The metallo‐activated complexes of all of these compounds were isolated and structurally defined.This study establishes a new trans‐metal‐trapping (TMT) procedure based on a mixture of LiTMP (the base) and tris(trimethylsilylmethyl)gallium [Ga(CH Whereas alkyllithium reactions are generally irreversible, LiTMP reactions tend to be pKa‐dependent equilibria. In TMT, these equilibria are shifted towards the desired lithiated substrate product by its interception by a trapping agent  procedure based on a mixture of LiTMP and tris(trimethylsilylmethyl)gallium  1 .Pyrazine previously required four molar equivalents of LiTMP in THF at −75 °C, but only to afford modest yields of 2‐substituted derivatives  mixed in some cases with 2,5‐disubstituted species (16 %).2‐2,5‐{(GaR3)}2C4H2N2] 2 cleanly as crystals isolated in a 44 % yield LiZn(TMP)tBu2], which operates through a synchronized bimetallic synergy distinct to that of stepwise TMT, as it affords only the 2,5‐disubstituted pyrazine even with a 1:1, base:substrate stoichiometry.2⋅TMEDA/1.5 LiTMP in THF produced 59 % of the isolated 2‐iodopyrazine that reportedly decomposes at room temperature, but in hexane a significant amount of coupled dimer product was also seen.4The role of PMDETA is to aid crystallization and stabilization of the sensitive metallo species by chelating to lithium, but it is added at a later stage to avoid undergoing a competing TMT deprotonation itself. Stoichiometric control was also evident when the base:trap:substrate ratio was doubled to 2:2:1, giving the 2,5‐digallated pyrazine  3. Interestingly, 1H NMR monitoring of the reaction revealed an important effect of the order of metal reagent addition on reaction regioselectivity. Thus, when LiTMP is added as a solid to the hexane solution of GaR3 and pyridazine, 3 is obtained in a 78 % yield, along with small amounts of the C4‐gallated regioisomer (16 % yield). Contrastingly, if the substrate is added to the hexane suspension of LiTMP and GaR3, the yield of 3 decreases to 50 %, and more C4 metalated product is seen (36 %). These contrasting results suggest an activating effect of the GaR3 component, which perhaps can initially coordinate to the Lewis basic N atoms of the heterocycle, facilitating its lithiation at C3. The crystal structure of 3 . A novel feature is the Li(PMDETA) unit bridging the two diazine N atoms [Li−N bond lengths 2.093(5) and 2.043(5) Å for the non‐disordered molecule of the Z′=2 structure] leading to a 1, “2.5”‐separation. Consequently, the spiro Li center, connecting the 3‐ and 2×5‐atom rings, has a 5‐coordinate geometry.With its 1,2‐placement of N atoms, pyridazine offers a choice of metalation sites. Site selectivity in its metalation is exceptionally challenging, as evident from previous work using excess LiTMP in 0.5 ZnCl1–3, TMT was demonstrated tangibly through isolation and crystallographic characterization of a metalated derivative, here [1‐(PMDETA)Li‐6‐(GaR3)‐(C4H3N2)], 4. This was made in a 27 % isolated crystalline yield, though NMR monitoring of the reaction shows that under the conditions studied 4 forms in a higher 59 % yield. Its structure  exhibits many of the features in 1–3 with the proximal 1,6‐separation of its GaR3 and (PMDETA)Li akin to that of those in dimetalated pyrazine 2.Because pyrimidine was found to be totally inert to LiTMP from 0 °C to reflux temperatures,13C NMR studies in D8‐THF at −75 °C, we extended the TMT study to this fused heterocycle.3)‐3‐{Li(PMDETA)}C6H4NCS] 5 thiophenolate isomer detected. Deprotonative gallation of the C2 center was evident from its downfield resonance at 209.5 ppm in the 13C NMR spectrum. In the crystal, 5 follows the pattern in the TMT diazine series with the GaR3 and Li(PMDETA) units adjacent on deprotonated C and N atoms, respectively, with a Ga1‐C13‐N1‐Li1 torsion angle of −13.9(4)°  base equivalents for a 97 % yield of 2‐iodobenzothiazole after I2 quenching, though no metallo intermediate was identified.In view of the fact that the 2‐lithiated derivative of the related nitrogen–sulfur heterocycle benzothiazole is known to exist simultaneously in ring‐closed and ring‐open forms, as best evidenced by Boche's 5 to methyl trifluoromethanesulfonate  we obtained a yield of 62 % of the desired methylated product and did not observe any opening of the azole ring. In contrast, though this strong electrophile did quench the monogallated pyrazine 1, it proved too aggressive and decomposition ensued. Therefore, we turned to a second electrophile in trimethylsilyl chloride, which offers another potential pitfall in being too bulky. Reassuringly, this pitfall proved unfounded and the heterocyclic rings of both 1 and 5 were successfully converted to Me3Si derivatives at their C−Ga sites . These promising results demand that a full systematic, optimized study of all of these new gallated compounds is now undertaken with a series of electrophiles.For synthetic campaigns, it is important that the new C−Ga bonds in these systems are accessible to electrophiles. In preliminary NMR spectroscopic experiments (see the Supporting Information for full details), on subjecting the gallated benzothiazole In summary, because LiTMP trans‐metal‐trapping via a gallium alkyl has been substantiated here with challenging sensitive unactivated diazines and benzothiazoles, this can potentially open the floodgates to a general improvement in many other metallation reactions with various sensitive and non‐sensitive substrates where LiTMP and related bulky bases give only low‐to‐moderate yields of products.CCDC 1494979 (1), 1494980 (2), 1494981 (3), 1494982 (4), and 1494983 (5) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre.Full experimental details and copies of NMR spectra are included in the Supporting Information. 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.


These spin–orbit entangled states can host exotic quantum phases such as topological Mott state, unconventional superconductivity, and quantum spin liquid. The lacunar spinel GaTa4Se8 was theoretically predicted to form the molecular Jeff = 3/2 ground state. Experimental verification of its existence is an important first step to exploring the consequences of the Jeff = 3/2 state. Here, we report direct experimental evidence of the Jeff = 3/2 state in GaTa4Se8 by means of excitation spectra of resonant inelastic X-ray scattering at the Ta L3 and L2 edges. We find that the excitations involving the Jeff = 1/2 molecular orbital are absent only at the Ta L2 edge, manifesting the realization of the molecular Jeff = 3/2 ground state in GaTa4Se8.Strong spin–orbit coupling lifts the degeneracy of  d oxides can lead to exotic electronic ground states. Here the authors use resonant inelastic X-ray scattering to demonstrate that the theoretically proposed Jeff = 3/2 state is realised in GaTa4Se8.The strong interaction between electron spin and orbital degrees of freedom in 5 Examples include some cubic perovskite compounds of early 3d transition metals, in which the degeneracy of t2g orbitals is large and the oxygen octahedra are only weakly distorted. For heavy 5d electrons, the strong spin–orbit coupling (SOC) can reduce the degeneracy by splitting the t2g orbitals into a Kramers doublet (Jeff = 1/2) and quartet (Jeff = 3/2), and recovers the orbital angular momentum4. Recently, iridates with 5d5 have drawn much attention because the half-filled state near the Fermi level (EF) is a Kramers doublet and a relatively weak electron correlation leads to the Jeff = 1/2 Mott ground state7. This state offers opportunities to explore quantum phases such as a topological Mott insulator8, unconventional superconductivity13 and quantum spin liquid17.The quantum effects of electronic orbitals are pronounced in degenerate systems where the orbital degrees of freedom have to be considered on equal footing with spins as in, e.g., the Kugel–Khomskii modelJeff ground state systems are still emerging. Beyond the well-known 5d5 iridates, the main challenge in other 5d electron systems is to build a concrete understanding of the exotic quantum effects with the Jeff state. A relatively simple, but more interesting case is the 5d1 system, which results in a Jeff = 3/2 effective moment. Examples can be found in double perovskites such as Sr2MgReO618, Ba2YMoO621 and Ba2NaOsO623. In the ionic limit, the magnetic moment of Jeff = 3/2 vanishes because the orbital component cancels the spin component4. The spin–orbital entanglement leads to a strong multipolar exchange of the same order as the ordinary bilinear exchange4, giving access to a variety of exotic phenomena in multipolar systems such as 4f-/5f- heavy Fermion compounds24. While recent advanced X-ray spectroscopic studies showed clear signatures in Sr2IrO4 and the other iridates29 for the case of Jeff = 1/2, the physics of the Jeff = 3/2 state has to date remained elusive in experiment.At present, theories, modeling constructs and experimental investigations of relativistic 4Se8, was suggested as a model system for the molecular Jeff = 3/2 Mott insulating ground state30. As shown in Fig. 4Se4, or simply so-called Ta4 cluster. The short intra-cluster having a Ta–Ta distance of ≤3 Å naturally induces the molecular orbital (MO) states residing on the cluster31. The MO calculation for the Ta–Ta bonding orbitals of Ta4 cluster and the ab-initio band structure calculation found that one electron occupies the MO states with t2  symmetry near EF35]. The strong SOC of the Ta atom splits the three-fold degenerate t2 MO states into Kramers doublet (Jeff = 1/2 MO states) and quartet (Jeff = 3/2 MO states), and the quarter-filled state near EF is the Kramers quartet as shown in Fig. Jeff = 3/2 MO states is small (~0.7 eV) and the relative strength of on-site Coulomb correlation, i.e., U (~ 2 eV), is sizable, rendering GaTa4Se8 a rare example of a molecular Jeff = 3/2 Mott insulator30.Recently, a lacunar spinel compound, GaTaJeff state is necessary to understand the underlying mechanisms of quantum phenomena that have been reported and speculated for this material and others42. At low temperature, for example, GaTa4Se8 exhibits an intriguing transition40 towards a non-magnetic and possibly spin singlet state, which are presumably related to a peculiar bump observed in the susceptibility and specific heat40. Furthermore, this non-trivial magnetic and electronic behavior could be related to superconductivity observed under pressure38. Considering that the previous studies do not take the SOC into account30, determining the nature of its magnetic moment is essential to elucidate the physics of GaTa4Se8 and to address the related issues that have been theoretically discussed largely for the 5d oxides.Experimental identification of the relativistic Jeff = 1/2 ground state in Sr2IrO4, the salient experimental evidence has been that the magnetic resonant X-ray scattering (MRXS) intensity of the Néel-ordered state is nearly absent at the L2 edge6. The destructive quantum interference at L2 edge only occurs in the complex Jeff = 1/2 state S = 1/2 states of real wave functions. A few magnetically ordered iridium compounds were found to show the same phenomenon29. On the other hand, verifying the Jeff = 3/2 state in non-magnetic GaTa4Se8 is a greater challenge. MRXS analysis, which is only possible for magnetically ordered materials, cannot be exploited.In the case of the Jeff = 3/2 state. At the Ta L edges, dipole transitions give rise to the direct RIXS via 2p→5d absorption and subsequent 5d→2p decay, which directly probes the valence and conduction band states43. As depicted in Fig. Jeff = 1/2 MO level is branched off from the atomic j = 5/2 and the absorption transition between L2 (2p1/2) and j = 5/2 is naturally dipole-forbidden44. In contrast, the Jeff = 3/2 MO states are composed of both the atomic j = 5/2 and j = 3/2 states. Therefore, we were able to establish the molecular Jeff = 3/2 ground state in GaTa4Se8 by examining the excitation spectra at both the Ta L3 and L2 absorption edges. This is the first spectroscopic evidence for a molecular Jeff = 3/2 ground state in a real material.Here we used the high-resolution resonant inelastic X-ray scattering (RIXS) technique to explore the 3- and L2-edge X-ray absorption spectroscopy (XAS) spectra, respectively, which were measured in the partial yield mode by recording the shallow core–hole emissions . Both RIXS spectra show basically the same resonant profiles. It should be noted that broad excitation peaks ~3.5 and 7 eV are resonantly enhanced when Ei is tuned near to the primary XAS peak. On the other hand, the narrow excitation peaks below 2 eV are resonantly enhanced when Ei is tuned to the ~ 2 eV below the XAS maximum.Figure t2g and eg symmetry orbitals from the ab-initio band structure calculations. A quite large portion of the unoccupied states is located above 2 eV and has a mixed character of t2g and eg symmetry, which explains the overall XAS feature and the broad high-energy RIXS peaks above the 2 eV energy loss. Regarding these broad peaks, there is no distinct difference between the Ta L3- and L2-edge RIXS spectra.The XAS and RIXS spectra clearly reveal the overall structure of the unoccupied states guided by insights from the band structure calculations. Figure t2g symmetry character dominates the energy range near EF (±2 eV). In the XAS spectra, excitations to these t2g states, including the possible relativistic Jeff states, do not show up as a distinct peak but are located in the lower-energy shoulder region of the large XAS peak. The narrow RIXS peaks below the 2 eV energy loss are assigned to orbital excitations within these t2g manifolds. In the case of the L3-edge RIXS  excitations along (hhh) high-symmetry direction. Three orbital excitation peaks at the 0.27, 0.7 and 1.3 eV energy loss positions are clearly identified for all momentum transfers with some intensity modulations. Within the instrument energy resolution (~ 100 meV), no dispersion is observed for these three excitations.Figure 2-edge RIXS (Ei = 11.133 keV) excitations along the (hhh) high-symmetry direction. Like the L3-edge RIXS excitations  dependence of the L3-edge RIXS excitations where the scattering angle (2θ) is fixed to 90°. When a grazing angle (θ = 30°) is used, the 1.3 eV peak is largely enhanced while the 0.27 eV peak is suppressed. Figure θ dependence of the L2-edge RIXS excitations where 2θ is fixed to 90°. For all θ angles, no peak structure shows up in the 1.3 eV energy loss region  and a broad upper Hubbard band (UHB). In this case, a broad orbital excitation between LHB and UHB is expected. Importantly, no contrast between the L3- and L2-edge RIXS spectra is expected. On the other hand, with a strong SOC, the well-defined Jeff = 1/2 MO band is branched off, leaving out the Jeff = 3/2 MO band near EF. A moderate U opens a gap, making it a molecular Jeff = 3/2 Mott insulator. Multiple orbital excitations are expected between the occupied bands , and the unoccupied Jeff = 3/2 and Jeff = 1/2 bands.Having the solid experimental observation that there is no 1.3 eV peak only in the Lright) and DOS (middle) near EF, which were projected onto the low-energy MO states  formed in the Ta4 tetrahedron cluster  and the dipole approximationJeff = 3/2 MO states (peaks A and B) are also revealed in the calculated L2-edge RIXS spectrum in Fig. 3-edge RIXS calculations, the intensity of peak B is much weaker than that of peak A. This is understood because peak B contains the excitation from the Jeff = 3/2 MO states to the Jeff = 1/2 MO states, and its spectral weight is partially suppressed for the L2-edge RIXS excitations in the following reason. For the RIXS process to occur, both photon absorption and emission must be the allowed transitions43. In the L2-edge RIXS excitations, the photon absorption between 2p1/2 and the Jeff = 1/2 MO states is naturally dipole-forbidden because the Jeff = 1/2 MO states mostly comprise the Ta atomic j = 5/2 states as shown in Fig. 4b44. This is clearly seen in peaks C and D, which are totally absent in the calculated L2-edge RIXS spectrum in the Fig. Jeff = 1/2 MO states was totally suppressed at the L2 edge. Hence, the total absence of the 1.3 eV peak in the L2-edge RIXS spectra of GaTa4Se8 can be identified as arising from the destructive interference at the L2 edge of the Jeff = 1/2 MO states, thereby establishing the molecular Jeff = 3/2 ground state in GaTa4Se8.The excitations involving the unoccupied Jeff states. With the help of the band structure and the cluster model calculations, the RIXS excitation spectra taken at L3 and L2 edges provide clear evidence that the Jeff = 1/2 MO band is well separated from the Jeff = 3/2 MO band and the excitations involving the Jeff = 1/2 MO band are totally suppressed only at L2 edge, verifing the molecular Jeff = 3/2 ground state in GaTa4Se8. Unlike MRXS, the RIXS technique can be useful even for a system with no long-range magnetic order  as demonstrated in the current study. Considering a strong SOC (~ 0.5 eV) of 5d orbital, this type of study is possible with a moderate energy resolution of ~ 100 meV, which is easily achievable for all 5d transition-metal L edges in the current state-of-the-art RIXS spectrometer46.We have focused on spectroscopic evidence in search for the destructive quantum interference of Jeff = 3/2 nature of GaTa4Se8 does not only just provide the opportunities for investigating Jeff physics but also elucidates the current important issues in GaTa4Se8 and its close cousins such as GaNb4 X 8 and GaMo4 X 8 . In GaTa4Se8, for example, the underlying mechanism is not clearly understood for the ‘paramagnetic’ insulator to metal transition and the superconductivity under pressure38. Furthermore, the magnetic behavior of this material at low temperatures does not seem to support a simple ‘paramagnetic’ Mott phase34. It should be emphasized that the relativistic Jeff state has not been identified in the previous studies, since it is a recent theoretical finding30 and is experimentally established in this study. On the basis of the current spectroscopic evidence, one can consider the ground state of GaTa4Se8 as the manifestation of the frustrated magnetic phase emerging from the non-trivial interactions among the relativistic Jeff = 3/2 moments21. Moreover, we speculate that the superconductivity reported in this material is also related to this phase. We note that the other related lacunar spinel compound, GaNb4Se8 which is also expected to have the molecular Jeff = 3/2 nature30, exhibits a quite similar low-temperature behavior and becomes superconducting. On the other hand, the molecular Jeff = 1/2 material with basically the same structure, GaMo4 X8 , is well understood as a ferromagnet41 and does not exhibit superconductivity47. Our total energy calculation shows that the molecular Jeff = 3/2 moments of the Ta4 cluster are in fact antiferromagnetically coupled between neighboring clusters (EAFM-FM = −7.4 meV per cluster). Considering the fcc arrangement of this cluster unit, this strongly suggests magnetic frustration49. In this regard, our current study may indicate that the molecular Jeff moments are frustrated in this material. This is compatible with recent experimental observations of the specific heat and magnetic susceptibility, interpreted as a formation of dimerization and a spin singlet state39.Establishing the molecular 3- and L2-edge XAS spectra by analyzing resonant emission signals. The incident photon bandpass is ~0.8 eV. In the case of the L3 edge, the Lβ2 emission, which leaves out a shallow (~ 230 eV) core–hole of 4d, was analyzed by the Ge (555) analyzer, which was on the 1 m Rowland circle. Because of a long lifetime of the shallow core–hole, a high-resolution (<2 eV) XAS was obtained50. In the case of the L2 edge, the Lγ1 emission, which leaves out a shallow (~ 230 eV) core–hole of 4d, was analyzed by the Si (466) analyzer. Note that the use of the analyzer is essential for the L2-edge XAS because a poor resolution of an energy-resolving detector cannot totally eliminate the Ga K-edge emission (~ 10.2 keV) from the Ta Lγ1 emission (10.9 keV).Diced spherical analyzers were used to record L46 of the Advanced Photon Source. X-rays were monochromatized to a bandwidth of 70 meV, and focused to have a beam size of 40(H) × 15(V) μm2. A horizontal scattering geometry was used with the incident photon polarization in the scattering plane. For the L3-edge RIXS, a Si (066) diced spherical analyzer with 4 inches radius and a position-sensitive silicon microstrip detector were used in the Rowland geometry. For the L2-edge RIXS, a Si (466) diced spherical analyzer with 4 inches radius was used. The overall energy resolution of the RIXS spectrometer at both edges was 100 meV, as determined from the full-width-half-maximum of the elastic peak.The sample grown by the vapor transport method in a sealed quartz tube was mounted in a displex closed-cycle cryostat and measured at 15 K. The RIXS measurements were performed using the MERIX spectrometer at the 27-ID B beamline4Se8 were obtained by the selenium transport method51. The pure powders of GaTa4Se8 were placed in an evacuated silica tube with a small excess of Se and heated at 950 °C for 24 h and then slowly cooled (2 °C h−1) to room temperature.Single crystal samples of GaTa52, which is based on the linear combination of pseudo-atomic-orbital basis formalism. The exchange-correlation energy was calculated within the LDA  functional53. The energy cutoff The exchange-correlation energy was calculated within the LDA  functional of 400 Rydberg was used for the real-space integration and the 8 × 8 × 4 Monkhorst-Pack k-point grid was used for the momentum-space integration. The SOC was treated within the fully relativistic j-dependent pseudopotential and non-collinear scheme54. DFT + U  formalism by Dudarev et al.56 was adopted for our calculations. Our main result is based on Ueff = U−J = 2.3 eV, and we found that our conclusion and discussion are valid for different Ueff in a reasonable range  order has been taken to present the electronic structure. We found that the magnetic order does not change the band characters or their relative positions, and therefore, it does not affect any of our conclusion or discussion.Electronic structure calculations were performed by OpenMX software package57. We have employed the Kramers–Heisenberg formula of the RIXS scattering operator45 and calculated the RIXS spectra by using the continued fraction method58. The RIXS scattering operator was determined with the zeroth order of the ultrashort core–hole lifetime expansion and the dipole approximation was applied with taking the experimental X-ray beam geometry  on reasonable request.Supplementary InformationPeer Review File


Carbon (C) surface segregation from bulk stabilizes the Fe(0 18 1) vicinal surface by forming a c(3 One dimensional (1D) magnetism was realized using stepped surfaces, even in non-magnetic semiconductors3. Control of the magnetic anisotropy of magnetic materials has been realized using stepped surfaces. Using this route, 1D chains with perpendicular magnetic anisotropy4 (PMA) or exotic PMA in Fe(110) ultrathin films on stepped W(110) were produced5. Nevertheless, little is known about the modification of magnetic surface states on stepped terraces, probably due to the fragility of the clean surfaces against adsorption of overlayers . Most of the studies were performed with scanning tunneling microscopy12. Monoatomic steps of Fe films on Au display an enhancement of intensity at the step edges. It has been understood as scattering of surface state (SS) electrons by step edges10. Scattering effects have been used to partially explain interlayer exchange coupling and tunnel magnetoresistance. In this context, magnetic surface states can impact the performance of Fe/Ag/Fe spin valves14 or Fe/MgO/Fe magneto-tunnel junctions (MTJ). The high magnetoresistance depends on the electronic properties at the interfaces17. In both examples passivation of the outermost layer is required. In the latter case, intercalation of a carbon (C) layer at the Fe-MgO interface has shown to improve the performance of the junctions19. The observed behavior can be understood assuming that carbon passivates the surface and as a consequence it prevents the intermixing and oxidation of Fe with the MgO. Indeed, evidences of passivation of the Fe(0 0 1) surface by carbon have been observed at the C exhibits the largest spin polarization of the surface states31. A miscut of 2.7° results in terraces that determine lateral confinement of the surface states33. The terraces have their normal parallel to the [0 0 1] direction and, as a consequence, the surface states are those well known of the Fe(0 0 1) surface, but modified by lateral confinement.The Fe(0 18 1) surface termination of the sample was obtained from a Fe single crystal cut with an offset angle of 2.7° from the Fe(0 0 1) with the steps aligned parallel to the [1 0 0] direction 1(a). The choice of this surface was done based on the fact that among all the low index surfaces of Fe, extensively studiedD~30 Å. This value corresponds to a miscut angle of 2.7°, in agreement with the expected value. Besides the main spots faint spots at  fractional beams positions and weak halos around them can be clearly distinguished  high symmetry direction collinear with the normal emission (NE) direction and the axis of the photoelectron energy and momentum analyzer. In this geometry, the band dispersion along the [±1 0 0] and [0 ±1 0] surface high symmetry directions was measured in a large angular range with both LH and LV polarizations  correspond to an azimuthal angle Φ = (0° ≡ 180°) and that along the [0 −1 0] direction (k|| perpendicular to the steps) to the Φ = (90° ≡ 270°). A double peak structure close to the Fermi level is well distinct in the data measured with LH (Φ = 0°) while a single peak dominates the spectra measured in the Φ = 90° configuration. A reversed behavior can be observed for LV polarization: the double peak appears for the (Φ = 90°) configuration and the single one for (Φ = 0°). The splitting of the surface state measured for LH-(Φ = 0°) and LV-(Φ = 90°) is consistent with the carbon reconstruction on flat Fe(0 0 1). Furthermore, the lineshape of the EDCs measured with LH-(Φ = 90°) and LV-(Φ = 0°) for which only one state can be observed is more similar to that measured for clean Fe(0 0 1) at the same photon energy  and (Φ = 90°) have been carried out as a function of the photon energy in the range from 50 eV to 35 eV. The results, displayed in Fig. A) of LV light lies on the surface and it is perpendicular to the steps for Φ = 0°(Φ = 90°). The LH polarized light can be decomposed into one component parallel to the surface and another perpendicular to it. The intensity of each component is equally distributed in both cases due to the 45° incidence angle of the experiment. In this case the in-plane A component is parallel(perpendicular) to the steps for Φ = 0°(Φ = 90°). This 90° rotation explains the fact that the double(single) peaks are observed for different azimuths with LV and LH polarizations. Since these states are symmetric with respect to A for Φ = 0° and Φ = 90°, the observation of a double(single) peak dispersion depending on whether A is parallel(perpendicular) to the steps must the consequence of the existence of steps at the surface.The azimuthal angle dependence of the double peak is unexpected since in the experimental configuration used the two azimuths are equivalent for each polarization. Therefore, the same dispersion of the electronic states was expected. Moreover, we observe that the spectral shape for LH (Φ = 0°) and LV (Φ = 90°) is the same except for the higher relative intensity for LV photons. A single state close to the Fermi energy can be observed for LH (Φ = 90°) and LV (Φ = 0°). This can be explained by the geometrical configurations used for LH and LV  is different, with the high binding energy peak having a flatter k|| dispersion. The single state measured with A perpendicular to the steps  disperses and has the same binding energy as the low binding energy state measured for the other azimuth, thus pointing to a common origin. States seen with both LH and LV polarization have mixed symmetry. Slight differences in the dispersion for the two polarizations suggests interaction with bulk states20. The data for the C vectors, thus for both emission planes in our geometry  are even with respect to a plane containing the x(y) vector and can be excited with LH light when the latter has an Ax(Ay) component. On the opposite, the states are odd with respect to a plane containing the y(x) vector and therefore are observed with LV polarized light having Ay(Ax) components. Our experiments with LH polarization probe the dxz(dyz) for Φ = 0°(Φ = 90°) and with LV polarization both the dxy and dyz for Φ = 0°. For Φ = 90° the dxy and dxz orbitals are measured. The fact that for LH(LV) and Φ = 90°(Φ = 0°) only one state is observed could be interpreted with a high binding energy state of pure dxy character, whereas the low binding energy state should have dxy + dxz + dyz character. This orbital assignment is questioned by calculations on the reconstructed C-c and carbon reconstructed surface, the nature of the 3 dxz + dyz as expected for clean Fe25. The fact that only one state is seen when the potential vector is perpendicular to the steps suggests that Fe-like states are located at the step-edges and the c where Au chains are located on the terraces and the step-edges have a Si honey-comb structure with spin-split surface states3.The results for the low binding energy state are compatible with a mixture of 20. In the present case we observe not just passivation but stabilization of the surface electronic states of the clean Fe surface. The robustness of the passivation was tested by depositing Ag on top of the C:c(3 in situ deposition of 2.2 monolayers of Ag -Deposition of Ag has been chosen due to the well known growth of Ag on Fe(0 0 1)38. This interface has been extensively studied after the observation of quantum confinement effects40. Metal coverage of stepped surfaces generally uptakes from the step edges. The permanence of the Fe surface state signature indicates that the surface electronic structure of the present system is indeed effectively protected3. No signature of quantum confinement was observed. This is different from other transition metal stepped surfaces where confinement of surface states in the direction perpendicular to the terrace edges has been observed34. In the present case the apparent lack of 1D confinement can be understood in connection with the interaction between the cross-linked chains, at 45-degrees with respect to the step edges, that determine a 2D network of surface states. The energy position of the states after Ag deposition shifted to 0.137 eV higher binding energies. The presence of the photoemission surface peaks after Ag deposition confirms the passivation role of the carbon termination and supports the interaction of the surface states with bulk states. The effective protection of the surface electronic structure of iron by carbon and by nanostructuring  indicates that suitable surface engineering could become relevant in, e.g. MTJ technology. The problems related to interface magnetically dead-layers, or with the loss of the surface-enhanced magnetic moments, are potentially overcome if a suitable surface nano-engineering is developed exploiting both structural, morphological and chemical protection. One very interesting possibility would be to realize Fe/MgO/Fe(0 18 1) junctions or Fe/Ag/Fe(0 18 1) spin valves to explore the possibility to enhance, by this architecture, the performance of these systems.The C:c surface followed by annealing procedures are expected to give similar results.in-situ at the APE-LE beamline of IOM-CNR at Elettra Sincrotrone-Trieste. The ARPES experiments were done using linearly polarized undulator radiation in the 10–100 eV energy range. The total energy resolution of the SCIENTA-2002 analyser and the monochromator was kept around the thermal broadening value, kB · T ~ 25 meV. The STM data were measured at room temperature. Typical currents between 100 to −550 pA were used for the images measured with negative voltage. For the positive voltage images the currents used were in the 700 pA to −350 pA.To characterize the properties of the obtained surface we used three different techniques: LEED (low energy electron diffraction), STM (scanning tunneling microscopy) and ARPES (angle resolved photoemission spectroscopy). All experiments were performed Supplementary information


It is even better than the benchmarking platinum catalyst. The molecule-like bimetallic nanocluster represents a class of catalysts that bridge homogeneous and heterogeneous catalysis and may provide a platform for the discovery of finely optimized catalysts.The theoretically predicted volcano plot for hydrogen production shows the best catalyst as the one that ensures that the hydrogen binding step is thermodynamically neutral. However, the experimental realization of this concept has suffered from the inherent surface heterogeneity of solid catalysts. It is even more challenging for molecular catalysts because of their complex chemical environment. Here, we report that the thermoneutral catalyst can be prepared by simple doping of a platinum atom into a molecule-like gold nanocluster. The catalytic activity of the resulting bimetallic nanocluster, PtAu Volcano plots for electrocatalytic hydrogen production show the best catalysts as those ensuring the hydrogen binding step is thermodynamically neutral. Here, the authors report fabrication of a highly active thermoneutral electrocatalyst via doping of a single platinum atom into a gold nanocluster. This cluster catalyst represents a special type of catalyst that bridges homogeneous and heterogeneous catalysis.Ligand-protected gold and silver nanoclusters have received much attention as materials with many practical applications because of promises offered by their unique optical, electrochemical and catalytic properties192021232125 cluster, leading to a single Pt-doped bimetallic cluster  corresponds to the intact ion with a chemical composition of Au25(SC6H13)18. The peak at m/z ∼7,032 Da observed for the Pt-doped gold cluster is however almost superimposed with that of Au25(SC6H13)18 due to their small mass difference. The isotope pattern of the Pt-doped gold cluster (blue) shown in the inset of 25(SC6H13)18 (red) and matches well with the simulated isotope pattern (black) of PtAu24(SC6H13)18, manifesting that one Au atom in the Au25 cluster has been cleanly replaced by a Pt atom. Hereafter, these clusters will be abbreviated as Au25 and PtAu24, respectively. The average core size of both clusters is found to be around 1.1 nm  and 2.1 eV (γ) and the disappearance of the band at 1.8 eV (β) for [PtAu24]0 in 24.The ultraviolet-vis-near-infrared (NIR) absorption spectra in 25 cluster is drastically altered on doping of a Pt atom. To further unravel the electronic structure of the PtAu24 cluster, square-wave voltammetry (SWV) of Au25 and PtAu24 clusters were conducted. Voltammetry has been effectively used to study the electron transfer properties and the electronic structures of gold nanoclusters1925 cluster, indicating that the Au25 cluster is in anionic form, that is, [Au25]−. Therefore, the current peaks observed at −0.39 (O1), −0.04 (O2) and 0.69 (O3) correspond to the first, second and third oxidation peaks of the [Au25]− cluster, while the peak at −2.06 V (R1) corresponds to the first reduction peak. The electrochemical gap determined from the difference between the first oxidation (O1) and reduction (R1) potentials is found to be 1.67 V. The HOMO-LUMO gap for [Au25]− is determined to be 1.32 V by subtracting the charging energy term (O1–O2) of 0.35 V from the O1–R1 gap.The optical measurements show that the electronic structure of Au25 cluster, PtAu24 is charge neutral with the open-circuit potential found at −0.49 V. Thus, the current peaks observed at −0.03 (O1) and 0.41 (O2) correspond to the first and second oxidation peaks of the PtAu24 cluster, while −0.76 (R1) and −1.10 V (R2) correspond to the first and second reduction peaks. The observed O1–R1 gap and the deduced HOMO-LUMO gaps from the SWV in 24]0 shifted positively by nearly 1 V compared to that of [Au25]−, potentially offering the possibility to lower overpotentials for reductive electrocatalysis.The redox potentials are drastically altered on doping of a Pt atom in the core as can be seen in 4NPF6) solution containing 1.0 M trifluoroacetic acid (TFA) in the absence (black) and presence of Au25 (red) and PtAu24 (blue) clusters at a glassy carbon electrode (GCE). Compared to the blank current, the current for the proton reduction is significantly increased in the presence of Au25. The current further increases in the presence of PtAu24. The onset potential (Eonset) of catalytic current is observed at −1.10 V for Au25, positively shifted by 150 mV compared to that of the blank GCE. In the presence of PtAu24, the onset was found at −0.89 V, positively shifted by 360 mV. The thermodynamic reduction potential of proton is estimated to be −0.82 V in THF (1.0 M TFA) and thus the onset potential of −0.89 observed for PtAu24 corresponds to an overpotential of 70 mV  observed in 24. When the catalytic activity of Au25 was examined with increasing concentration of TFA, the current increase at a more negative potential (−1.1 V) and the reduction current associated with HER was found to be much smaller , 2 is the number of electrons involved in the catalytic reaction, R is the ideal gas constant, T is the temperature in Kelvin, F is Faraday's constant and v is the scan rate. As can be seen in Ic increases with increasing acid concentration and levels off above 1.0 M TFA. At the acid concentration of 1.0 M, kobs was then obtained by plotting Ic/Ip as a function of v−1/2 as shown in kobs value calculated from the slope of the fit line (−1 at −1.5 V (η=650 mV). Under the same experimental condition, the kobs value calculated in the presence of Au25 is only 8,000 s−1. The rate constant observed for PtAu24 is also significantly higher than those reported to date for highly active molecular electrocatalysts; compared to cobalt complexes (700 s−1 at η=890 mV)−1 at η=720 mV)kobs value  thus far reported was observed at η=650 mV for a nickel complex that catalyses H2 formation with pendant amines that act as proton relayskobs value obtained for PtAu24 is also higher than that.where fit line  is 121,0−1.5 V η=50 mV. Un24, we compared kobs as a function of potential. As can be seen in kobs value found at −0.89 V is 1,300 s−1 and sharply increases to 62,000 s−1 at −1.1 V, followed by a gradual increase to 186,000 s−1 at −2.0 V. Interestingly, the potential where the drastic increase occurs matches well with the second reduction wave [PtAu24]−/2−. By contrast, the kobs value calculated for Au25 is found to be relatively small  until −2.1 V, where [Au25]− is reduced to [Au25]2−.To understand the origin of the extraordinary catalytic activity observed for PtAu24. That is, the catalytic currents observed at potentials negative to the [PtAu24]−/2− wave  all exhibit linear correlation with [PtAu24] and [TFA]1/2, respectively, which is consistent with the heterolytic HER mechanism H]0 intermediate24 observed in this work clearly display the characteristic of molecular catalysts.This result is very reasonable considering the charge state of PtAu24]2− with [Au25]2−, the kobs value calculated for [PtAu24]2− is 229,000 s−1, more than 10-fold higher than that of [Au25]2−  at −2.2 V at which both clusters are present as a dianionic form. The vastly different catalytic activity can be understood by considering the reduction potential match between the catalyst and the substrate (proton). That is, when the reduction potential of catalyst matches closely with the thermodynamic potential of proton, the catalyst can act as an effective electron transfer mediator and enhance its activity dramatically4145The charge state is not the only factor affecting the HER activity of the clusters. Comparing 2− is rather thermodynamically neutral (−0.059 eV), whereas that for [Au25]− is 0.539 eV. This result clearly suggests the initial hydrogen binding is energetically favourable on [PtAu24]2−, but is endothermic on [Au25]−, explaining the high HER activity observed for [PtAu24]2−. However, the second H adsorption on [PtAu24]2− and [Au25]− is found to be both endothermic; 0.369 and 1.21 eV for [H-PtAu24]−  and [H-Au25]0, respectively. Thus, the DFT calculations predict that hydrogen evolution occurs on PtAu24 via the coupling of the adsorbed hydrogen with another proton from the solution  with an energy change of −0.155 eV . By contrast, the most thermodynamically favourable H2 generation occurs on Au25 via the homolytic pathway , indicating that the adsorbed H atom forms H-Pt chemical bond with the central Pt. In the DFT calculations, we initially placed the H atom on the intact cluster surface away from the Au atoms; during geometry optimization, we found that the H atom spontaneously moves into the subsurface to interact directly with the central Pt atom while breaking some surface Au-Au bonds. This result suggests that the H-Pt bond formation is a downhill process with no transition state. The H-Pt bond is also manifested in the local electronic density of states  with PtAu24 in THF containing 1.0 M TFA in an H-type cell. 2 detected by gas chromatography analysis at each overpotential. The clusters display symptoms of decomposition after prolonged CPE experiments and thus the electrolysis was conducted for 15 min. As can be seen in the figure, production of H2 is first detected at an overpotential of 0.2 V and increases with increasing overpotential. Comparison of the amount of H2 produced with the charge consumed indicates that the current efficiency for H2 production is > 97% when the overpotential is 0.4 V or higher. After subtracting the H2 production from the blank solution, the TOF at η=0.6 V is found to be 4.8 mol H2 (mol cat)−1 s−1. To the best of our knowledge, this value is considerably higher than any other molecular catalysts reported for HER in similar conditions6789122 (mol cat)−1 s−1 obtained for a copper complex at a higher overpotential of 0.75 V −1 s−1 at η=0.92 V  and Nafion was dropcast on a gas diffusion layer (GDL) electrode (PtAu24/C/GDL). We found that the clusters immobilized on the carbon black with the Nafion binder exhibit higher stability during electrolysis. 2 detected after 60 min CPE at each overpotential in 1.0 M Brinton–Robinson buffer solution (pH 3). As can be seen in the figure, H2 generation was achieved near the thermodynamic potential (η=70 mV), which also matches very well with the Eonset found in 2 generation increased rapidly with increasing overpotential and the current efficiency for HER went up to nearly 100% when the overpotential is 100 mV or higher. At η=400 mV, the average current density increases above 12 mA cm−2. Having immobilized such well-defined clusters on the electrode, we were able to calculate the TOF from the CPE experiments. As can be seen in 2 (mol cat)−1 s−1 at η=0.6 V in aqueous media after subtracting the contribution from the blank C/GDL electrode. This value is again much higher than those obtained in comparable conditions. For comparison, a TOF of 2.2 mol H2 (mol cat)−1 s−1 was obtained at η=0.59 V in acetate buffer (pH 4.5) for a cobalt catalyst immobilized on carbon nanotubesWhile PtAu24/C/GDL electrode has prompted us to compare its activity with the benchmarking Pt/C  dropcast on a GDL electrode (1 cm2). Since TOF could not be determined for the Pt/C, we have compared the H2 production rate per mass of metals  in the catalyst composites (mol H2 g−1 h−1). As can be seen in 24/C/GDL and Pt/C/GDL electrodes start to produce H2 near the thermodynamic potential, but the H2 production rate determined for the PtAu24/C/GDL is much higher than that for the Pt/C/GDL. For instance, the H2 production rate determined for the PtAu24/C/GDL is 25 mol H2 g−1 h−1, which is more than two times higher than that for the Pt/C/GDL catalyst (11 mol H2 g−1 h−1) at the same overpotential of 0.6 V.Although it is not straightforward to directly compare the TOF values obtained from the homogeneous  and hete2 production; significantly higher than any other molecular catalysts reported thus far, to the best of our knowledge, and even higher than the benchmarking platinum catalyst. Mechanistic investigations have revealed that hydrogen binding step on the bimetallic cluster is thermodynamically neutral and the central Pt atom forms Pt-H chemical bond, pointing to a key role of the dopant. The molecule-like bimetallic cluster operates efficiently in both homogeneous and heterogeneous conditions, representing a special type of electrocatalyst. The molecule-like bimetallic cluster may thus provide a platform for the discovery of finely tuned catalysts, which could have broad implications for catalysis beyond H2 production.Here we show that the electronic structure and the catalytic activity of a cluster catalyst can be fine controlled by doping a Pt atom into a stable gold cluster. The bimetallic cluster is molecule-like and exhibits excellent catalytic activity for H2Cl2 (SWV) or THF (LSV) containing 0.1 M Bu4NPF6 as a supporting electrolyte that was degassed and blanketed with a high-purity Ar gas. A GCE (3 mm diameter) was used as a working electrode, a Pt wire as the counter electrode and Ag/AgNO3 (0.1 M AgNO3 in CH3CN) as the reference electrode for the LSV experiments. SWV was carried out with a Pt disk (0.4 mm diameter) working electrode at 100 mV s−1 with a pulse height and a width of 20 mV and 20 ms, respectively. Ferrocene (Fc+/0) was added as an internal reference for Ag/AgNO3.SWV and LSV were conducted with an electrochemical workstation  in CH4NPF6) that was degassed and blanketed with Ar gas. Electrolysis was conducted for 15 min under vigorous stirring with an electrochemical workstation  equipped with three electrodes consisting of glassy carbon plate working electrode (area=1 cm2), a GDL  counter electrode (1 cm2) and a Ag/AgNO3 reference electrode. CPE experiments in heterogeneous condition were carried out for 60 min under vigorous stirring with a ZIVE MP1 potentiostat  in an H-type cell that was equipped with a composite working electrode and a Ag/AgCl (3 M NaCl) reference electrode in one compartment and a platinum plate (1.68 cm2) counter electrode in the other compartment. The working electrode was separated from the counter electrode by a proton exchange membrane . Both compartments were filled with 60 ml of 1.0 M Brinton–Robinson buffer (pH 3.0) that was degassed and blanketed with Ar gas. The composite working electrode was fabricated by spreading a catalyst ink prepared by mixing 16 μg of the cluster catalyst, 200 μg of carbon black (Vulcan XC-72) and 1.5 μl of Nafion solution  in 50 μl of THF on a GDL  electrode (1 cm2). For comparison, CPE experiments were conducted under the same condition using a commercial Pt/C  catalyst that was mixed with Nafion and dropcast on a GDL electrode (1 cm2). The average size of Pt particle was 3.5±0.5 nm. The amount of H2 evolved was quantified from an analysis of the headspace using an Agilent 7890B gas chromatography equipped with a thermal conductivity detector.CPE experiments in homogeneous condition were conducted in a 5 ml cell containing 2.5 μM cluster and 1.0 M TFA dissolved in 2.0 ml THF (0.5 M Bu25(SCH3)18 and PtAu24(SCH3)18 clusters were performed with the quantum chemistry program Turbomole V6.5. The def2-SV(P) basis sets were used for C, S and H, while effective core potentials which include scalar relativistic corrections and 19 (18) valence electrons were used for Au (Pt). Geometry optimization was done with the TPSS  functional. In our calculations, solvent effects were treated implicitly by making use of the conductor-like screening model (COSMO) as implemented in Turbomole.DFT calculations of the HER on AuThe authors declare that all the other data supporting the findings of this study are available within the article and its How to cite this article: Kwak, K. et al. A molecule-like PtAu24(SC6H13)18 nanocluster as an electrocatalyst for hydrogen production. Nat. Commun.8, 14723 doi: 10.1038/ncomms14723 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Notes


Brassica napus L.), which has yellow flowers, is both an important oil crop and a traditional tourism resource in China, whereas the Orychophragmus violaceus, which has purple flowers, likely possesses a candidate gene or genes to alter the flower colour of oilseed rape. A previously established B. napus line has a particular pair of O. violaceus chromosomes (M4) and exhibits slightly red petals. In this study, the transcriptomic analysis of M4, B. napus (H3), and O. violaceus with purple petals (OvP) and with white petals (OvW) revealed that most anthocyanin biosynthesis genes were up‐regulated in both M4 and OvP. Read assembly and sequence alignment identified a homolog of PAP2At in M4, which produced the O. violaceus transcript (PAP2Ov). The overexpression of PAP2Ov via the CaMV35S promoter in Arabidopsis thaliana led to different levels of anthocyanin accumulation in most organs, including the petals. However, the B. napus overexpression plants showed anthocyanin accumulation primarily in the anthers, but not the petals. However, when PAP2Ov was driven by the petal‐specific promoter XY355, the transgenic B. napus plants produced red anthers and red petals. The results of metabolomic experiments showed that specific anthocyanins accumulated to high levels in the red petals. This study illustrates the feasibility of producing red‐flowered oilseed rape, thereby enhancing its ornamental value, via the ectopic expression of the PAP2Ov gene. Moreover, the practical application of this study for insect pest management in the crop is discussed.Oilseed rape ( The EBGs are regulated by individual R2R3 MYB transcription factors , whereas the LBGs are regulated by a MYB‐bHLH‐WD40 (MBW) complex, which is also found in many other dicot species, such as Petunia hybrida, Zea mays and Antirrhinum majus , enhancer of glabrous (EGL3) and transparent testa 8 (TT8) from the basic‐helix‐loop‐helix (bHLH) family; production of anthocyanin pigment 1 (PAP1), production of anthocyanin pigment 2 (PAP2), MYB113 and MYB114 from the MYB family; and transparent testa glabrous (TTG1), a WD40‐type protein, were found to play a role , cover many acres worldwide and are important sources of edible oil and biodiesel  O. E. Schulz , which has large purple flowers, is widely cultivated as an ornamental plant in China. One disomic addition line with all the chromosomes of B. napus and a specific pair of O. violaceus chromosomes produced flowers with a red colour  in the anthocyanin biosynthetic pathway was identified as being expressed in the petals of the addition line. The ectopic expression of OvPAP2 driven by a Brassica petal‐specific promoter, rather than its own promoter or the 35S promoter, successfully produced a red colour on the yellow petals of B. napus. These results could contribute to the production of OSR with entirely red flowers, optimizing OSR tourism resources and generating strategies for pest control. The potential regulation of anthocyanin accumulation in the petals of Brassica species is discussed.However, to date, there are no reports on other flower colour variations  caused by the accumulation of anthocyanins in the petals, although red or purple colours are common in the leaves, stems, sepals and pods of these species (Mushtaq B. napus cv. Huashuang 3 (H3), at the early vegetative growth stage, except for the purple colour on some of the youngest leaves and buds. At bolting, the stature of the M4 plants was smaller than that of H3, and the flowers displayed variable degrees of red colour on the yellow background of the petals. In M4 plants, red pigments were observed at the basal portion of petals on half‐open or open flowers that overlapped with the yellow background, which generated ‘orange‐red’ parts of the yellow petals  slowly faded to lilac; in contrast, its natural mutant (OvW) had completely white petals  plants with red petals had a phenotype that was nearly the same as that of the donor, s Figure a; by cons Figure a, suggess Figure b–d.O. violaceus, we performed petal transcriptome analysis. In total, 14 G of raw data and 115 million high‐quality clean reads were obtained. For each of the four samples, approximately 30 million reads were obtained, 89% of which were clean reads   Table . To simp4 Figure , which w4 Figure . NotablyH3 Table .PAP1 and EGL3, which showed higher expression in O. violaceus than in B. napus. Moreover, most genes showed higher expression in M4 than in H3 but almost the same expression in OvW and OvP , showed significantly different expression in O. violaceus, with its fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) value being reduced from 15.13 in OvP to 0 in OvW (Table PAP1 (Bra039763), which had a slightly lower expression, seven other positive regulators (MYB12 (Bra004456), MYB111 (Bra037419), PAP1 (Bra001917 and Bra004162), TT8 (Bra037887), GL3 (Bra025508), EGL3 (Bra027653)) were up‐regulated. Moreover, two negative regulators (MYBL2 (Bra016164), LBD37 (Bra031833)) were down‐regulated, consistent with the colour change in the petals of M4  and long BLAST lengths (>750 bp) between M4 and O. violaceus. This result indicated that these unigenes, which were expressed in M4, might be transcribed from the foreign chromosome from O. violaceus. Specific primers were designed to amplify the homologs of PAP2 from M4 and O. violaceus , Brassica oleracea (Bol042409) and A. thaliana (AT1G66390) showed that the sequence from M4 was identical to that of O. violaceus , which was expressed in the petals, but the homologous PAP2 genes from the B. napus genome were not expressed or were expressed at a very low level. The OvPAP2 cDNA encoded a 238‐amino acid protein containing an R2R3 MYB domain with a signature motif for interaction with bHLH proteins from the 3f clade , four transgenic plants with obvious purple phenotypes were obtained. The OvPAP2‐overexpressing plants showed varying levels of purple pigmentation on the stems and leaves, especially the pistils  and of purple on the youngest leaves and buds, but no purple or red colour was observed in the petals or other organs  analysis of petals and anthers from Westar and P6 (CaMV35S::OvPAP2 plant with red anthers) .We then transformed the vector into two ) Figure . PAP2, FB. napus (H3) according to sequence information  and the petals  petals was more stable and consistent among different flowers than was that of the M4 petals. Similarly, the red pigments mainly accumulated at the base of the petals and were most obvious in the semi‐open or open flowers  of the XY355::OvPAP2 plants produced much redder flowers, which generated more red pigments at the base of the petals as well as in other parts, especially the vascular tissue, resulting in petals with a red texture  produced slightly red/purple pigments both on the anther walls and at the base of the petals , in addition to P6 and XYP3  PAP2 expression was up‐regulated in XY355::OvPAP2 petals and anthers but was much lower than in M4  and P6 ; nevertheless, both DFR and ANS were up‐regulated in the two tissue types and were expressed more highly in XY355::OvPAP2 petals and anthers than in M4  and P6 , corresponding to the depth of the red colour in these materials; and (iv) PAP2 expression increased slightly in the anthers of the P4 plant, but the other genes were more or less the same in the anthers of H3, suggesting that the level of ectopic OvPAP2 expression was important for regulating the structural genes in anthers. Based on these results, OvPAP2 was considered important for anthocyanin biosynthesis in the red petals of M4, as it could up‐regulate several important structural genes in some tissues. However, only when both DFR and ANS were up‐regulated could the tissues produce red pigments, becoming redder with higher expression. Moreover, in the petals of transgenic plants, high OvPAP2 expression under the petal‐specific promoter XY355 but not the CaMV35S promoter could up‐regulate both DFR and ANS, but the reason why is unclear.To further observe and study the reason for the different phenotypes of the different materials, the ten ABGs were analysed by qRT‐PCR in the petals and/or anthers of H3, M4 and P4  of the profiling data sets separated the four samples from each other Figure a. The mem/z 1005; MS/MS, m/z 757/535/287) indicated that it was a known anthocyanin, cyanidin 3‐coumaroyldiglucoside‐5‐malonylglucoside , but this identification requires further confirmation by other methods. These results indicated that red petals contained more anthocyanins than yellow petals and that some anthocyanins were red petal‐specific, which is in accordance with the higher expression of F3H, F3′H, DFR and ANS, which are involved in the key steps of anthocyanin production, in the petals of M4 and XYP3.To identify the metabolites that significantly differentiated the red petals (M4 and XYP3) from the yellow petals (H3 and P6), orthogonal projection to latent structure‐discriminant analysis (OPLS‐DA) modelling was performed on the profiling data sets Figure c. S‐plote Figure   analysis between M4 and H3 and subsequent gene cloning and sequencing comparisons identified a homolog of AtPAP2 from O. violaceus in M4. AtPAP2 is a member of the R2R3 MYB gene family and is also a component of the MBW complex. The overexpression of either PAP1 or PAP2 could cause different degrees of anthocyanin production in the leaves, stems and flowers of Arabidopsis . However, delphinidin‐based anthocyanins were previously found in B. rapa (purple bok choy) , which encodes a key regulator in the MBW complex, was isolated from an O. violaceus chromosome added to B. napus by comparative transcriptomic analysis. The overexpression of OvPAP2 via the 35S promoter in Arabidospis led to pigment accumulation in various organs, but in B. napus, it produced only red anthers. The petal‐specific promoter‐mediated overexpression of OvPAP2 in B. napus successfully changed the yellow and white petals into red‐yellow/red petals due to the accumulation of known and new anthocyanins. The present study illustrated the feasibility of diversifying the flower colours of B. napus by introducing key gene(s) from other species.Here, a homolog of B. napus and one specific pair of O. violaceus chromosomes producing slightly red petals (designated M4) was obtained previously , the wild‐type O. violaceus with purple petals (OvP) and its natural mutant with white petals (OvW) were grown in the greenhouse of the Huazhong Agricultural University in the autumn of 2011. The petals of half‐open flowers were collected, and total RNA was extracted using the guanidine isothiocyanate method. The quality and quantity of the purified RNA were determined by measuring the 260 nm/280 nm absorbance (A260/A280) using a spectrophotometer . RNA integrity was further verified by 1.5% agarose gel electrophoresis. The total RNA of all samples was assessed based on A260/A280 ≈ 1.7, quantity > 10 μg and good integrity.A disomic addition line with all the chromosomes of B. rapa (http://brassicadb.org/brad/downloadOverview.php) using Tophat . After sequencing, the high‐quality clean reads were obtained by filtering the dirty reads with adaptors or two Ns and the low‐quality reads. The clean reads from each sample were mapped to the genome of De novo assembly and comparative transcriptomic analysis were performed: all clean reads generated from two O. violaceus samples were assembled together as transcripts of O. violaceus using Trinity with default parameters . The same unigenes expressed in M4 and O. violaceus were identified by performing an all against all DNA sequence similarity  BLAST.B. napus and A. thaliana, respectively, specific primers  was used for all fragment amplifications. The PCR products were cloned into pMD™18‐T  and sequenced. The obtained sequences were BLASTed using SeqMan to identify the correct CDS of PAP2, which was mapped from different materials and species using ClustalW2 . The sequence alignment figures were made using GeneDoc.Based on the assembled sequence and the genome sequence of rs Table  were desArabidopsis were used to calculate distance estimates for a neighbour‐joining tree with MEGA6 software  to identify bHLH protein binding sites and conserved motifs defining the members of R2R3 MYB subgroup 6.The CDSs of MYBs belonging to subgroups 2, 4, 5, 6 and 7 from et al., OvPAP2  was cloned from the cDNA of M4 into the pMDC83 containing the CaMV35S promoter using the traditional digestion and ligation method with a restriction endonuclease and T4 ligase  Lithuania). The amplification primers were PAP2‐XF and PAP2‐BR  and B. napus (H3 and Westar). Another vector (pCAMBIA2300) containing OvPAP2 was constructed (primers: PAP2‐PF and PAP2‐KR) with the OSR petal‐specific promoter XY355 (355 bp), which was amplified (primers: XY355‐HF and XY355‐PR) from the genome of B. napus (H3) according to sequence information  according to the user manual. First‐strand cDNA synthesis was performed with two micrograms of total RNA from each sample using a RevertAid™ First Strand cDNA Synthesis Kit  Lithuania) according to the manufacturer's protocol. The specific quantitative primers for ten ABGs were designed using Primer 3.0; all primer sequences are listed in Table The petals of newly fully opened flowers from H3, M4 and the overexpression plants were collected in liquid nitrogen. A total of 0.1 g of frozen powder was placed into a 2‐mL screw‐cap tube with 1 mL of methanol/water/acetic acid . The sample was then put on ice in the dark with sonication for 30 min and then vortexed at 4 °C in the dark overnight. The supernatant was collected after centrifuging at 12 000 × g for 10 min and was further diluted appropriately and filtered twice using a 0.22 μm PTFE filter  before injection for metabolomics analysis.et al., Metabolomics analysis was performed on a Waters Acquity UPLC system connected to a Synapt G2‐XS QToF mass spectrometer, which was equipped with an electrospray ionization source , according to methods previously described  and Orychophragmus violaceus (OvW and OvP).Figure S2 Comparison of the FPKM values of the ABGs in the phenylpropanoid pathway in Brassica napus (H3 and M4) and Orychophragmus violaceus (OvW and OvP).Figure S3 Comparison of the FPKM values of the regulatory genes acting on the anthocyanin biosynthesis pathway in Brassica napus (H3 and M4) and Orychophragmus violaceus (OvW and OvP). Note that here, PAP1 represents all homologs of AtPAP1, AtPAP2, AtMYB113 and AtMYB114 in Brassica rapa that have high sequence similarity.Figure S4 The sequence alignment of PAP2 in each of the samples or species.Figure S5 Protein features encoded by OvPAP2.Figure S6 Phenotypes of the offspring  of the XY355::OvPAP2 Brassica napus plant.Figure S7 Relative abundance and mass spectrum of one biomarker that is differentially accumulated in the red (M4 and XYP3) and yellow (H3 and P6) petals of Brassica napus.Click here for additional data file.Table S1 Summary of alignment statistics from the RNA‐Seq of Brassica napus (H3 and M4) and Orychophragmus violaceus (OvW and OvP).Table S2 The expression levels of anthocyanin biosynthesis genes in Brassica napus (H3 and M4) and Orychophragmus violaceus (OvW and OvP).Table S4 The primers for gene cloning and vector construction.Table S5 Identified anthocyanin biomarkers that were differentially accumulated in the red and yellow petals of Brassica napus.Table S6 qRT‐PCR primers.Click here for additional data file.Table S3 A comparison of the unigenes between M4 and Orychophragmus violceus annotated as anthocyanin biosynthesis genes.Click here for additional data file.


The authors regret that a typographical error appeared in the first version of their paper, where IL-1 was cited in place of IL-17A. The affected paragraph appears below with the corrected text:+ T cells expressing the marker CD69 in the spleen and mesenteric arteries, suggesting that γ/δ T cells might have an initiating role in hypertension. Likewise, antibody clearance of γ/δ T cells reduced the hypertensive response to Ang II. The precise mechanisms by which these cells promote hypertension remain to be defined, but these cells are also major sources of IL-17A, which has prohypertensive actions in both the kidney and vasculature .Although the majority of T cell receptors are composed of α/β chains, a small percentage possess γ/δ receptors, and recent evidence suggests that these also contribute to hypertension. Caillon et al. (2017) recently showed that γ/δ T cells are important in hypertension, and that mice lacking these cells exhibit a markedly blunted rise in blood pressure and preserved endothelial function in response to Ang II infusion. γ/δ T cell–deficient mice also exhibited fewer activated CD4The online HTML and PDF versions of this paper have been corrected. The error remains only in the print version.


IRIS) was initiated in 2008 to study the emergence of neuraminidase inhibitor (NAI) resistance and the clinical course of influenza in immunocompetent treated and untreated patients.The Influenza Resistance Information Study (NAIs. RT‐PCR‐positive samples were cultured and tested for NAI resistance by specific RT‐PCR and phenotypic testing. Scores for influenza symptoms were recorded on diary cards (Days 1‐10). This study focuses on influenza A‐infected cases only.Patients had throat/nose swabs collected on days 1, 3, 6 and 10 for analyses of influenza type, subtype and virus susceptibility to RT‐PCR‐positive patients, 2316 had influenza A of whom 1216 received oseltamivir monotherapy within 2 days of symptom onset . Except for 9 patients with naturally resistant seasonal H1N1 (2008/9), no resistance was detected in Day 1 samples. Emergence of resistance (post‐Day 1) was detected in 43/1207 (3.56%) oseltamivir‐treated influenza A‐infected patients, with a higher frequency in 1‐ to 5‐year‐olds (11.8%) vs >5‐year‐olds (1.4%). All N1‐ and N2‐resistant viruses had H275Y (n = 27) or R292K (n = 16) substitutions, respectively. For 43 patients, virus clearance was significantly delayed vs treated patients with susceptible viruses , and 11 (23.2%) remained RT‐PCR positive for influenza at Day 10. However, their symptoms resolved by Day 6 or earlier.Among 3230 Oseltamivir resistance was only detected during antiviral treatment, with the highest incidence occurring among 1‐ to 5‐year‐olds. Resistance delayed viral clearance, but had no impact on symptom resolution. In this context, a global observational study was initiated in 2008, the Influenza Resistance Information Study (IRIS), to study the emergence of NAI resistance and the clinical course of influenza in immunocompetent treated and untreated patients.The primary objective of the IRIS study was to assist with early detection of influenza resistance to antivirals and describe the clinical course and outcome of patients with influenza according to subtype and antiviral susceptibility.Influenza Resistance Information Study is a prospective, multicentre, information‐gathering study (NCT00884117). It is the largest study of its type that has collected sequential clinical and virological data during the course of infection, using sensitive RT‐PCR detection methods for both detection of the virus and follow‐up of substitutions associated with oseltamivir resistance in H1N1 and H3N2 viruses. Major findings of the first 3 years of this study have already been reported.This article reports the first 5 years of surveillance carried out through IRIS, with a specific focus on the description of the emergence of influenza A‐resistant viruses in treated patients, including the timeline of the emergence of the resistant viruses and the identification of the substitutions associated with this resistance.22.1Influenza Resistance Information Study  is a 7‐year prospective, multicentre, observational study. Recruitment started in December 2008 (Year 1), continued throughout the 2009‐10 A/H1N1 influenza pandemic and until March 2013 (Year 5). After the 5th season, the study design was modified to continue for 2 additional years (Years 6 and 7 until March 2015) with a different objective (focus on immunocompromised children only).During the first 5 years of the study, inclusion centres were located in Europe , USA, China (Hong Kong) and Australia. Enrolment was carried out during 5 northern and 4 Southern Hemisphere influenza seasons. The study was performed in compliance with the principles of the Declaration of Helsinki and its amendments, and in accordance with Good Clinical Practice. The study protocol and amendments were approved by independent ethics committees and institutional review boards at each centre.2.250 values performed on the viruses by a chemiluminescent assay (NA‐Star), and the measure of the fold increase observed as compared to IC50 values of susceptible strains, according to the common procedure.During this study period, the criteria for inclusion were as previously described.All Influenza‐positive samples by RT‐PCR were cultured on MDCK cells, and subsequently sequenced (haemagglutinin [HA] and neuraminidase [NA] gene segments) and phenotypically tested for NAI resistance when possible, as previously described.2.3Scores for 7 cardinal influenza symptoms  were recorded daily on diary cards by the patient , checked by the physician (days 1‐10), and summed to produce a total symptom score as previously described.2.4Kaplan‐Meier plots were generated for time to viral RNA clearance and time to resolution of all diary card symptoms (no single symptom score of >1 on diary card). The statistical analysis was carried out by either Kruskal‐Wallis or Wilcoxon signed‐rank tests.3During the 5‐year surveillance period, 3230 patients with a single influenza strain infection were recruited. All study centres enrolled patients, the majority of whom (75.3%) were from the Northern Hemisphere Figure . Of 3230Among the 2316 influenza A‐positive patients registered, 2147 were eligible for analysis and 1216 received oseltamivir monotherapy (52.5%) within 2 days of symptom onset  , antiviral resistance was detected by mutation‐specific RT‐PCR in Day 1 viruses from neither the 1207 treated patients nor the 910 non‐treated patients. However, emergence of resistance (post‐Day 1) was detected by RT‐PCR in 43/1207 of the oseltamivir‐treated influenza A H1N1pdm09 or H3N2 patients  or the R292K substitution for N2 viruses (n = 16). No other substitutions known to be associated with reduced sensitivity to NAI in human influenza viruses were detected when the Na‐gene segment was sequenced from viruses obtained in culture on MDCK cells .50 values associated with either RI or HRI, likely because of mixed sensitive/resistant populations  of samples in which resistance first appeared, viral loads were too low for phenotyping (Table In a significant number of cases (24/27 H1N1pdm2009 and 15/16 H3N2), the resistant virus detected by the mutation‐specific RT‐PCR, contained a mixed population of H275/Y275 or R292/K292 for at least one time point (Table P < .0001) and 10.9 days for the treated patients with a resistant virus (P < .0001)  were still RT‐PCR positive at Day 10 . This low rate of detection is consistent with the literature and recent reports from WHO, where resistance in initial samples is rarely reported.In our study, the proportion of R292K substitutions in H3N2 viruses isolated  was relatively low as compared to Kiso et al.50, while 3 had a normal IC50. This lack of detection of reduced inhibition may be due to the use of the Na‐Star system (chemiluminescent assay) that is less sensitive than the gold standard MUNANA‐based fluorescent assay.According to analysis of the sequential specimens collected in these patients, resistant viruses emerged by Day 3 of treatment from susceptible strains and were selected by oseltamivir. It is known that the fitness of R292K H3N2 viruses is putatively severely impaired.The H275Y resistance in H1N1pdm09 was the most reported in this study (27/43 R‐Viruses). This is consistent with the various reports about oseltamivir resistance in N1 viruses in general. This resistance emerged from Day 3 in 1/3 of the 27 cases, not only in children , when the viral load was significantly low. We can speculate that the risk of transmission is correlated with the viral shedding and concentration at the site of replication. So, even if we also observed that virus detection (and shedding) was longer as compared with non‐treated and treated patients . The above authors are members of the IRIS Advisory Board. JSN‐V‐T is a named author on this manuscript and EIC of Influenza andother respiratory viruses. However all Editorial processes and decisions relating to this manuscript were handled independently by Dr. Rebecca Jane Cox,Associate Editor. JSN‐V‐T played no role in any decisions related to revisionor acceptance of the manuscript.


ZF) protein motifs, stabilized by binding of Zn(II), typically function as interaction modules that bind nucleic acids, proteins and other molecules. The elucidation of the redox states of ZF proteins in cellular conditions, which depend on their midpoint redox potentials, is important for understanding of ZF functioning. In the present study we determined the midpoint redox potentials for representatives of Cys2His2 and Cys4 types of ZF proteins in apo and Zn(II)‐bound forms using electrospray ionization mass spectrometry. The midpoint redox potentials of the apo forms of Cys2His2 and Cys4ZF proteins were −326 and −365 mV (pH 7.5), respectively. These values are close to the cytosolic redox potential of approx. −350 mV (pH 7.5) and thus we can conclude that the apo form of Cys2His2‐type ZF proteins is predominantly reduced but apo forms of Cys4‐type ZF proteins should be substantially oxidized in the cytoplasm. As expected, Zn(II) binding stabilized the reduced forms of both ZF proteins: the corresponding redox potential values were −284 and −301 mV, respectively. Consequently, binding of Zn(II) ions to ZF motifs can act as a sensitive switch that activates the functioning of the ZF motifs within the cell, and also protects them from oxidation and can function as part of a redox‐sensitive regulation mechanism of cellular functions.Zinc finger ( Thiol groups in ZF can exist in three major states: thiol‐thiolate, Zn(II)‐bound thiolate and oxidized disulfide. The shift of the redox balance in the cells or cell compartments to the oxidative side may result in oxidation of Cys residues to disulfide and the release of Zn(II) ions with concomitant loss of ZF functions Em values, which helps in understanding functioning and redox regulation of ZFs. Namely, by comparing Em values with the cellular redox potential values we can conclude in which redox state ZFs exist under changing redox conditions within cells. For our studies we have chosen two representatives of ZF proteins with different Zn(II)‐coordinating ligands. The first one, ZF‐1, (PDB ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2el5) is the 18th Cys2His2‐type ZF domain of human DNA‐binding ZF protein 268 (ZNF268). ZNF268 is involved in cell development during embryogenesis and the postnatal period in higher mammals. Disorders in ZNF268 expression and processing may cause cancer and leukemia, but not all functions of ZNF268 are yet known. The second protein, ZF‐2, (PDB ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2c6a) is the Cys4‐type ZF domain of a ubiquitin E3 ligase murine double minute 2 protein, MDM2. MDM2 plays a key role in tumorigenesis through p53 tumor suppressor inactivation The aim of the current study was a quantitative description of redox properties of ZFs through determination of their in vitroElectrospray ionization mass spectrometry (ESI‐MS) has proved to be a suitable method for determination of the redox properties of various peptides and small proteins 2His2 (ZF‐1) and Cys4 (ZF‐2) types of ZF proteins in apo and Zn(II)‐bound forms were determined using ESI‐MS. Based on the results obtained we can conclude that the apo form of the Cys2His2 type of ZF proteins is predominantly reduced but the apo form of the Cys4 type of ZF proteins should be substantially oxidized in the cytoplasm, and Zn(II) ions protect both ZF proteins from oxidation and ensure their functioning.In the present study the midpoint redox potentials for representatives of Cysox) and ammonium hydroxide solution (25%) were from Sigma‐Aldrich ; Zn(II) acetate was from Scharlau ; and formic acid was from Riedel‐de Häen . Oxidized DTT (DTTox) was synthesized from reduced DTT according to the protocol described in ®II UV/UF ultrapure water system, Barnstead, Lake Balboa, CA, USA) was used for preparation of solutions.Human ZF peptides ZF‐1 (GENPYECSECGKAFNRKDQLISHQRTHAGES) and ZF‐2 (SFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKG), of purity 95%, were obtained from Peptide 2.0 Inc. ; dithiotreitol (DTT); β‐mercaptoethanol (BME), oxidized BME , to prepare 200 μm stock solutions. The volatile compounds BME and BMEox were used for redox buffering since this pair is more suitable for ESI‐MS measurements than biological system containing GSH/GSSG Weighted solid samples of ZF‐1 and ZF‐2 were dissolved in 75 mm peptide was incubated with 25 mm BME at 25 °C; 50 μL aliquots were taken at different time points  and injected by a syringe pump into a QSTAR Elite ESI quadrupole time of flight (Q‐TOF) mass spectrometer  at flow rate 10 μL·min−1.Apo‐ZF‐1 was completely oxidized in the stock solution. For the kinetic measurements of apo‐ZF‐1 reduction, 5 μm apo‐ZF‐2 samples were incubated in 75 mm ammonium formate buffer, pH 7.5 at 25 °C; 50 μL aliquots of the sample were injected into the mass spectrometer as described for apo‐ZF‐1. For kinetic measurements, 5 μm apo‐ZF‐2 samples containing 50 mm BME as a reducing agent or 25 mm BMEox as an oxidizing agent were incubated at 40 °C for 3 h; 50 μL aliquots of each reaction mixture were injected into the mass spectrometer as described above. All mass spectra were recorded in the positive mode for 2–3 min in the m/z region 700–2000 Da. The working parameters of the instrument were: ion spray voltage, 5.5 kV; source gas, 55 L·min−1; curtain gas, 20 L·min−1; declustering potential 1, 60 V; declustering potential 2, 15 V; focusing potential, 320 V; ion release delay, 6; ion release width, 5; ion energy, 2; channel electron multiplier, 2450V; detector voltage, 2.3 kV. The spectra were deconvoluted and analyzed with the bioanalyst 2.0 program (Applied Biosystems).Apo‐ZF‐2 was partially oxidized in the stock solution. In order to obtain information about the air oxidation of the diluted ZF‐2, 5 and 10 μorigin 8.0 .All data were analyzed by using m of apo‐peptides were incubated in BME/BMEox redox buffers with different redox potential values generated by different ratio of BME/BMEox (0–100%) in order to achieve the redox equilibrium between the protein forms . Argon‐purged 75 mm ammonium formate, pH 7.5, was used in all experiments.For the measurements of the midpoint redox potentials of the apo forms of the peptides, 5 μox redox buffers with total concentrations ([BME] + 2[BMEox]) equal to 25 mm at 25 °C in the case of ZF‐1 and 50 mm at 40 °C in the case of ZF‐2. Conditions for ESI‐MS measurements were the same as described above. The relative content of the reduced form of the peptide, referred to as fractional content (y), was calculated from the experimentally determined average molecular mass (m) of the mixture of reduced and oxidized peptides in different redox buffers taking into account all charge states present in the spectrum by using the following equation:The equilibrium ratios of apo‐peptides were determined by ESI‐MS after 30 min incubation in BME/BMEx is the difference in molecular masses of reduced ZF and fully oxidized ZF peptides (x = 2 Da in the case of ZF‐1 and x = 4 Da in the case of ZF‐2), mZFS–S is the calculated molecular mass of the fully oxidized peptide, and mC is the experimentally determined average molecular mass of the mixture of reduced and oxidized peptides under particular redox conditions.where The redox potential of the buffer was created by varying the ratio of reduced and oxidized forms of BME. Corresponding redox potential values were calculated according to the Nernst equation:Eʹ0(BME) = −0.231 V (pH 7.0 and 25 °C) R is the gas constant (8.315 J·K−1·mol−1), n is the number of electrons transferred in the reaction, and F is the Faraday constant (9.6485 × 104 C·mol−1). Eʹ0(BME) values for 40 °C were calculated using Eqn where E/ΔpH is equal to −60.1 mV where Δy) from the environmental redox potential (x) by fitting it to the Boltzmann equation:A1 and A2 are constants, and x0 is the midpoint redox potential (Em). Data were fitted using an A1 of 0  and an A2 of 1  by using origin 8.0.The midpoint redox potentials for the apo‐ZFs were determined from the dependence of fractional content of fully reduced apo‐peptide (m apo‐peptides with 7 μm Zn(II) acetate were incubated with 25 mm BMEox at 25 or 40 °C for 3 h; 50 μL aliquots were injected into the QSTAR Elite ESI‐Q‐TOF mass spectrometer at various time points  and the MS spectra were recorded as described above. Fresh stock solution of Zn(II) acetate was prepared before measurements.For determination of the release of Zn(II) ions from the Zn(II)‐loaded forms of the ZF peptides, the mixtures of 5 μ1S–S/ZF‐10S–S and ZF‐22S–S/ZF‐20S–S pairs, the midpoint redox potential of ZF peptides was determined also in the presence of the freshly prepared 7 μm Zn(II) acetate in the 50 mm BME/BMEox redox buffers. Samples were incubated for 30 min at 25 °C in the case of ZF‐1 and for 3 h at 40 °C in the case of ZF‐2 and ESI‐MS spectra were measured as described above. Fractional content (y) of Zn1 peptides in the mixture of oxidized apo‐ZF and Zn1ZF was determined from areas of MS peaks for oxidized apo‐ZF (SapoZF) and Zn1ZF (SZnZF) forms by using the following equation:For the estimation of the effect of Zn(II) ions on the redox equilibrium of ZF‐11 peptides were determined by fitting the fractional content of Zn1 peptides in the mixture of oxidized apo‐ZF and Zn1ZF to Eqn Midpoint redox potentials of Znm of freshly dissolved ZF‐1 was 3490.71 Da, which corresponded to the m of oxidized peptide with a disulfide bond   already at 25 °C  corresponded to the mixture of oxidized and reduced forms of the peptide . Therefore, the kinetics of apo‐ZF‐2 oxidation in the presence of oxidized DTT and BME was also studied. Mixing of apo‐ZF‐2 with 25 mm DTT resulted in almost instant reduction of the peptide, but oxidation at the same concentration of DTTox was slow and only partial oxidation of apo‐ZF‐2 was achieved after 3 h of incubation at 40 °C. The treatment of apo‐ZF‐2 with 50 mm BME and 25 mm BMEox resulted in fast and complete reduction and oxidation of the apo‐peptide with half‐life <5 min at 40 °C  and oxidized (5408.87 Da) forms of the peptide, respectively.Electrospray ionization mass spectrometry spectra of apo‐ZF‐2 displayed two major peaks with charges +4 and +5. The isotopic resolution of the peaks was also at the baseline level. In contrast to apo‐ZF‐1, apo‐ZF‐2 was not fully oxidized: the experimental average m BME/BMEox redox pair and the samples were equilibrated at 25 °C for 30 min, which was sufficient for reaching an equilibrium. For determination of the midpoint redox potential of ZF‐2, the samples were incubated in 50 mm BME/BMEox at 40 °C for 30 min.Considering the results of kinetic measurements, the determination of the midpoint redox potential of apo‐ZF‐1 was carried out in the presence of 25 mox in different ratios at total concentration of [BME] + 2[BMEox] of 25 mm. The determined redox potential value, Em(apo‐ZF‐1), at pH 7.5 was equal to −326 ± 2 mV  at pH 7.5 , we concluded that presumably ZF‐2 binds trace amounts of Zn(II) ions from the tubing of the ESI‐MS system. The redox potential value for apo‐ZF‐20S–S at pH 7.5 was −365 ± 1 mV.The midpoint redox potential of apo‐ZF‐2 was determined from the shift in equilibrium of apo‐ZF‐27.5 Fig. Bc. A minm BME in the presence of Zn(II) ions resulted in an immediate appearance of Zn1ZF‐1, indicative for binding of Zn(II) ions by reduced peptide form. Figure 1ZF‐1 incubated for 30 min in 25 mm BME/BMEox buffers. Apo‐ZF‐11S–S was the only form present in the oxidizing conditions  ions Fig. Aa, but im Zn(II) showed that Zn(II) ions immediately bound to reduced apo‐ZF‐2 at both 25 and 40 °C  ions as only a minor apo‐ZF‐22S–S peak was detected in the spectra . During all experiments, the size of the K+ adduct peaks remained proportional to all ZF‐2 peaks  and (BME)2) and therefore the K+ adducts were not taken into account in the calculations. The (BME)2 adduct, which appeared only at high concentrations of BMEox, was also not taken into account in the calculations.Up to three KEm of Zn1ZF‐1 at pH 7.5 from the dependence of fractional content of Zn1ZF‐1 in the mixture of apo‐ZF‐11S–S and Zn1ZF‐1 at equilibrium against Eh of BME/BMEox redox buffer; its value was −284 ± 1 mV (pH 7.5 and 25 °C)  has a more positive Em value compared with the Cys4 type of ZF (ZF‐2), which speaks for their lower tendency to form disulfide in the cellular environment. Disulfides in the Cys4 type of ZF (ZF‐2) form cooperatively and require higher reducing capacity for reduction. In order to understand the biological significance of differences of midpoint redox potential values of ZFs they should be compared with the redox potential values in cellular conditions. The redox potential value in the cytosol of eukaryotic cells, determined in situ, is approximately −350 mV (pH 7.5) 2His2 type of ZFs are predominantly reduced in these conditions, whereas the Cys4 type of ZFs is substantially (approx. 70%) oxidized. However, both reduced and oxidized forms of apo‐ZFs are apparently present in cellular conditions.Table Em value in the case of ZF‐2 is in agreement with the observation that Cys4 ZFs tend to have higher affinity towards Zn(II) ions than the Cys2His2 ZFs Binding of Zn(II) induced a substantial shift in the redox potential value of both ZF peptides (42 mV for ZF‐1 and 64 mV for ZF‐2 at pH 7.5) indicating that Zn(II) peptides are more resistant to oxidation as compared with apo‐peptides. As the process of the oxidation of the zinc forms involves the release of Zn(II) ions, the protection from oxidation should depend on the affinity of ZF towards Zn(II). The larger shift in the Em values for Zn(II) forms of both types of ZF peptides are similar indicating that under normal cellular conditions the Zn(II) forms of ZFs are preferably reduced, but even a small shift in the environmental redox potential could result in partial oxidation of the ZF peptides and release of Zn(II) ions, which indicates that ZF proteins might function as redox switches. Such a switching might occur during cellular differentiation and proliferation, which are connected with substantial changes towards more oxidative cellular redox potential values PP and JS designed the research. JS and EK performed the experiments. JS, EK, PP and VT analyzed the results; the manuscript was written with contributions from all authors. All authors gave their approval to the final version of the manuscript.