In order to realize topologically nontrivial spin structures in SrRuO3, the interface-driven DMI is required as discussed in the above-mentioned skyrmion case. The ratio of J/D is inversely correlated with ntopo for any type of topological spin textures. The consequence therefore remains the same; positive (negative) gate voltage should increase (decrease) J/D.We examine the other possibilities of the underlying spin texture. One of the candidates is the conical spin structure, which is topologically trivial and is reported to give rise to THE-like signalρTHE change is independent of temperature as shown in Fig. HC) is shifted from 0.73 T (VG = 200 V) to 0.76 T (VG = −180 V) at 30 K  and THE (ΔρTHE/ρTHE (0 V)) with different thickness of SrRuO3 films are shown in Fig. ρxx variation normalized by zero-bias value (Δρxx/ρxx (0 V)) at various temperatures  ranges from 1.2% in SRO5/SIO2/Sub to 6.6% in SRO3/SIO2/Sub at 10 K. Since ρxx is inversely proportional to carrier density (n), the order of magnitude is consistent with the expected electron accumulation by a SrTiO3-back-gate transistor (Supplementary Note ρAHE in bilayers indicates that the gate-bias control of AHE has the specific tendency regardless of the film thickness. The same plots of SrRuO3 single layers (SROm/Sub) are also depicted for comparison. They have negligible change in AHE, while their ρxx variation is in the same range with that in bilayers with the same SrRuO3 thickness. As already pointed out, these results demonstrate the importance of inserting the SrIrO3 layer rather than the EF shift in SrRuO3. According to the equation  but also to RH. We show the calculated variation of ρTHE contributed from RH, i.e., n variation (ΔRH i.e. Δn = n(VG = 200 V) − n(VG = −180 V)) with the red dash-dotted line in Fig. n  and VG-independent electron mobility. All the modulations in heterostructures are located far above the red dash-dotted line. Therefore, most of the observed ΔρTHE is attributed to the change in size of the topological spin texture, i.e., DMI. These pronounced modulations of the AHE and THE are both achieved not by carrier-density variation in SrRuO3 but by modification of SOC at the interface of SrRuO3 and SrIrO3.The observed modulations of the AHE and THE are not simply explained by the variation of carrier density. In order to show this clearly, the variations of AHE . This thickness dependence is probably attributed to band structure deformation as observed in the thin limit of SrRuO332; the density of states near EF shrinks as SrRuO3 gets thinner.Recent theoretical calculations33. In contrast to the conventional Rashba systems, however, we have to take into account of the strong SOC of SrIrO3, the magnetic properties of SrRuO3, and their strong hybridization. Furthermore, since the itinerant-electron systems including SrRuO3 and SrIrO3 have complicated distribution of band-anti-crossings in their band structures35, the quantitative evaluation of the electric field-induced modulation in the heterostructures requires elaborate theoretical investigation. Nevertheless, the present observations clearly indicate that the electric field applied to the thin SrIrO3 plays the crucial role in SOC at the interface and even brings about the significant modification of magnetic properties in the neighboring itinerant ferromagnet. The method of inserting a thin nonmagnetic material with strong SOC between a ferromagnet and a gate dielectric may be applicable to tuning many intriguing spin-orbit coupled phenomena such as magnetic anisotropy15, domain wall motion36 and the DMI iteself37.One of the plausible pathways in the electric field-induced change of SOC is through the interface potential gradient, which is observed as the electrical control of Rashba-type band splitting in a semiconductor quantum well3 and SrRuO3, and single layer films of SrRuO3 were deposited on SrTiO3(001) substrates by pulsed laser deposition using a KrF excimer laser (λ = 248 nm). The substrate temperatures during the growth of SrRuO3 and SrIrO3 were 730 °C and 600 °C, respectively, where oxygen partial pressure was 120 mTorr. The laser fluence was 1.2 J/cm2 for SrRuO3 and 2.6 J/cm2 for SrIrO3.The epitaxial bilayers composed of SrIrO3(001). Magneto-optic Kerr effect was measured with a laser at 690 nm wavelength in polar geometry by using a photoelastic modulator.The magnetization data were recorded by a SQUID magnetometer with a magnetic field applied perpendicularly to the film plane and along the magnetic easy axis grown on SrTiO6 A/m2 for SRO5/SIO2/Sub. Back-gate transistors were fabricated using 0.5-mm-thick SrTiO3 substrates as a gate dielectric and silver paste as a gate electrode at the opposite side of the deposited films. Antisymmetrizations were performed for both the Hall resistivity and the Kerr rotation angle. Ordinary Hall term was subtracted from the Hall resistivity by linear fitting in a higher magnetic field region.Transport properties were measured in Hall bars cut by a diamond wheel saw (1 mm × 2.5 mm) and ultrasonically bonded with Al wires. The applied current was 10 μA, which corresponds to 3.6 × 10The data that support the findings of this study are available from the corresponding author upon request.Supplementary Information


According to the analysis of prognosis, this subset was significantly associated with poor 5‐year overall survival and worse disease‐free survival in HCCs, indicating that pNrf2 and Keap1 were two‐functional biomolecules, not only the oxidative stress markers but also biomarkers for prognosis of HCCs.In this paper, variation tendency of phosphorylated Nrf2, as the activated form of native Nrf2, was studied in 107 primary hepatocellular carcinoma (HCC) specimens treated by curative hepatectomy. Moreover, the coexpression of oxidative stress markers Keap1 and pNrf2, and their association with pathological features were also evaluated based on those specimens. The results showed that preserved cytoplasmic Keap1 expression of cancer cells was observed in 59 HCCs, while reduced Keap1 expression was determined in remaining 48 ones. With regarding to nuclear pNrf2 expression, 75 HCCs were defined as high and the other 32 ones as low. There was a significant association between Keap1 and pNrf2 expression in HCCs. Higher pNrf2 expression was observed, at a more substantial proportion, in those specimens with reduced Keap1 expression, compared to those with preserved Keap1 expression. The subset with higher pNrf2 and reduced Keap1 expression was defined as pNrf2 Hepatocellular carcinoma (HCC) ranks second among the most common cancer‐related mortality in the world Multiple etiological factors have been reported to be associated with the development of HCC. Among these factors, reactive oxygen species (ROS) has been a very hot topic. Relatedly, oxidative stress results from an elevation of ROS production or a decline of ROS‐scavenging capacity attracts a mass of focus from many researchers Huang et al. investigated the correlation of Keap1 and Nrf2 expression and pathological features in oral squamous‐cell carcinoma (OSCC) and elucidated that the oxidative stress markers, Keap1, Nrf2, Prdx6, and CD147 were significantly correlated with each other and had prognostic value as potential therapeutic targets in OSCC In this study, to understand the role of Keap1 and pNrf2 in HCC and to investigate the association between their expression and prognosis of HCCs, we analyzed their expression status in paratumor and tumor and elucidate their clinical implication.A total of 107 specimens from primary HCC patients treated by curative hepatectomy at Sir Run Run Shaw hospital , from 2008 to 2013 were used in this study. Patients were performed with irregular/segment/hemi‐ hepatectomy. Tumor tissues were dissected from macro‐ or microscopic confirmed HCC tissues, and their corresponding paratumors were isolated from liver tissues at least 2 cm away from tumors and with no microscopic tumor cells μm sections were deparaffinized in xylenes, and hydrated in alcohols. Endogenous peroxidase activity was blocked using 3% H2O2 in methanol for 10 min. The sections were then washed and antigens were retrieved using antigen‐specific methods. Then these sections were treated with 10% normal goat serum and incubated overnight at 4°C with mouse anti‐Keap1 antibody , rabbit anti‐Nrf2 antibody , or rabbit anti‐pNrf2 antibody . The bound primary antibody was visualized with streptavidin–biotin peroxidase  according to the manufacturer's instructions. Tumor specimens that were expected to have a high expression of pNrf2, which react against the specific antibodies were used as positive control tissue sections. Tumor specimens incubated with nonimmune serum were used as negative controls, to ensure that secondary antibodies did not produce background stain.Immunohistochemical studies were performed on 107 tumor and paratumor specimens. 5‐Five fields of each specimen were selected to evaluate the results by immunohistochemical staining. Specimens were scored by two independent pathologists. Staining intensities were assessed with high‐power (×400) microscopy, and was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The proportions of tumor cells positively stained with Keap1 and pNrf2 were scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The Q‐score was the sum of the intensity and proportion scores and ranged from 0 to 7. A Q‐score≤2 was considered negative, or reduced expression, and a Q‐score>2 was considered positive, or preserved expression. Accordingly, cytoplasmic Keap1 and nuclear pNrf2 immunostaining were assessed in 107 HCC specimens.Total RNA was extracted from tissues using Trizol . Reverse transcription was performed according to the protocol of PrimeScript RT reagent kit . Quantitative PCR was performed using SYBR premix Ex Taq . Gene expression in each sample was normalized with the house keeping gene (GAPDH) expression. Relative quantification of target gene expression was evaluated using the comparative CT method. The primers used for this study were listed in Table μm nitrocellulose membranes (Millipore). The membranes were blocked with 5% fat‐free dry milk in TBST for 2 h, then incubated with primary antibodies, mouse anti‐Keap1 antibody ; rabbit anti‐Nrf2 antibody ; rabbit anti‐pNrf2 antibody  or mouse anti‐Tubulin antibody . Tubulin was used as internal control. The immunoreactive blots were visualized using an enhanced chemiluminescence reagent  according to the manufacturer's instructions.Total protein was extracted using 1× RIPA buffer (sigma). The lysis buffer was supplemented with protease inhibitor cocktail (Roche). According to protein concentrations, equal amounts of protein were separated on 12% polyacrylamide gels and transferred onto 0.45 χ2 test or the Student's t‐test was used to analyze group differences. Kaplan–Meier method and the log‐rank test were used for overall survival and disease‐free survival analysis. Univariate analyses were used to examine the prognostic factors . A P < 0.05 was considered to be statistically significant.The data were analyzed by SPSS version 17.0 . The P = 0.002), vascular invasion (P = 0.002), and metastasis (P = 0.005), as depicted in Table P = 0.0103).A total of 107 patients with HCC met inclusion criteria in this cohort. The characteristics of patients and clinical characteristics of HCC included in the study were summarized in Table P = 0.082; hazard ratio [HR] = 2.24) and worse disease‐free survival , and a similar result was observed in Figure − patients and less than 40% of the subset of pNrf2+ Keap1− patients lived more than 80 months after surgery. According to the analysis of prognosis, it indicated that reduced expression of cytoplasmic Keap1 was significantly associated with poor 5‐year overall survival and worse disease‐free survival in HCCs, especially with upregulated pNrf2 expression.Keap1 protein was mainly expressed in cytoplasm Fig. . In addiP < 0.05; HR = 0.313) and DFS . Furthermore, comparing the OS and DFS of pNrf2+ Keap1− subset and other subset, it was observed that patients with high pNrf2 and reduced Keap1 expression demonstrated worse prognosis, OS  and DFS   was 6% and inactivating mutations of KEAP1 was 4% in human HCC NFE2L2 and inactivating mutations of KEAP1NFE2L2 and KEAP1 mutations were only observed in advanced HCC suggesting that these mutations were late events in human liver carcinogenesis Keap1 mutations; among those mutated Keap1, only 25% (1/4) were false positive Keap1 inactivated mutation, indicating that the rate of high nonfunctional keap1 expression caused by Keap1‐inactivated mutation is even low Keap1 mutations Some of the somatic mutations of AKR1B10, NQO1, AKR1C1, GCLM, and GCLC were involved in this signaling pathway. MacLeod et al. and Agyeman et al. found aldo‐ keto reductase (AKR) 1B10 and AKR1C1 to be major target genes of Nrf2 in human cells Additionally, on the way to explore the mechanism underlying Keap1‐Nrf2 pathway, the expression of Nrf2‐target genes, as well as pNrf2 protein, play an informative role, considering their potential prognostic significance. Various downstream genes, + Keap1− subset was significantly associated with poor 5‐year overall survival and worse disease‐free survival in HCCs, indicating that pNrf2 and Keap1 were two‐functional biomolecules, not only the oxidative stress markers but also biomarkers for prognosis of HCCs.In this manuscript, the coexpression of oxidative stress markers Keap1 and pNrf2, and their association with pathological features were evaluated in HCC. The result showed that there was a significant association between Keap1 and pNrf2 expression in HCCs. Higher pNrf2 expression was observed, at a more substantial proportion, in those specimens with reduced Keap1 expression, compared to those with preserved Keap1 expression. According to the analysis of prognosis, the pNrf2According to all these recent findings, the Keap1‐Nrf2 system plays a crucial role in cellular defense against oxidative stress, which has been reported to promote cancer development and act as a biomarker to anticipate prognosis of HCCs. But little of the specific mechanisms are known about its association with carcinogenesis of HCC.+ Keap1− subset was significantly associated with poor 5‐year overall survival and worse disease‐free survival in HCCs, indicating that pNrf2 and Keap1 were two‐functional biomolecules, not only the oxidative stress markers but also biomarkers for prognosis of HCCs.In this paper, variation tendency of pNrf2, as the activated form of native Nrf2, was studied in 107 primary hepatocellular carcinoma (HCC) specimens treated by curative hepatectomy. Moreover, the coexpression of oxidative stress markers Keap1 and pNrf2, and their association with pathological features were also evaluated. The correlation between Keap1 and pNrf2 expression and clinical outcome was assessed. The result showed that there was a significant association between Keap1 and pNrf2 expression in HCCs. Higher pNrf2 expression was more likely observed in those specimens with reduced Keap1 expression. The pNrf2None declared.Figure S1. The mRNA levels of AKR1C1(A) and GCLC(B) were assessed by qPCR in tumor(n = 14) and paratumor(n = 14). Typical staining for high and low nuclear pNrf2 or Keap1 expression in paratumor(C). The negative and positive control for the pNrf2 (Ser40) antibody (D)Click here for additional data file.


The membrane insertase YidC catalyzes the entrance of newly synthesized proteins into the lipid bilayer. As an integral membrane protein itself, YidC can be found as a monomer, a dimer or also as a member of the holotranslocase SecYEGDF-YajC-YidC. To investigate whether the dimeric YidC is functional and whether two copies cooperate to insert a single substrate, we constructed a fusion protein where two copies of YidC are connected by a short linker peptide. The 120 kDa protein is stable and functional as it supports the membrane insertion of the M13 procoat protein, the C-tailed protein SciP and the fusion protein Pf3-Lep. Mutations that inhibit either protomer do not inactivate the insertase and rather keep it functional. When both protomers are defective, the substrate proteins accumulate in the cytoplasm. This suggests that the dimeric YidC operates as two insertases. Consistent with this, we show that the dimeric YidC can bind two substrate proteins simultaneously, suggesting that YidC indeed functions as a monomer. During their biosynthesis, most of these proteins insert into the membrane in an unfolded state by interaction with YidC or with the Sec translocase2. Under certain conditions, the Sec translocase can form a large complex consisting of SecYEG, and SecDF, YajC and YidC3. In this Sec holo-translocon, the peripheral SecA protein may participate using ATP hydrolysis to promote the translocation of large periplasmic substrate domains4.The plasma membranes of bacteria contain about a thousand different integral proteins with various topologies5. Membrane proteins are released from SecY through its lateral gate7. The membrane insertase YidC of E. coli is a 6-spanning membrane protein that is capable to catalyse membrane insertion of small proteins, e.g. the coat proteins of the filamentous phage M13 and Pf312. In contrast to the Sec translocase, YidC does not possess a transmembrane channel. Instead, it has a hydrophilic groove in the inner leaflet of the bilayer and a transmembrane hydrophobic slide15. An open question is how a substrate protein interacts with YidC to achieve its integration into the membrane bilayer.The 10-spanning SecY protein of the SecYEG complex has an hour glass-like structure with a hydrophobic ring at its center that controls the passage of a protein chain through the membrane17 suggested a dimeric arrangement, possibly in a dynamic equilibrium with the monomer18. In addition, YidC and its mitochondrial homolog Oxa1 were found in dimeric complexes by cryo-electron microscopy19. Furthermore, Boy and Koch (2009) purified YidC from inner membrane vesicles as a 140 kDa protein and concluded that YidC appears mainly as a dimer in the cell20.Whereas the molecular structure of both SecYEG and YidC show a single protein globule, they still may be present as oligomers in the membrane. Cryo-electron microscopy of SecYEG23. When one of the protomers is inactivated by a mutation, the dimeric YidC still retains activity, showing one active protomer is sufficient for YidC activity. Finally, we show that a YidC dimer can bind two substrate molecules simultaneously, suggesting that the functional unit of the insertase is a monomer.To test whether a dimeric YidC is functional and whether two copies functionally cooperate to insert a single substrate protein, we artificially constructed a YidC dimer by gene fusion. We show here that the YidC dimer is functional and can insert M13 procoat into the membrane. Also, the dimer is capable of inserting the C-tail protein SciP and the Pf3-Lep model protein that were previously characterized as YidC substrate proteinsyidC gene was duplicated and the two copies of the gene were connected by a linker coding for a flexible 13 amino acid peptide  showed the expected size of 120 kDa on a Western blot and no monomeric YidC was present (compare size with YidC0 control). A coexpressed M13 procoat protein was readily cleaved by signal peptidase (with its active site on the periplasmic side of the membrane) and hence inserted by either the monomeric YidC or the YidC dimer, respectively, proving that the dimer was a functional membrane insertase . Also, the purified YidC dimer was examined by electron microscopy  and second, a mutation in residue 362 that changes the threonine into an alanine24 were investigated. The two YidC mutants did not complement the chromosomal depletion strain MK6 overnight using an agar plating assay , where the periplasmic exposure of a cysteine results in an increased molecular weight of 0.5 kDa. The shift of the SciP protein was only observed for the wild-type  in one protomer was combined with a mutation of T362A expected to perturb the hydrophilic groove within the transmembrane domainsay Fig. . In all say Fig. .Figure 6The above complementation and activity results with a YidC dimer possessing one inactive protomer suggest that the two protomers operate on their own. However, it is still possible that two dimers come together to contribute their functional subunits to form a complex conferring insertase activity. To rule this latter possibility out, we examined substrate binding within the dimeric YidC. The contact site mutation 427 C was introduced into the first, the second or both protomers of YidC and coexpressed with M13 procoat 33 C Fig. , lane 2.To investigate the substrates that are crosslinked, the dYidC was purified and tested whether it has a substrate bound. Since the two YidC protomers are linked with a peptide encoding a recognition site for the prescission protease Prp, the purified dimer was cleaved after incubation for 3 h at 4 °C with the protease Fig. . After c14 and dimeric forms28 have been isolated. In addition, YidC that is linked to SecYEGDFYajC has been documented-methionine was added for 1 min, followed by the addition of 10% TCA.The wild-type M13 procoat expressed from a pMS119EH derivative was co-expressed with one of the various defective YidC mutants to test for their capability of inserting a substrate. Overnight cultures of MK6 harboring two plasmids, one encoding the YidC defective mutants or the YidC controls (pGZ119EH) and the other the wild-type M13 procoat (pMS119EH), were grown in LB  overnight at 37 °C. The cells were washed twice with LB medium and then inoculated 1:100 in fresh LB medium  and grown under YidC depletion conditions to an ODThe TCA precipitated cells were washed with acetone and centrifuged for 5 min at 4 °C. After drying at 95 °C for 5 min, 50 µL of resuspension buffer  was added and the pellet was incubated at 95 °C for 5 min. 1 mL of TEN-TX buffer  and 20 µL of Staph-A (Zymed) were added and incubated at 4 °C on a rotating wheel for 30 min. The samples were then centrifuged for 5 min at 4 °C to pellet the Staph-A. The supernatant was transferred to a new tube containing His tag (or M13) antibody for immunoprecipitation. The samples were incubated on the rotation wheel overnight at 4 °C before adding 20 µL of Staph-A for 1 h. Staph-A was pelleted by centrifugation for 1 min at 4 °C, washed twice with TEN-TX buffer and once with TEN-buffer . The pellets were resuspended in 50 µL gel loading buffer with or without 100 mM DTT and heated at 95 °C for 5 min. The samples were centrifuged for 2 min and the supernatant loaded on a SDS polyacrylamide gel without disturbing the Staph-A pellet.22 was co-expressed with one of the various defective YidC mutants in MK6 cells in LB  under YidC depletion conditions to an OD600 = 0.5 at 37 °C. After resuspension in M9 medium lacking methionine the cells were grown for another 45 min, induced with 1 mM IPTG for 10 min. 2.5 mM AMS (Molecular Probes) was added for 1 min and then labeled with 15 µCi 35S-methionine for 2 min. Nonradioactive L-methionine was added for 10 min, and the AMS reaction was quenched by the addition of 10 mM DTT for further 10 min. The cells were acid-precipitated and immunoprecipitated (his tag antibody) as previously described.The plasmid-encoded SciP-C35S]-methionine for 1 min. Cells were collected by pelleting in a microfuge at 4 °C and resuspended in 40% sucrose and 33 mM Tris-HCl (pH 8.0). Cells were converted into spheroplasts by treatment with lysozyme (5 µg/mL) and 1 mM EDTA (pH 8.0) on ice for 30 min. The spheroplasts were treated with or without proteinase K  for 1 h on ice. 5 mM PMSF  was added to stop the protease reaction. Pf3-Lep was immunoprecipitated with leader peptidase antibody and samples analyzed by SDS-PAGE and phosphorimaging.pMS119 bearing Pf3-Lep was expressed with one of the YidC constructs to examine N-tail translocation. Overnight cultures of the YidC depletion strain MK6 bearing two plasmids, one encoding the YidC defective mutant or the YidC blank control (pGZ119EH) and the other Pf3-Lep (pMS119), were grown in LB  overnight at 37 °C. After washing the cells twice in LB medium, the cells were inoculated 1:100 in fresh LB medium  and grown under YidC depletion conditions for 6 h. In the last 10 min of the 6 h period, 50 µM IPTG was added to induce the expression of YidC defective mutant. The cells were then washed with M9 medium and transferred into M9 lacking methionine, and grown for 30 min. The YidC construct and Pf3-Lep was induced with 1 mM IPTG for 2 min, and then pulse-labeled with [The band intensity (without gel background) of the inserted protein was quantified using the phosphorimaging Aida Analyzer . For quantification of the membrane insertion of M13 coat protein we took into consideration that the membrane inserted and processed coat has only one methionine whereas the non-processed procoat has 3 methionines. For Pf3-Lep, the proteolytic fragment has 6 methionines, whereas the intact Pf3-Lep has 7.600 = 0.7 and growth of the culture was continued for 4.5 h. The cultures were then harvested and resuspended in buffer A  followed by disruption with a One Shot cell disruption system (Constant Systems LTD). The lysed cells were centrifuged  to precipitate cell debris. The supernatant was transferred to an ultracentrifuge tube and the membrane vesicles spun down at 140,000 × g for 2 h at 4 °C. The membrane pellet was resuspended in buffer A and loaded on a three step sucrose gradient  in 50 mM Tris-HCl, pH 8, 1 mM EDTA to separate the inner and outer membrane vesicles. The resuspended membrane vesicles were loaded on the gradient and centrifuged at 110,000 × g for 16 h. The inner membrane vesicles (IMVs) concentrate at the separation layer between sucrose step 1 and 2 while the outer membrane vesicles (OMVs) are between step 2 and 3. The IMVs were carefully collected with a syringe and mixed 1:4 with sucrose-free Tris buffer . The IMVs were then centrifuged at 140,000 × g for 2 h at 4 °C and resuspended in buffer B . IMVs were solubilized with 2% DDM for 2 h and centrifuged at 100,000 × g for 1 h at 4 °C. YidC in the supernatant was then purified by immobilized metal ion chromatography (IMAC). Ni-Sepharose beads were prepared by washing twice with 10 column volumes (CV) with buffer C . 20 mM of imidazole was also added to the solubilized membranes before adding the Ni-Sepharose. After 2 h of incubation on a rotary wheel at 4 °C, the column was run by gravity flow, washed with 40 CV of buffer D  and then eluted with the buffer E . The protein was further purified by size exclusion chromatography using a Superdex 200 10/300 at RT in 10% glycerol, 10 mM Ada (acetamidoimino-diacetic acid), pH 6, 300 mM NaCl, 0.02% DDM, 0.01% Cymal6). The collected fractions were analyzed by SDS-PAGE and visualized by Coomassie staining.For purification, tryptone medium was used to grow the MK6 cells bearing pGZ119-dYidC. 0.5 mM IPTG was added at an ODFor testing the accessibility of dYidC to the prescission protease, two versions (one with and one without the prescission protease site) of the YidC dimer were purified as described above, except the sucrose centrifugation and gel filtration step. Prescission protease was added to the purified YidC in 300 mM NaCl, 50 mM Tris-HCl, pH 8, 1 mM EDTA and incubated at 4 °C for 3 h prior to terminating the reaction by the addition of 75% methanol.600 = 0.5 at 37 °C. 1 mM IPTG was added for 10 min, followed by the addition of 200 µM DTNB for 3 h. Then, 20% TCA was added to the cultures and stored overnight at 4 °C followed by processing for PAGE and immunodetection (his tag antibody). For cleavage with prescission protease of the dYidC-substrate complexes, the proteins were purified and cleaved as described above and subsequently analyzed via PAGE and immunodetection.Overnight MK6 cultures in LB , harboring plasmids encoding cysteine mutants of YidC (pGZ119EH) and M13-H5+33C procoat (pMS119EH) were inoculated 1:100 into fresh LB medium and grown under depletion conditions to an ODDataset 1


Specifications TableValue of the data•Data on transesterification kinetic also is useful to determine time temperature and composition to obtain high-quality biodiesel from waste frying oil;•Data on Biodiesel greenhouse gases emission is useful to compare to emission of other fuels;•Data on biodiesel energy efficiency is a good indicator to choose the best biodiesel blends to use in power generation.1The data showed in this article is related to the research article by Silva Filho et al. 2A) of waste frying oil. This way, adjustments were made to find the kinetic constant (k) and the initial concentration (CA0). After determination of the kinetic constants, activation energy (Ea) of the transesterification reaction was calculated based on the linearised Arrhenius equation. All fitting model were evaluated of according to This analysis was performed in a jacketed reactor and 100 mL total volume, into which we added 50 mL of oil and 8.33 mL of alcohol and mixed them in the reactor (6:1 volumetric ratio). The reactions proceeded at temperatures of 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C, with 0.1% NaOH (m/v) as a catalyst during constant stirring for up to 120 min. Measurement of the biodiesel amount formed was carried out every 10 min by decanting into a separating funnel; the light phase was washed with petroleum ether and separated in a Soxhlet extractor The energy data generated after the combustion of the oil in the generator, as well as fuel consumption and efficiencies are shown in 2, and SO2 amounts were measured every 2 s via insertion of the BEA720 gas analyser sensor (Bosch Ltd®) into the gas outlet duct of the power generator engine. Measurements were taken during power generation using diesel oil and biodiesel blends of 10, 20, 30, 40, 50, 75 and 100% purity. These measurements were used to compare the gaseous emissions of biodiesel samples with the control (diesel oil), as shown in Silva et al.


It is found that as the BaTiO3 NF aspect ratio and volume fraction increased the permittivity and maximum electric displacement of the nanocomposites increased, while the breakdown strength decreased. The nanocomposites with the highest aspect ratio BaTiO3 NFs exhibited the highest energy storage density at the same electric field. However, the nanocomposites with the lowest aspect ratio BaTiO3 NFs achieved the maximal energy storage density of 15.48 J/cm3 due to its higher breakdown strength. This contribution provides a potential route to prepare and tailor the properties of high energy density capacitor nanocomposites.There is a growing need for high energy density capacitors in modern electric power supplies. The creation of nanocomposite systems based on one-dimensional nanofibers has shown great potential in achieving a high energy density since they can optimize the energy density by exploiting both the high permittivity of ceramic fillers and the high breakdown strength of the polymer matrix. In this paper, BaTiO There have been a variety of reported methods for synthesizing BaTiO3 NFs, such as the two-step hydrothermal method, one-step hydrothermal method, topochemical solid-state reaction and anodic aluminum oxide template method2531323 NFs on the dielectric constant (relative permittivity), breakdown strength and energy storage density of the nanocomposites were investigated systematically. On increasing the BaTiO3 NF volume fraction or aspect ratio, the dielectric constant and maximum electric displacement of the nanocomposites increased monotonically while the breakdown strength decreased monotonically. Under the same electric field, the nanocomposites with higher volume fraction or higher aspect ratio BaTiO3 NFs possessed higher discharged energy density. The maximal energy storage density reached 15.48 J/cm3 in nanocomposites containing 7.5 vol% BaTiO3 NFs synthesized at 210 °C for 2 h under the electric field of 300 kV/mm.In this study, we have prepared P(VDF-HFP) nanocomposites with a range of aspect ratio BaTiO3 nanofibers (BT NFs), an initial hydrothermal reaction was used to synthesize Na2Ti3O7 nanofibers (NT NFs) due to its extensive research history and easily controlled nanofiber morphology2Ti3O7 nanofibers (NT NFs) which exhibited a high aspect ratio and favorable dispersibility. 2Ti3O7 (PDF card NO. 31–1329). The corresponding TEM images were shown in 2Ti3O7 nanofibers (HT NFs), which retained the morphology of the Na2Ti3O7 nanofibers.In order to obtain BaTiO3 NFs by a second hydrothermal reaction. The H2Ti3O7 phase is a layered titanate, which is a good precursor for soft chemical synthesis because of its open structure that enables ion exchange and topochemical transformation. 2Ti3O7 and BaTiO3, respectively. During the second hydrothermal reaction, the Ba2+ ions diffuse into the host lattice of H2Ti3O7 by ion exchange with H+, which leads to a rearrangement of the octahedra sharing and structural transformation to perovskite BaTiO3. The Ba2+ ions possess a higher positive charge compared with H+, thereby promoting the structural transformation. In addition, it is known that the edge sharing octahedra were driven into vertex sharing octahedra during the topochemical transformation3 NFs.The HT NFs were transformed to BaTiO3 NFs hydrothermally synthesized at 210 °C for 2–12 h, respectively. The corresponding XRD patterns are shown in 3 (PDF card NO. 75–0462) without any impurity phase can be obtained at 210 °C with a reaction time ranging from 2 h to 12 h and the crystallization of BaTiO3 was improved at longer reaction times. When the reaction time was 2 h, the products were a mass of nanoparticles and few nanofibers, as shown in 3 NFs synthesized at 210 °C for 12 h. It can be observed that the surface of BaTiO3 NFs was rough and a HRTEM image of a typical BaTiO3 NF synthesized at 210 °C for 12 h is shown in 3 respectively, which revealed that the NFs grew in the [001] direction. The corresponding selected area diffraction pattern (SADP) also exhibited the characteristics of a single crystal.3 NFs increased to a much greater extent compared to the diameter, leading to an increase in the fibre aspect ratio. The length and diameter of the BaTiO3 NFs synthesized at 210 °C for 2 h, 6 h and 12 h were analyzed using SEM images by ImageJ software, as shown in 3 NFs calculated from 3 NFs increased with an increase of reaction time.It was worth noting that with an increase of reaction time, the length of the BaTiO3 NFs in the P(VDF-HFP) matrix, the BaTiO3 NFs were surface functionalized by dopamine. As highlighted in the introduction, the breakdown strength of the nanocomposite is equally important as the dielectric constant with regard to the energy density and the breakdown strength tends to decrease with increasing volume fraction of ceramic filler. In order to improve the dielectric properties of nanocomposites and maintain a high breakdown strength, the nanocomposites were fabricated at a relatively low volume fraction of BaTiO3 NFs in this study, varying from 2.5% to 7.5%. The upper-surface morphology of the BaTiO3 NFs/P(VDF-HFP) nanocomposites are shown in 3 NFs synthesized at 210 °C for 2 h, 6 h and 12 h, respectively where it can be seen that the aspect ratio of the BaTiO3 NFs in nanocomposites gradually increased. 3 NFs synthesized at 210 °C for 12 h. The visibility of the BaTiO3 NFs in nanocomposite became more pronounced with increasing BaTiO3 NFs volume fraction. It can be observed that the BaTiO3 NFs exhibited good compatibility and dispersibility in the P(VDF-HFP) matrix and the nanocomposites exhibited limited defects as the volume fraction of BaTiO3 NFs approached 7.5%. This can be attributed to that the fact that the BaTiO3 NFs were surface modified by dopamine and therefore formed a strong adhesive bonding force with the polymer matrix.To improve the compatibility and dispersibility of BaTiO3 NFs aspect ratio and volume fraction for a frequency range of 1 kHz to 10 MHz. The dielectric constant of the nancomposites increased with the increase of volume fraction of BaTiO3 NFs since BaTiO3 possess a higher dielectric constant compared with pure P(VDF-HFP) polymer. At the measurement frequency of 1 kHz, the dielectric constant of the nanocomposite with 7.5 vol% BaTiO3 NFs synthesized at 210 °C for 12 h (aspect ratio 21.0) reached 23.4 while the dielectric constant of the samples with 7.5 vol% BaTiO3 NFs synthesized at 210 °C for 6 h (aspect ratio 7.4) and 2 h (aspect ratio 3.5) was 17.8 and 14 respectively. This demonstrates that on increasing the aspect ratio of the BaTiO3 NFs, the dielectric constant of the nancomposites was significantly improved. There are a number of previous reports and theoretical models that have demonstrated the increased dielectric constant as a result of using high aspect ratio ceramic fillers142535i is known as the depolarization factor of ellipsoids in the x, y, z direction. For BaTiO3 NFs, where the radii ax > ay = az. Ni can be expressed as equation 2where N3 NFs.The Maxwell-Garnet model indicates that the dielectric constant of the nanocomposites will increase on increasing the aspect ratio of BaTiO3 NFs. For example, the dielectric loss of the nanocomposites with 7.5 vol% BaTiO3 NFs synthesized at 210 °C for 2 h, 6 h and 12 h was 0.032, 0.037 and 0.046 at 1 kHz, respectively. The dielectric loss remained less than 0.06 (<100 kHz) for all samples which was attributed to the relatively low volume fraction of BaTiO3 NFs and their good compatibility and dispersion in the polymer matrix. The dielectric properties of nanocomposites were efficiently improved by increasing the aspect ratio of the BaTiO3 NFs, without the need for additional fillers.However, there was no apparent variation about the dielectric loss on increasing the aspect ratio of BaTiO3 NFs ranged from 2.5% to 7.5% and the aspect ratio of the BaTiO3 NFs were 3.5, 7.4 and 21.0 respectively. As can be seen from max) increased monotonically with an increase of the volume fraction of BaTiO3 NFs. A similar trend can also be observed in max of nanocomposites with 7.5 vol% for various aspect ratios of BaTiO3 NFs and the Dmax was enhanced with increasing the electric field. 3 NFs, the Dmax of nanocomposites improved significantly. For instance, the Dmax of the nanocomposites with 7.5 vol% and higher aspect ratio (AR) BaTiO3 NFs (AR = 21) reached 5.4 μC/cm2 while the value was only 3.9 μC/cm2 for lower aspect ratio BaTiO3 NFs (AR = 3.5) under an electric field of 160 kV/mm. It has been demonstrated in 0εrE, where ε0 and εr are free space and the relative of the permittivity of the nanocomposites respectively, then under the same electric field (E) an increase in Dmax can result from an increase of dielectric constant of the nanocomposites on increasing the volume fraction or aspect ratio of the BaTiO3 NFs.The electric breakdown strength of the nanocomposites was analyzed by a two parameter Weibull cumulative probability function, as shown in P(E) is the cumulative probability of electric failure, E is the experimental breakdown strength, EB is a scale parameter that refers to the breakdown strength at the cumulative failure probability of 63.2% which is regarded as the characteristic breakdown strength, and β is the Weibull modulus associated with the linear regressive fit of the distribution. 3 NFs. The characteristic breakdown strength (EB) is summarized in 3 NFs, the EB of the nanocomposites decreased; see B of the nanocomposites with 2.5 vol% BaTiO3 NFs and aspect ratio of 21 was 268.7 kV/mm, which reduced to 257.4 kV/mm and 188.4 kV/mm when the volume fraction of BaTiO3 NFs increased to 5.0% and 7.5%, respectively. The introduction of more BaTiO3 NFs into the polymer matrix resulted in defects and electric field concentrations, which increased the risk of failure and decreased the breakdown strength. The nature of the field concentrations with respect to volume fraction and aspect ratio are discussed later. It also can be demonstrated in B of the nanocomposites decreased with an increase of the aspect ratio of the BaTiO3 NFs.where The energy storage density of the nanocomposites were calculated from D-E loops according to the 3 NFs synthesized at 210 °C for 2 h as a function of electric field and volume fraction of BaTiO3 NFs. This indicates that the energy storage density increased with an increase of electric field. Moreover, the nanocomposites with the higher volume fraction of BaTiO3 NFs possessed the higher energy storage density under the same electric field. 3 NFs as a function of electric field and aspect ratio of the BaTiO3 NFs. Similarly, the nanocomposites with the higher aspect ratio BaTiO3 NFs exhibited a higher energy storage density under the same electric field. The higher energy storage density can be attributed to the significantly enhanced dielectric constant and Dmax of the nanocomposites on increasing the volume fraction and aspect ratio of the BaTiO3 NFs. 3 NFs. Under an electric field of 300 kV/mm, the maximal discharged energy density of 15.48 J/cm3 was obtained in the nanocomposites with 7.5 vol% on low aspect ratio BaTiO3 NFs synthesized at 210 °C for 2 h.3 NF volume fraction and aspect ratio, finite element modelling (Ansys APDL v15.0) was employed. A two dimensional electrostatic analysis of a single high permittivity BaTiO3 NF  embedded in a low permittivity P(VDF-HFP) matrix (εr = 7) was used to investigate the effect of fiber aspect ratio and angle (with respect to applied field direction) on the maximum localized electric field. BaTiO3 composites in the range of the experimentally produced aspect ratios (3.5 to 20.1) and volume fractions (2.5 vol.% to 7.5 vol.%) were analyzed.To provide a deeper understanding of the decrease in breakdown strength and increase in dielectric constant with an increase in BaTiO3 inclusion leads to the electric field concentrating in the low permittivity matrix, and such field concentrations are likely to be sites for initiating dielectric breakdown within these nanocomposites. This can be seen in 3 fiber with an aspect ratio of 3.5 at 2.5 vol.%. The blue contours are low field regions and red contours indicate high field concentrations at the tips of the fiber. High aspect ratio fibers aligned almost perpendicular to the applied field direction (angle > 85°) produced slightly lower electric field concentrations compared to lower aspect ratios, however at angles below 85° the high aspect ratio fibers result in local electric fields that are significantly higher than the applied field, see 3 aspect ratio and volume fraction on the relative permittivity (dielectric constant) and field concentration, where an increase in local electric field leads to a reduced breakdown strength. The relative permittivity  within a P(VDF-HFP) matrix was used to demonstrate the effect of separation distance on electric field concentrations within the matrix. The ‘worst-case’ in terms of field concentration was occurred when two particles were aligned in the direction of the field, as in High BaTiO3 nanofibers (BT NF) with a variety of aspect ratios were synthesized by a two-step hydrothermal method. The effects of the aspect ratio and volume fraction of the BT NF on dielectric properties and energy storage densities of the P(VDF-HFP) based one-dimensional nanocomposites were investigated and modeled in detail. As the aspect ratio and volume fraction of the BaTiO3 NFs was increased, the dielectric constant and Dmax of the nanocomposites were both increased monotonically while the breakdown strength decreased. The nanocomposites with highest aspect ratio and volume fraction of BT NFs exhibited the highest energy storage density under the same electric field. The maximal energy storage density reached 15.48 J/cm3 in the nanocomposites with 7.5 vol% BT NFs synthesized at 210 °C for 2 h under the electric field of 300 kV/mm. This work provides a potential new route to prepare and tailor the properties of novel high energy density capacitor nanocomposites.In this study, BaTiO3 nanofibers (BT NFs) were synthesized by a two-step hydrothermal method. Firstly, sodium titanate nanofibers  were synthesized. A 1.446 g mass of titanium oxide  was added to 70 ml NaOH solution (10 M) and the mixture was stirred for 2 h to form a homogeneous suspension. Hydrothermal reactions were carried out at 210 °C under an auto-generated pressure for 24 h in a 100 ml Teflon-lined autoclave. The products were washed by distilled water and then soaked in diluted 0.2 M hydrochloric acid  for 4 hours to obtain hydrogen titanate nanofibers (H2Ti3O7 NFs). The BT NFs were synthesized by a second hydrothermal reaction where 0.150 g of H2Ti3O7 NFs were dispersed in 70 ml Ba(OH)2∙8H2O solution and the mixture was sonicated for 10 min. The hydrothermal reactions were carried out at 210 °C under an auto-generated pressure for 2–24 h in a 100 ml Teflon-lined autoclave to obtain BT NFs at a range of aspect ratios. The products were soaked in 0.2 M HCl solution briefly, then washed using distilled water several times and dried at 80 °C in an oven.The BaTiON,N-dimethylformamide (DMF) by stirring and sonicating to form a homogeneous suspension. The suspension was then cast onto a clean glass and dried at 80 °C for 12 h under vacuum. The dried nanocomposite sheets were then compressed into films at 200 °C under a pressure of approximately 15 MPa. Gold electrodes were sputtered on both sides of the film using a mask with 2 mm diameter eyelets.The BT NFs were mixed with a solution of P(VDF-HFP) in The crystalline phases of the NFs were evaluated by X-ray diffraction  with Cu-Kα radiation (λ = 1.5406 Å) at room temperature. The size and morphology of the NFs and the nanocomposites were observed using a scanning electron microscope . High-resolution transmission electron microscopy (HRTEM) images of the NFs were taken with a Titan G2 60–300 instrument, using an accelerating voltage of 300 kV. The frequency-dependent dielectric constant and dielectric loss were measured using an Agilent 4294 A LCR meter with frequency ranging from 1 kHz to 10 MHz. The electric displacement-electric field loops and leakage current were measured by a precision Premier II ferroelectric polarization tester  at room temperature and 10 Hz.How to cite this article: Zhang, D. et al. Significantly Enhanced Energy Storage Density by Modulating the Aspect Ratio of BaTiO3 Nanofibers. Sci. Rep.7, 45179; doi: 10.1038/srep45179 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Specifications tableValue of the Data•These data provide the micrographics, chemical composition and crystallinity data of CNC and CNF from blue agave waste.•These data provide further information about NMR analyses of the different cellulose nanoentities.•These data allow researchers to extend the comprehension of the related article.1gave tequilana Weber var. azul) waste. For more information, please refer to Robles et al. The data in this article contains information on the chemical composition , visual 2SEM images were obtained with a Scanning electron microscope Hitachi S-3400N with field emission cathode, with a lateral resolution of 10–11 Å at 20 kV.Chemical characterization was done according to standard methods 13C NMR spectrometry was performed at a frequency of 250 MHz with an acquisition time of 0.011 s, at room temperature. The spectrum was recorded over 32 scans and water was used as solvent for all the nanocelluloses.CrystallinityCrystallinity indexes were calculated as follows:Segal Index I200 corresponds to the main crystalline domain at around 23°, and IAM is the scatter of the amorphous cellulose, which has its highest intensity around 2θ = 18°.In which Peak fitting:θ1 and 2θ2 the limits of the fitted signal for the corresponding crystalline domains ; while Stot corresponds to the total area R2 ≥ 0.997 was achieved, which corresponds to a 99.7% accurate fitting.In which the sum of the areas correspondent to the diffraction of crystalline planes is assumed to be the area of the crystalline region, being 2C4-NMR:In which Scrys corresponds to the crystalline region of the C4 spectra (from 87 to 93 ppm) while Stot corresponds to the total area of the C4 region which includes crystalline and amorphous contribution.hklδ) were estimated with the Scherrer equation Crystallite domain sizes (


The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2‐oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC‐KDM inhibitors. Demethylation of a trimethylated H3K9 peptide by K241A KDM4A was also observed, although the catalysis was markedly less efficient than for wild‐type KDM4A, and demethylation activity was not increased upon increasing the O2 concentration. Binding analyses by fluorescence polarisation indicate similar binding efficiencies of the histone substrate to both the WT enzyme and the K241A variant. Overall, the results imply that K241 is important in KDM4A catalysis; although it does have a role in coupling 2OG oxidation and demethylation, it is unlikely to be involved in regulating O2 binding.Here, we report biochemical studies assessing the role of K241 in KDM4A catalysis. The results reveal that the K241A KDM4A variant is capable of catalysing substrate‐uncoupled oxidative decarboxylation of 2OG at comparable efficiency to wild‐type KDM4A, thus implying a similarly efficient reaction with OEscherichia coli BL21(DE3) cells; a truncated wild‐type sequence (KDM4A WT), or a variant with an alanine in place of Lys241 (K241A KDM4A). Studies were then carried out to evaluate the ability of the two KDM4A proteins to catalyse the demethylation of a histone peptide substrate. The two proteins (1 μm) were individually incubated with 2OG ,m), ferrous iron (10 μm) and a 15‐residue H3K9me3 peptide  in HEPES buffer  at 37 °C; the extent of the reaction was determined by using MALDI‐TOF mass spectrometry after 40 min. Substantial demethylation of the H3K9me3 peptide was observed in the sample with WT KDM4A, resulting in formation of both di‐ and monomethylated products . These findings suggest that K241 is required for efficient KDM activity, thus supporting previous work that also employed MS.Initially, we produced two recombinant variants of KDM4A residues 1–359) in –359 in E2 binding/recruitment proposed by Chen et al.,2. 1H NMR analyses were therefore carried out to assess the ability of the WT and K241A KDM4A to catalyse substrate‐uncoupled turnover of 2OG to succinate, a process that requires O2 binding. Samples containing either WT or K241A (20.5 μm), an excess of 2OG (2 mm), ascorbate (1 mm), and ferrous iron (100 μm) in ammonium formate buffer  were subjected to 1H NMR analysis at 1.5 min intervals over a 30‐min reaction. With both WT and K241A KDM4A, the triplet 1H resonance at δH=2.4 ppm (corresponding to 2OG) decreased in intensity during the analysis, while a new singlet 1H resonance at δH=2.3 ppm, corresponding to succinate emerged ,KD(JIB‐04)=22±5 and 7±2 μm for WT and K241A, respectively, Figures Nϵ‐trimethyllysine at position 9 . Initial succinate formation rates were markedly increased for both the WT and K241A enzymes; this implied binding of the histone fragment, which is reported to stimulate succinate formation by KDM4,1H resonances corresponding to lysyl methyl groups were obscured by residual glycerol and HEPES from the enzyme stocks; this hindered the detection of KDM activity. Stimulation of succinate formation was greater for WT than for K241A KDM4A.Given the role of K241 in O2 concentration. KDM assays  were carried out with the use of a Mass Flow Controller (Brooks Instruments) to equilibrate the reactions at either 20 or 80 % O2, as described;2 concentration  was incubated with the 2OG analogue N‐oxalylglycine (1 mm),2 (10 μm) and various concentrations of the WT and K241A enzymes (0–20 μm). The fluorescence emission spectra of the samples parallel and perpendicular to the excitation plane were then measured, and used to calculate the fluorescence polarisation at each enzyme concentration. The apparent binding constants for peptide binding were near identical for both enzymes , thus indicating that mutation of the K241 residue to alanine does not alter the affinity of KDM4A for the peptide substrate ‐oxo intermediate before methyl C−H bond oxidation can occur. Targeting K241 is of interest both with respect to developing classical tightly binding inhibitors and to developing inhibitors that decouple 2OG and substrate oxidation. Such inhibitors are of interest because uncoupling could cause inactivation; in the absence of “prime” substrate, self‐oxidation of 2OG oxygenases can occur, either directly or through the production of reactive oxygen species.The overall MS, NMR spectroscopy and fluorescence polarisation studies imply that K241 has a role in KDM4A catalysis. However, the observation that the K241A variant catalyses uncoupled succinate formation with comparable efficiency to the WT implies that the role(s) of K241 during catalysis do not involve  binding of OThe 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.SupplementaryClick here for additional data file.