In the Akita-Niigata region – which corresponds to the slope stretching from the volcanic-arc to the back-arc –a thermogenic signature characterize the gases, with prevalence of heavy carbon isotopes, low CH4/C2H6 and CH4/3He ratios. Natural gases from mud volcanoes in South Taiwan at the collision zone show heavy carbon isotopes, middle CH4/C2H6 ratios and low CH4/3He ratios. On the other hand, those from the Tokara Islands situated on the volcanic front of Southwest Japan show the heaviest carbon isotopes, middle CH4/C2H6 ratios and the lowest CH4/3He ratios. The observed geochemical signatures of natural gases are clearly explained by a mixing of microbial, thermogenic and abiotic methane. An increasing contribution of abiotic methane towards more tectonically active regions of the plate boundary is suggested.Methane emission from the geosphere is generally characterized by a radiocarbon-free signature and might preserve information on the deep carbon cycle on Earth. Here we report a clear relationship between the origin of methane-rich natural gases and the geodynamic setting of the West Pacific convergent plate boundary. Natural gases in the frontal arc basin  show a typical microbial signature with light carbon isotopes, high CH If the geological carbon cycle is well documented for its oxide forms, such as CO25, much work is needed to describe the geological cycle of its reduced forms, such as methane (CH4)8. The shallower biological methane cycle is relatively well quantified, such as biological release from wetlands, rice paddies, animals and termites9 and its impact on the greenhouse gas budget of the atmosphere10. The deep geological methane cycle still has to be well constrained8. Much attention has recently focused on the sources of radiocarbon-free geological methane  in the domain of petroleum geochemistry and microbiology13.Volatile elements are transported from the Earth’s interior to the hydrosphere and the atmosphere through volcanic and hydrothermal systems in addition to micro- and macro-seepages from active tectonic areas16, and (b) methane derived from hydrocarbon generation processes in sedimentary basins7. In principle, the chemical and isotopic composition of this methane and the associated natural gases should correlate with the geodynamic setting of the region where these gases originate20.There are two types of geological methane; (a) abiotic methane originated in volcanic and geothermal systems25, which is characterized by geophysical parameters such as terrestrial heat flow, Bouguer gravity anomaly, seismic velocity (P-wave perturbation), crustal deformation and magnetic anomaly. Therefore, the CH4/3He ratios of natural gases are expected to provide constraints on the origin of methane. In addition, we measured the nitrogen isotope composition of the sampled natural gases, which may provide further insights about their formation.In order to elucidate the origin of geological methane and its relationship with the geodynamic setting of the explored area, we collected natural gases in the West Pacific region Fig.  and meas27. Except for three CO2-rich samples in South Taiwan , natural gas samples are dominated by hydrocarbon species such as methane and ethane (C2H6). Akita-Niigata natural gas samples show appreciable amounts of ethane and propane (C3H8), while the samples from the South Kanto gas field are dominated by an almost pure methane phase. Nitrogen is a minor component. South Taiwan samples have intermediate characteristics compared to the samples from the Akita-Niigata and South Kanto gas fields. Nitrogen content varies significantly from 0.1% to 21%, while oxygen abundances are generally close to 0.1%, suggesting that air contamination is negligible. All argon contents are much lower than the air value of 0.934%, while helium abundances are ranging from 2.7 ppm to 285 ppm, mostly higher than air value of 5.24 ppm, confirming an elemental signature that differs from atmospheric noble gas.Chemical compositions of 21 natural gas samples collected and analyzed in this work are listed in Supplementary Table 13C/12C) in methane, nitrogen (15N/14N), helium (3He/4He) and argon (40Ar/36Ar) of the acquired natural gas samples are listed in Supplementary Table 4He/20Ne, N2/36Ar, CH4/3He and CH4/(C2H6 + C3H8). C1, C2 and C3 in figures and tables correspond to CH4, C2H6 and C3H8, respectively. Supplementary Table 27. Carbon and nitrogen isotopes are expressed in the delta (δ) notation, as parts per thousand deviation  from the international standard PDB and atmospheric nitrogen, respectively. Helium isotopes are given in the Ra notation, where Ra is the atmospheric 3He/4He ratio of 1.382 × 10−6 (ref.28). The δ13C values of methane in this work vary significantly from −69.6‰ to −35.9‰. This range of values is consistent with previous work in the area27. Generally Akita-Niigata natural gas samples have δ13C values consistent with those of South Taiwan, both showing carbon isotope signatures being heavier than those determined in the South Kanto samples. δ15N values are ranging from −4.2‰ to + 2.8‰ in this work, indicating minor fractionation with respect to the air value of 0‰. There is no apparent discrepancy of the nitrogen isotope signature among Akita-Niigata, South Kanto and South Taiwan natural gases. Helium isotope ratios vary significantly from 0.06Ra to 6.29Ra . The lowest value of 0.06 ± 0.05Ra is typical for crustal radiogenic helium, while the highest of 6.29 ± 0.14Ra indicates the presence of He with a typical subduction-like magmatic signature of 7.4 ± 1.3Ra23. Generally, the 3He/4He ratios of the South Kano samples are lower than the atmospheric air value of 1Ra, while those of Akita-Niigata show ratios higher than this value, indicating the occurrence of different proportions of mantle helium. South Taiwan samples are variable compared to those of South Kanto and Akita-Niigata. The northernmost samples  show higher 3He/4He ratios than those in south, probable due to a mostly pure mantle source for gases in the northern Taiwan31. Most of the measured argon isotope ratios are similar to the air value of 298.6 28 within the experimental error. A few samples from South Taiwan (CL-2 and WSD) show excess radiogenic 40Ar* with the 40Ar/36Ar ratios higher than air, but these ratios are much smaller than those observed in natural gases of geotectonically stable regions33.Isotopic compositions of carbon  ratio, in the so-called Bernard plot. Figure 13C values higher than −50‰ and CH4/(C2H6 + C3H8) ratios lower than 100. On the other hand, microbial methane is produced at temperatures lower than 50 °C in shallower environments such as wetlands, by methanogenic archaea. Methanogenesis consists either of CO2 reduction or fermentation of methylated substrates36. Microbial methane has lighter δ13C values from −60‰ down to −110‰ and CH4/(C2H6 + C3H8) ratios higher than 1000.Geological methane is classified into two groups according to its origin: abiotic origin related to magma and biogenic origin from sedimentary organic matterthe CH4/CH6 + C3H813C values . In agreement with these authors, we suggest that methane in the South Taiwan samples is of thermogenic origin with some secondary modification by molecular fractionation. Even though Hong et al.41 carried out an intense sampling to obtain a mapping of the methane flux in the region, molecular fractionation was not taken into account as a possible process affecting the isotopic composition of methane in the area.It is noted that all South Kanto samples are located within the area of microbial origin, while most Akita-Niigata samples are of thermogenic origin except for two samples (Nos 16 and 17) with lighter δues Fig. . Therefosee Fig. . Etiope 13C values than those of South Taiwan  ratios between 100 and 3200 as compiled from data obtained in several submarine hydrothermal systems of the EPR43 and those compiled by Sano and Fischer23. Tokara samples are well overlapping with the EPR region in Fig. 34. Therefore, based on the above arguments and the geodynamic setting of the sampling sites characterized by parameters such as heat flow, gravity anomaly and seismic velocity, the order of contribution of abiotic methane is estimated as follows: Tokara > South Taiwan = Akita-Niigata > South Kanto. This relation is comparable to the one set by the measured helium isotope ratios: the Tokara samples (up to 4Ra) show much higher 3He/4He values than the South Kanto samples (<0.3 Ra), while those of the South Taiwan and Akita-Niigata samples are variable  ratio. The CH4/3He ratio is a possible candidate, since 3He traces univocally mantle contribution to fluids45. Since the CO2/3He ratio was successfully adopted as discriminant ratio for volcanic gases from island arcs worldwide44, the CH4/3He ratio may be also useful. Figure 13C values of CH4 and the CH4/3He ratios. There are three independent end members in Fig. 4 to 3He in an EPR hydrothermal system, 5 × 10−6 (ref.20), is representative of abiotic methane derived from mid-ocean ridges associated to a δ13C value of −23 ± 6‰43. On the other hand, the CH4/3He ratio of the biogenic and the crustal end members was estimated to be 5 × 1012 by Wakita et al.26 and 1 × 1013 by Sakata et al.27. These values are very similar to the CO2/3He ratio of 1 × 1013 in limestone and sedimentary end members when Sano and Marty46 discussed the origin of carbon in fumarolic gases from island arcs. In this work, we assume that both microbial  and thermogenic  methane have the CH4/3He ratio of 1 × 1013. We take a δ13C value of −25 ± 5‰ for the thermogenic end-member based on the most mature gas in the Akita-Niigata region47 and methane in submarine sediment-associated system48. On the other hand, we adopted a microbial δ13C end member of −75 ± 5‰ from Wakita et al.26.Even though abiotic (EPR-type) methane is discriminated from thermogenic and microbial methane in the Bernard plot, it is difficult to calculate quantitatively the abiotic contribution because the mixing line is complicated by the location of each end member. We need another parameter to discriminate the abiotic component in a more straightforward way than the CH/C2H6 + CH8 ratio./C2H6 + CH8 ratio.Meas, EPR, Ther and Micr refer to measured methane values, EPR-type abiotic methane, thermogenic methane, and microbial methane, respectively. With δ13CEPR = −23 ± 6‰, δ13CTher = −25 ± 5‰, δ13CMicr = −75 ± 5‰, (CH4/3He)EPR = 5 × 106, (CH4/3He)Ther = 1 × 1013 and (CH4/3He)Micr = 1 × 1013, we can calculate the contributions of the three components E (abiogenic), T (thermogenic), and M  in the samples. The deconvolution results of the lower and upper ranges at each site are listed in Supplementary Table 4/3He ratios and the isotopic fractionation indicated by the δ13C values during the fluid migration from the gas source to the sampling site are not taken into account. Etiope et al.40 suggested that isotopic fractionation resulting in higher δ13C values in mud volcano samples is less than 5‰ due to a segregation process or a chromatographic effect during fluid migration towards the planet’s surface. On the other hand, the molecular ratio CH4/(C2H6 + C3H8) of some mud volcano samples may increase two orders of magnitude during advective fluid migration40. If this is the case, the original CH4/3He ratios may be altered by the migration from the gas source to the sampling site. There are two samples in South Taiwan (WSD and MP) characterized by high CH4/(C2H6 + C3H8) ratios of 5000 ~ 10000 with thermogenic δ13C values of −36‰ ~ −38‰  remain smaller than the average EPR-type methane contribution of the South Taiwan samples  with a high EPR-type contribution are also characterized by thermogenic proportions of 50–70% of the total, suggesting heat source in crust.Natural gases from mud volcanoes in South Taiwan show a thermogenic contribution of approximately 65% and a microbial share of 35%, which is very similar to those of Akita-Niigata samples  with a contribution of EPR-type abiotic methane of only 3.2% of the total  are distributed within the mixing area delimited by the three end members  suggesting that the mixing hypothesis is likely valid. Hence, it is possible to deconvolve each contribution using the simple equations described by Sano et al.63. With the end members δ15NMantle = −5‰, δ15NSediment = + 7‰, δ15NAir = 0‰, (N2/36Ar)Mantle = 6 × 106, (N2/36Ar)Sediment = 6 × 106, and (N2/36Ar)Air = 1.4 × 104 , we can calculate the percentage of the three components, i.e. mantle, sedimentary and atmospheric nitrogen, in the samples. In this calculation, the elemental fractionation of the N2/36Ar ratio and the nitrogen isotope fractionation from the gas source to the sampling site are not taken into account, which sometimes has a significant effect on methane-rich gases. Two mud-volcano samples (WSD and MP) of South Taiwan are probably affected by advective fluid migration resulting in high CH4/(C2H6 + C3H8) ratios as discussed in a previous section. Note that the WSD sample shows a very negative δ15N value, possibly generated by isotope fractionation due to migration. At the same time, the N2/36Ar ratios are expected to increase significantly because of the preferential enrichment in lighter atoms/molecules. Except for a couple of samples that seem to show a migration effect, the distribution of the natural gas data in Fig. 64. This distribution is also similar to the one observed for the Tatun Volcanic Group in North Taiwan65.Figure 15N values and the N2/36Ar ratios, these data are consistent with the atmospheric composition, suggesting that actual contributions of other end members such as mantle and sediment are significantly smaller. This agrees well with the results of methane end-member deconvolution showing negligible abiotic contribution and may reflect the geotectonic setting characterized by a non-volcanic environment. On the other hand, the average mantle contributions to the Akita-Niigata and South Taiwan samples are 27% and 36%, respectively. The northernmost samples in South Taiwan  show the highest proportions of mantle nitrogen up to 45%. These estimates are larger than those obtained for high-temperature volcanic gases of island arcs4 that are probably affected by fractionation during the migration from the gas source to the sampling site. Due to the fluid migration process, δ15N values may decrease, while N2/36Ar ratios would increase  and CH4/3He ratios, atmospheric δ15N values and N2/36Ar ratios, that are well explained by a microbial origin characterized by low-temperature processes and a non-volcanic signature typical of a fore-arc basin. The Akita-Niigata samples show heavy δ13C values, low CH4/(C2H6 + C3H8) and CH4/3He ratios, variable δ15N values and N2/36Ar ratios, suggesting a thermogenic origin possibly related to magmatic heat in a volcanic-arc region. The South Taiwan samples are characterized by heavy δ13C values, intermediate CH4/(C2H6 + C3H8) ratios, low CH4/3He ratios, variable δ15N values and N2/36Ar ratios, probably related to the collision tectonics with some magmatic heat and active fault. The largest contribution of abiotic methane is in South Taiwan samples with 0.34% on average. A slightly smaller average proportion of 0.21% is observed in the Akita-Niigata samples. The abiotic contribution in the South Kanto samples is negligibly small . These trends are consistent with the tectonic settings of the plate characterized by variations in heat flow, seismic velocity and gravity anomaly.We have measured the elemental and isotopic compositions of carbon, nitrogen, argon and helium in methane-rich natural gases from the West Pacific convergent plate boundary. A relationship between origin of methane-rich natural gas and geographical and geodynamic settings is summarized in Fig. 66. The Kazusa Group reaches 2000–4000 m in thickness with several turbidite formations deposited in a deep basin, and it is located across a large area of the Tokyo Bay region. Four natural gas samples were collected from production wells at their wellheads. At the sampling site, a lead glass container with vacuum valves at both ends was filled with water to avoid air contamination, and one end was connected to the outlet of the well. After replacing the water in the container by collected gases, both valves were closed. The Akita-Niigata natural gas field is located in Northeast Japan, face to the Sea of Japan. The gas field lies on a basin developed in a transient slope from volcanic arc to back-arc region. The volcanic arc is attributable to magma generated by subduction of the Pacific Plate beneath the Eurasia Plate . The overall error of the analyses was approximately 10% at 2σ for major gases such as CH4 and N2, which is estimated by the reproducibility of the standard measurements. The error may be up to 30% for trace gases like He and Ar. Carbon isotopes (13C/12C ratios) of CH4 were measured using a continuous flow GC-IRMS system . The overall analytical error was approximately 0.3‰ at 2σ70. Nitrogen isotopes (15N/14N ratios) of N2 were measured by the same GC-IRMS system for samples with nitrogen abundances larger than 1%. For N2 abundances smaller than 1%, we used a nitrogen isotope mass spectrometer under static vacuum operation . The 15N/14N ratio was measured at masses 28 and 29 on a single Faraday cup using magnetic field cycling after separation and purification of nitrogen71. Helium and neon were purified using charcoal traps and Ti getters in an all-metal ultra-high vacuum extraction line. After the 4He/20Ne ratio measurement using a quadrupole mass spectrometer, helium was separated from neon using a cryogenic charcoal trap and the 3He/4He ratio was measured by a helium isotope mass spectrometer . The observed 3He/4He ratio was calibrated against standard atmospheric helium28. The 40Ar/36Ar ratios were measured by a quadrupole mass spectrometer  after separation and purification of argon.All measurements have been achieved at Atmosphere and Ocean Research Institute, the University of Tokyo. Major gas compositions were measured by a quadrupole mass spectrometer equipped with an electron multiplier detector . Abundances of CHSupplementary Information


Moreover, with the only exception of U87 exposed to Ni, our results suggest that metallic NPs have caused, on gene expression, similar effects to those caused by their corresponding ions. Copyright © 2015 The Authors. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.We have considered nanoparticles (NPs) of Fe, Co and Ni, three transition metals sharing similar chemical properties. NP dissolution, conducted by radioactive tracer method and inductively coupled plasma mass spectrometry, indicated that NiNPs and FeNPs released in the medium a much smaller amount of ions than that released by Co NPs. The two considered methodological approaches, however, gave comparable but not identical results. All NPs are readily internalized by the cells, but their quantity inside the cells is less than 5%. Cytotoxicity and gene expression experiments were performed on SKOV‐3 and U87 cells. In both cell lines, CoNPs and NiNPs were definitely more toxic than FeNPs. Real‐time polymerase chain reaction experiments aimed to evaluate modifications of the expression of genes involved in the cellular stress response , or susceptible to metal exposure (SDHB1 and MLL), or involved in specific cellular processes , gave different response patterns in the two cell lines. HSP70, for example, was highly upregulated by CoNPs and NiNPs, but only in SKOV‐3 cell lines. Overall, this work underlines the difficulties in predicting NP toxicological properties based only on their chemical characteristics. We, consequently, think that, at this stage of our knowledge, biological effects induced by metal‐based NPs should be examined on a case‐by‐case basis following studies on different  We have considered Fe, Co and Ni nanoparticles and studied their dissolution by radioactive tracer method and ICP‐MS. We have also performed cytotoxicity and gene expression experiments on two different cell lines. The obtained results convinced us that, at this stage of our knowledge, metal‐based nanoparticles should be examined on a case‐by‐case basis on different in vitro models. Moreover, our results suggest that metal‐based nanoparticles have caused similar effects to those caused by their corresponding ions. CoNPs are promising for nanotechnological applications such as pigments, catalysis, sensors, electrochemistry, magnetism, energy storage and magnetic fluids  with the following characteristics: FeNPs , NiNPs  and CoNPs (purity 99.5% aerodynamic particle size < 100 nm).et al., NPs were checked by transmission electron microscopy (TEM) using a JEOL‐1010 electron microscope  equipped with a CCD camera MORADA  operating at 90 kV. Samples for TEM were prepared placing 5 µl of a diluted suspension of each NP in ethanol on a formvar carbon‐coated copper grid /mg Fe), CoNPs /mg Co) and (58Co)‐NiNPs /mg Ni) were prepared by 12 or 96 h irradiation of 10 mg of dry Fe, Co and NiNPs in the thermal neutron flux of 2 × 1014 neutrons cm–2 s–1 of the nuclear reactor HFR . From 58Ni, the epithermal neutron activation process produces 58Co. This radioactive species has been successfully used as a tracer of Ni in rat tissues NP were resuspended in 2 ml MilliQ H2O to a final concentration of 100 μm, dispersed by an ultrasonic bath for 15 s, stirred for 30 min and ultracentrifuged for 30 min at 60 000 g at 4 °C. The 59Fe, 60Co and 58Co were measured in the supernatants. As a result, only a tiny fraction of < 0.8% of the three radiotracers was recovered in the supernatants indicating that 59Fe, 60Co and 58Co(Ni) radiolabels in the aqueous environment were firmly bound to the NPs and that the radioactive particles were suitable for the dissolution test.A portion of radiolabelled NPs was then submitted for a stability test to assess whether the m and incubated for 4 or 72 h at 37 °C, 5% CO2 and 95% humidity. After centrifugation at 25 000 g for 10 min, 59Fe, 60Co and 58Co in the supernatants were counted by the automatic integral γ‐counting system  equipped with a well type NaI (Tl) detector, using the windows of 950–1400 KeV (59Fe), 800–2000 KeV (60Co) and 600–1800 KeV (58Co). Experiments were performed in triplicate.To evaluate the dissolution, the radiolabelled NPs were resuspended in 10 ml RPMI culture medium to a final concentration of 100 μm NP suspension, sonicated for 15 min in a water bath, were diluted to a final concentration of 100 μm in RPMI‐1640 supplemented with 10% fetal bovine serum (FBS). Fifteen millilitres of each sample were placed in 10 cm Petri dishes and kept at 37 °C for 0 and 72 h in the presence of 5% CO2. After incubation, samples were centrifuged four times at 8000 g for 5 min and ultracentrifuged at 300 000 g for 2 h at 4 °C to remove undissolved NPs. The supernatants were filtered using a 0.22 µm pore size membrane .Ten millilitres of a 4 m3, by inductively coupled plasma mass spectrometry . The calibration curve was performed by the multi‐element calibration standard 3 method (Perkin‐Elmer) using a range of concentration of 10–700 ppb (µg l–1) for each element. The results were analysed by the NexION Instrument Control Session (Perkin‐Elmer).The analysis was performed, after a 1: 10 dilution of the samples with 2% ultrapure HNO2 atmosphere in RPMI‐1640 medium supplemented with 10% FBS, 1% l‐glutamine and 1% penicillin/streptomycin solution. The U87 (human glioblastoma) cell line was maintained, at the same condition, in DMEM medium supplemented with 10% FBS, 1% l‐glutamine, 1% sodium pyruvate and 1% penicillin/streptomycin solution. Cell lines, cell culture media and supplements were purchased from Sigma ; plastics were purchased by Euroclone SpA .SKOV‐3 (human ovarian carcinoma) cell line were maintained as adherent cells at 37 °C and a humidified 5% CO4, 1 × 104, 5 × 103 or 2.5 × 103 cells, depending on the exposure time) were seeded into 96‐well assay plates and cultivated for 24 h at 37 °C in 5% of CO2 to equilibrate and become attached before treatment. Cells were then exposed to 100 μL of increasing concentrations (0–160 µg) of CoNPs, FeNPs or NiNPs for 0.5, 1, 2, 24, 48 and 72 h. NP suspensions used in these experiments were freshly prepared each time, sonicated for 15 min and diluted according to the experimental protocol. The range of concentrations has been determined for each NP in preliminary experiments (range finding test) expressly run for this purpose. After the treatment, plates were equilibrated for 30 min at room temperature and then 100 µl of CellTiter‐Glo reagent was added to each well. Plates were shaken for 2 min and left at room temperature for 10 min before recording luminescent signals using the Infinite F200 plate reader . Each plotted value is the mean of three different experiments.Cell viability was determined measuring ATP content by the CellTiter‐Glo Assay  according to the manufacturer's instructions. Briefly, 200 µl of cell suspension  were added to the samples then placed in a microwave MARS5 express  at the following conditions: 1600 W, the temperature was raised to 175 °C ± 5 °C at 30 °C min–1 and maintained at 175 °C for 4 min and 30 s. Measurements were performed by NexION 300D ICPMS apparatus (Perkin‐Elmer) after 1/100 dilution of the samples with 2% ultrapure HNO3 as reported in the Dissolution experiments by inductively coupled plasma mass spectrometry.Quantification of the internalized metals was evaluated by ICP‐MS of mineralized cells. Therefore, cells exposed to NPs for 30 min were washed twice with PBS, collected in a Teflon tube and dried in a microwave oven at 105 °C. Twelve millilitres of ultrapure HNO6 SKOV‐3 and U87 cells were exposed at 5 µg 100 µl–1 (about 1 mm concentration that causes a 20–30% of ATP reduction) of CoNPs, FeNPs or NiNPs for 24 h. Exposed and not exposed cells were then washed twice with PBS and harvested. We have also arranged a set of samples in which cells were exposed, for the same time, to CoCl2.6H2O, FeCl3 or NiCl2 at a nominal concentration of 5 µg 100 µl–1 in terms of Co, Fe and Ni. Total RNA was extracted by the TRIzol method according to the manufacturer's instructions. The extracted RNA was quantified by spectrophotometer, its integrity was checked by gel electrophoresis and then it was retrotranscribed as previously reported  designed within coding sequence of selected genes, cytoplasmatic β‐actin was used as a housekeeping gene. For each sample, a set of PCR has been run without retrotranscription  was performed using 1 µl of cDNA and 1 μet al.  according to the manufacturer's instructions. The real‐time PCR was performed with specific TaqMan probes (Life Technologies) designed within the sequences of a panel of genes selected on the basis of the results of the qualitative PCR . Each reaction tube was set up according to Papis et al. , split iCt–ΔΔ (ΔCt = CtTarget – Ctβ‐actin and ΔΔCt = ΔCtExposed samples – ΔCtControl samples), was taken as dependent variables. Data analysis was performed by one‐way ANOVA analysis (α = 0.05), completed with Scheffé's test (P < 0.05) to determine which groups  were significantly different from control. Standard errors of real‐time experiments were computed by taking into account propagation of errors , CoNPs released a significant amount of metal  and this percentage increased to 44% after 72 h of incubation. NiNPs and FeNPs, instead, released a smaller amount of ions (6% and 4% of their mass). Similar behaviour was observed in experiments in which measurements were performed by ICP‐MS (data not shown).The dissolution of Co‐, Fe‐ and NiNPs is expressed as a percentage of ions released from the working suspension Fig. . Already–1), we have not been able to observe hormetic effects . The genes considered in this study are those generally involved in cellular stress response, such as HSP70 and MT2A, or susceptible to metal exposure, such as SDHB1 and MLL, or those more closely involved in specific cellular processes, such as CASP3, IQSEC1 and VMP1.First, we checked that the concentration used for CoCl3 did not seem to affect too much gene expression in SKOV‐3 with the exception of a downregulation of HSP70 and MT2A, as highlighted in Fig. 2+ , we have also determined, by radiotracers and ICP‐MS methods, the amount of NPs dissolved in culture media. Our experimental evidence reported that for FeNPs and NiNPs this amount was quite low, while CoNPs released a consistent amount of Co2+. The two considered methodological approaches gave comparable but not similar results, consequently to establish a protocol to evaluate NP dissolution is extremely complex, and further studies to clarify this key point are needed. These findings, supported by those obtained from the metal uptake . The SDH complex is located on the inner membrane of the mitochondria and participates in both the citric acid cycle and respiratory chain. SDHB is assessed to check mitochondrial metabolism. SDHB has been found perturbed by some metal compounds , or susceptible to metal exposure , or involved in specific cellular processes (CASP3), gave different response patterns in the two cell lines. This suggests that generalizing the induced cell response using a single cell line is not possible and strengthens the idea that the use of a combination of different The authors report no conflicts of interest.


Fischerella sp. was analysed for the effects of oxygen concentration and temperature on nitrogenase activity. Dark nitrogenase activity is directly related to aerobic respiration and was therefore used as a measure of the influx of oxygen into the heterocyst. Above 30% O2, the influx of oxygen was proportional to its external concentration. Below this concentration, the influx of oxygen was higher than expected from the external concentration. A higher or lower temperature also triggered the heterocyst to increase or decrease, respectively, dark nitrogenase activity while the external concentration of oxygen was kept constant. A higher dark nitrogenase activity requires a higher rate of respiration and therefore a higher flux of oxygen. Hence, the heterocyst of Fischerella sp. is capable of controlling the influx of air.Heterocysts are differentiated cells formed by some filamentous, diazotrophic (dinitrogen-fixing) cyanobacteria. The heterocyst is the site of dinitrogen fixation providing the oxygen-sensitive nitrogenase with a low-oxygen environment. The diffusion of air into the heterocyst is a compromise between the maximum influx of dinitrogen gas while oxygen is kept sufficiently low to allow nitrogenase activity. This investigation tested the hypothesis that the heterocyst is capable of controlling the influx of air. Here, the thermophilic heterocystous cyanobacterium  Another property that is shared by any nitrogenase is the extreme sensitivity of the enzyme for oxygen. Nitrogenase is irreversibly inactivated by exposure to even low concentrations of O22. Nitrogenase evolved during the pre-oxygenated state of the earth’s atmosphere3. With the appearance of oxygenic photosynthesis and the resulting oxygenation of the atmosphere and large parts of the earth’s biosphere, aerobic diazotrophic organisms had to evolve mechanisms to protect nitrogenase from inactivation by oxygen.Nitrogenase reduces N2 as a consequence of their photosynthetic mode of life, even though photosynthesis can satisfy the demand of energy and low-potential electrons for N2 fixation4. Diazotrophic cyanobacteria evolved a variety of strategies in order to provide a micro-oxic environment for nitrogenase and allow for the incompatible processes of oxygenic photosynthesis and N2 fixation. One of the most elegant solutions is the differentiation of a special cell, the heterocyst, for the fixation of N2 in certain filamentous cyanobacteria5. The heterocyst differentiates from a vegetative cell at semi-regular distances along the trichome through a complex chain of events6.Cyanobacteria are oxygenic photoautotrophic microorganisms. Many species are capable of fixing dinitrogen but this seems enigmatic in organisms that not only thrive in aerobic environments but also evolve O7. It is important to note that this glycolipid layer does not distinguish between O2 and N28 and does not serve as a specific O2 barrier. This means that the glycolipid layer should not stop gas diffusion completely because it would then also prevent N2 from coming in. Hence, the glycolipid layer of the heterocyst, serving as a gas diffusion barrier, must be a compromise between the maximum influx of N2 and the capacity to scavenge the O2 by respiration or otherwise. When the capacity of scavenging O2 is insufficient, oxygen will accumulate in the heterocyst and nitrogenase will be inactivated. Another property of the heterocyst is that it has lost the oxygenic photosystem II but retained photosystem I. The heterocyst is therefore capable of using light as energy source but depends on the neighbouring vegetative cells for reducing equivalents, which are imported as sucrose11. The imported reducing equivalents are partly used for respiratory scavenging of O2 and partly used to reduce ferredoxin, which serves as the electron donor to nitrogenase12.The heterocyst develops a special glycolipid layer which serves as a gas diffusion barrier2 fixation in the heterocyst can acclimate to environmental O2 concentrations14. It is assumed that this requires an alteration of the glycolipid layer of the heterocyst to become a more or less efficient gas diffusion barrier15. Indeed, depending on the environment from which they originated, heterocystous cyanobacteria exhibit different nitrogenase activity characteristics that are attributed to the gas diffusion property of the glycolipid layer of the heterocyst17. Theoretical considerations have indicated that a heterocystous cyanobacterium would maximize its daily N2 fixation by increasing its dark nitrogenase activity17. Although this might seem counter-intuitive it can be understood when considering that it would allow N2 fixation at any time and any place . Dark nitrogenase activity is driven by respiration. Hence, in order to maximize N2 fixation the heterocyst should maximize gas diffusion (to increase the influx of O2) to the extent of its maximum capacity to scavenge the O2. While the acclimation of the gas diffusion characteristics of the heterocyst glycolipid layer would be a slow process14, a more dynamic process would represent an advantage, especially under fluctuating environmental conditions. Walsby18 hypothesized that the regulation of the rate of gas exchange into the heterocyst could be accomplished through the opening of the pores that connects the heterocyst with the vegetative cell, analogous to stomata in plants. The aim of this work was to test whether the heterocyst is able to acclimate the influx of gas in order to optimize nitrogen fixation.It has long been known that N2 on nitrogenase activity (acetylene reduction) was tested at a fixed temperature of 30 °C. The total chlorophyll-specific nitrogenase activity (Ntot) (the sum of the maximum light-dependent (Nm) and light-independent (Nd) acetylene reduction) remained constant at 88 ± 4 µmol mg−1 h−1 in the concentration range of 0–30% O2  in the concentration range of 0–30% O2 was at its maximum rate at the given set of conditions. Above 30% O2, nitrogenase activity (Nm as well as Nd) decreased linearly with O2 concentration . At 12 and 60 °C there was low but still detectable nitrogenase activity . Examining the Ntot/Nd revealed that it remained constant in the temperature range of 21–39 °C at a value of ~2.2 ± 0.1  nitrogenase activity contribute equally to the total. The total nitrogenase activity increased in the temperature range 21–39 °C. Above 39 °C Nm rapidly declined while Nd remained fairly constant up to 50 °C. This resulted in Ntot/Nd approaching 1, meaning that nitrogenase activity was largely driven by respiration. Above 50 °C nitrogenase activity collapsed and no meaningful analyses of Nm and Nd could be made.The effect of temperature on nitrogenase activity was investigated at constant O2 fixation. For the fixation of dinitrogen, O2 concentration and temperature are particularly important and these two environmental parameters are also strongly interrelated. Temperature affects the solubility and diffusion rate of oxygen and also influences the rate of physiological (enzymatic) processes. Nitrogenase is irreversibly inactivated by O2 and therefore the organism has to provide an environment that is sufficiently low in O2 in order to be able to fix N2. This investigation aimed at testing the hypothesis that the heterocyst of Fischerella is capable of modulating the influx of gas in order to optimize N2 fixation. It was previously shown17 that natural populations of diazotrophic heterocystous cyanobacteria can improve the fixation of N2 best by increasing nitrogenase activity that occurs independently of light (Nd). It should be noted that increasing dark nitrogenase activity is only possible when a higher influx and respiration of O2 occurs in the heterocyst. However, this must be precisely controlled so that all O2 that enters the heterocyst is fully and instantaneously respired in order to prevent the inactivation of nitrogenase. Detection of nitrogenase activity is a proof that this condition is fulfilled. Maximizing the influx of O2 into the heterocyst also maximizes the influx of N2, guaranteeing the highest possible fixation of N2.Diazotrophic cyanobacteria are exposed to a suite of environmental factors that affect N2 nitrogenase activity was more or less constant at 88 ± 4 µmol ethylene (mg chlorophyll a)−1 h−1 . The linear decrease of Nd above 30% O2 . The transport of sucrose into the heterocyst must be sufficient to scavenge O2, otherwise we would not see nitrogenase activity. It is therefore not a limiting factor.The reason for the decrease of nitrogenase activity above 30% O O2 Fig.  indicateNd data obtained in the range of 30–90% O2 were linearly fitted using equation (a-specific acetylene reduction (µmol mg−1 h−1) and x = O2 (%), and the constants are a = −0.61 µmol mg−1 h−1 %−1 and b = 67.1 µmol mg−1 h−1.The equation 1\documen2 the theoretical supply of electrons (expressed as the rate of chlorophyll-specific acetylene reduction) would equal 67.1 µmol mg−1 h−1 (because no electrons would be required to reduce O2), and at 100% O2 the theoretical residual rate of chlorophyll-specific C2H2 reduction would equal 6.1 µmol mg−1 h−1. Per %O2 the electron supply (expressed as the rate of chlorophyll-specific acetylene reduction) required was 0.61 µmol mg−1 h−1 ((67.1–6.1)/100%), assuming only gas diffusion through the cell envelope proportional to the external O2 concentration.Hence, at 0% O2 concentration above 30% was linear and, hence, O2 diffusion was a function of its external concentration, below 30% O2Nd followed a dose-response model; equation . For the total nitrogenase activity (Ntot) as well as for Nm a value of 2.3 was calculated and 2.2 for Nd, hence Q10 was well within the expected range for a biochemical process. However, if Nd would only depend on the rate of diffusion of O2 into the heterocyst the Q10 is expected to be only marginally above 1 (1.084), that results from the temperature effects on O2 solubility and diffusion constant17. The Q10 of 2.2 for Nd means that respiration was not limited by O2 diffusion or availability and it reflects the temperature (kinetic) effect on the enzyme nitrogenase. In order to keep the heterocyst micro-oxic the influx of O2 must have been precisely controlled.In the temperature range 21–39 °C the 2 and N28. While on the one hand the heterocyst glycolipid layer will limit the rate of O2 influx, it must also allow sufficient influx of N2 for N2 fixation; on the other hand, influx of O2 is essential for generating energy through respiratory electron transport when light is unavailable and hence extends the period during which the organism is able to fix N2. It is also important to understand that by measuring nitrogenase activity one looks specifically at the processes in the heterocyst, rather than in the vegetative cells. For instance, by measuring the light-independent part of nitrogenase activity, this can be translated to respiration. In the dark, respiration is the only known source of energy for nitrogenase in the heterocyst, and, hence, dark nitrogenase activity can be interpreted as the influx of O2 into the heterocyst. When nitrogenase activity is detected one must conclude that the heterocyst is low in O2. The residual O2 concentration in the heterocyst can be as low as 0.6 µM, which is 0.2% of air saturation18 and depends on the Km(O2) of the O2-scavenging process.It is important to note that the heterocyst glycolipid layer forms a gas diffusion barrier that does not discriminate between OFischerella is capable of adjusting the influx of air into the heterocyst. First, the effect of the external O2 concentration on nitrogenase activity was measured at a constant temperature. Second, the effect of temperature on nitrogenase activity was measured at a constant external O2 concentration. Temperature affects the rate of metabolic processes, including respiration, but has a small effect on the rate of gas influx by diffusion. The gas influx is the combination of O2 solubility and diffusion. Whereas diffusion increases with temperature, solubility decreases and the two factors almost compensate each other (Q10 = 1.084)17.In this investigation, I demonstrate in two ways that Q10 in the range of 21–39 °C was 2.2. Nd can only increase when respiratory energy generation increases and this is only possible when the O2 influx increases. Since these experiments were done under a constant O2 concentration in the gas mixture, it was concluded that the heterocyst possesses a mechanism to increase the influx of O2. It is unlikely that the heterocyst glycolipid layer possesses the capacity to alter its gas diffusion properties in the short term of the experiment. One possibility that was proposed by Walsby18 is that the heterocyst terminal pores might perform this function   between the vegetative cell and the heterocyst is through the septal junctions (previously named microplasmodesmata) by molecular diffusion21. However, although gas molecules could diffuse together with the solutes, they may additionally also diffuse through the cell wall and cytoplasmic membrane which are impermeable for larger molecules such as sucrose.I observed that the light independent part of nitrogenase activity increased with temperature in the range from 12–42 °C. The es) Fig. . Canini Q10 is in the physiological range which means that it is related to the enzyme activity solely and that Nd is not limited  in the temperature range of 21–39 °C. Unless nitrogenase was replaced continuously and instantaneously, it was also unlikely that nitrogenase was inactivated by O2, because control experiments during which the cells were brought back to the initial conditions reproduced the original nitrogenase activity. This would require that the gas influx is tightly controlled to ensure that all O2 entering the heterocyst is respired. Another remarkable observation was that Nd was less sensitive to temperature than was the light-dependent part of nitrogenase activity, Nm. This could indicate that the photosystem is more thermosensitive than the respiratory system. Photosystem II is known as the most thermolabile component that determines the upper thermal tolerance of photosynthesis22. It has been shown that the D1 protein degrades faster at high temperature and is not re-synthesized23. It is not known whether a similar process affects photosystem I in the heterocyst and makes it more thermosensitive than the respiratory system24.The 2 concentrations at a constant temperature confirmed the presence of a control mechanism that determines the influx of gas into the heterocyst. Only above 30% O2 the supposed influx was a linear function of the external concentration and determined by the diffusion characteristics of the heterocyst envelope. Below 30% O2 a dose-response relationship was found. The lower the external concentration of O2 the higher the relative influx became. The gas diffusion through the heterocyst envelope would be an intrinsic property of the envelope and would be a linear function of the external O2 concentration at any O2 level. Subtracting this envelope gas diffusion from the Nd below 30% O2, I found an exponential decay of the additional (in excess of the envelope gas diffusion) chlorophyll-specific dark nitrogenase activity, hence O2 influx (measured as acetylene reduction), from ~60 µmol mg−1 h−1 at 0% O2 to ~0 µmol mg−1 h−1 at 30% O2 . As proposed here, the influx of a gas into the heterocyst through the pores would be equally controlled for acetylene and ethylene as it would be for air. Hence, it is unlikely that the use of the acetylene reduction assay instead of directly measuring N2 fixation would have led to different conclusions.The measurements reported here were carried out by the acetylene reduction assay. The reduction of acetylene to ethylene is an accepted measure of nitrogenase activity. It should be noted that the reduction of acetylene cannot be converted quantitatively directly into fixation of N2 concentration, such acclimation cannot be instantaneous and also depends on the properties of this glycolipid layer which should be genetically determined. For instance, it has been observed that heterocystous cyanobacteria that thrive in colder waters possess a heterocyst envelope with a stronger gas diffusion barrier than those that occur in warmer environments where respiration is faster. The results of this investigation support the idea of Walsby18 that heterocysts can also respond instantaneously to O2 and temperature as depicted in Fig. In conclusion, although it is known that heterocystous cyanobacteria can modify their heterocyst glycolipid layer in response to external OFischerella sp. strain MV11  was obtained from Prof. dr. W.R. Hess, University of Freiburg, Germany, and has been described in detail26. This is a true-branching, thermophilic, N2-fixing, heterocystous cyanobacterium belonging to the Stigonematales (section V) and was originally isolated from a geothermal site at a hot spring in Costa Rica. The organism was routinely grown at 27 °C in BG11o medium (devoid of any combined nitrogen)27 under a 12:12 hour light-dark cycle and 30 µmol m−2 s−1 warm-white fluorescent light.28. Here, I used the on-line set-up as described previously29. The set-up allows a highly sensitive measurement of ethylene  at a time resolution of 3–5 seconds and therefore provides almost real-time monitoring of nitrogenase activity. The sensitive and rapid monitoring of ethylene was done using a laser photoacoustic trace gas detector . A low amount (0.4–0.8 µg chlorophyll a) of Fischerella culture was gravity filtered on a Whatman GF/F glass fibre filter (47 mm). This low amount of culture prevented self-shading and also excessive ethylene production (which would result in an ‘overload’ of the ETD-300). The filter containing Fischerella was put in a custom-made measuring cell29. The cell was equipped with a Peltier temperature regulation unit . The filter was placed on a supporting grid underneath which growth medium was present that ensured that the filter was saturated with medium and that prevented desiccation. The incubation chamber was connected to a continuous flow of gas at 2 L h−1 that was led over the filter with Fischerella. The gas was a mixture of N2, O2, CO2 and C2H2, made up by mass flow controllers . Acetylene was kept at 10%. CO2 was premixed at 0.04% in the N2 and O2. The concentration of O2 was varied as required and the balance was made up with N2. O2 concentrations ranged from 0–90% . The gas was washed through water prior to entering the measuring cell. This served to humidify the gas and removed any acetone from the acetylene (which would interfere with the ethylene detection). Gas mixtures (O2 and N2 with 0.04% CO2) were purchased from Hoek-Loos (The Netherlands) and acetylene was obtained from Messer (The Netherlands). The gas mixture contained a very low background level of ethylene of ~10 ppbv due to contamination of the acetylene. Controls were run without Fischerella in the incubation chamber. Ethylene recordings of >100 ppbv were taken as positive for ethylene production. The whole set-up was placed in a temperature-controlled climate room. Illumination was provided by a cold-light 250 W halogen lamp and glass fibre light shower . A computer program written in Testpoint  controlled the gas mixtures, light intensity and temperature.Nitrogenase activity was monitored using the acetylene reduction assay2 the response of nitrogenase activity to light was measured as follows. Ten different photon irradiances were tested: 0, 1.4, 4, 7.1, 10.9, 15.5, 20.5, 26.5, 43.5 and 68.2 µmol m−2 s−1. Nitrogenase activity was monitored for 5 minutes at each light level. The measurement started with 15 minutes in the dark, followed by the series of light levels until the highest level tested which saturated nitrogenase activity at all conditions tested (light up), after which the same measurements were made while light levels decreased (light down). This was done to observe any hysteresis effects and to check that the original measurements could be reproduced. The system is robust and measurements were reproducible with high accuracy. A full light response measurement (light up and light down) took 105 minutes after which the computer adjusted the measuring cell to the next condition. The whole experiment ran unattended.At any given temperature and concentration of Oa, and in the second experiment (increasing temperature) it contained 0.7 µg chlorophyll a. The nitrogenase parameters at 27 °C from both experiments were similar and hence the data of the two experiments overlapped. In test runs it was demonstrated that cells that were lowered to 18 °C and subsequently brought back to 27 °C, reproduced the original nitrogenase activity and the same was true for cells that were increased to 54 °C and brought back to 27 °C. Below 18 °C (incubations at 15 °C and 12 °C) and above 54 °C (incubations at 57 °C and 60 °C) the cultures were to some extent negatively affected and also possessed low nitrogenase activity.The effect of temperature was measured in two series of experiments. In one series of experiments the temperature was decreased from 27 °C (growth temperature) in steps of 3 °C to 12 °C and in the second experiment the temperature was increased from 27 °C to 60 °C, also in steps of 3 °C. Both experiments were carried out immediately one after the other, using cells from the same stock culture. In the first experiment (decreasing temperature) the filter contained 0.4 µg chlorophyll 2 was measured in one series of measurements from 0–90% O2. The filter contained 0.8 µg chlorophyll a. The following O2 concentrations were tested and in this order: 0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, and 90%. The experiment was run at 30 °C. Before starting the experiment the culture was run at 0% O2, 20.5 µmol m−2 s−1 light and 30 °C for 30 minutes by which time a constant rate of nitrogenase activity was achieved. The system did not achieve a fully anoxic state. Previously, it was estimated that the setting 0% O2 may in fact represent a level of ~1% O230. This is in part because of leakage through the tubing used and the large volume of water (1 L) needed for washing the gas, as well as due to photosynthetic O2 production (in the light). Fully anoxic conditions in the dark would not have allowed any activity (since there would not be a possibility to generate energy). At each O2 concentration the light response of nitrogenase activity was recorded as described above  at the same 10 light levels as mentioned above for the recording of light-response curves. Test runs were performed before carrying out the final experiment reported here.The effect of O31; equation , the maximum light-dependent nitrogenase activity at light saturation, and the slope of the curve. From these fitted parameters, the total maximum nitrogenase activity, Ntot, was calculated as the sum of Nd and Nm. Ntot/Nd is a convenient parameter that is dimensionless and therefore biomass independent  and describes the contribution of light to nitrogenase activity. Ntot/Nd ≥ 1; when this ratio equals 1 no light-dependent nitrogenase activity is present. The light saturation coefficient Ik equals Nm/α, and represents the photon irradiance at which 0.5 Nm would have been reached when nitrogenase activity increases linearly with irradiance (Ks in Michaelis-Menten kinetics).The light response curves were fitted with the rectangular hyperbola modelequation  using thNI is the actual nitrogenase activity at light level I.where Q10) was fitted from the linear fit of the natural log transformed data of Nd, Nm, and Ntot with temperature32 using the software package Origin. Meaningful values (i.e. linear fits) of Q10 were obtained in the temperature range 18–39 °C. The temperature coefficient of O2 diffusion in the temperature range 18–39 °C was calculated from O2 solubility and diffusion coefficients that can be found in the tables provided by the company Unisense and can be downloaded from: http://www.unisense.com/files/PDF/Diverse/Seawater%20&%20Gases%20table.pdf.The temperature coefficient of nitrogenase activity (a was extracted from the GF/F filters in 2 ml 96% ethanol overnight at room temperature in the dark, immediately after the experiment was finished. The absorption coefficient of 72.3 ml mg−1 cm−1 was used to calculate the concentration of chlorophyll a.Chlorophyll


Devices based on the silver bismuth iodide deposited from solution on top of TiO2 and the conducting polymer poly (P3HT) as a hole‐transport layer are prepared and the photovoltaic performance is very promising with a power conversion efficiency over 2 %, which is higher than the performance of previously reported bismuth‐halide materials for solar cells. Photocurrent generation is observed between 350 and 700 nm, and the maximum external quantum efficiency is around 45 %. The results are compared to solar cells based on the previously reported material AgBi2I7, and we observe a clearly higher performance for the devices with the new silver and bismuth iodides composition and different crystal structure. The X‐ray diffraction spectrum of the most efficient silver bismuth iodide material shows a hexagonal crystal structure with space group R3‾m, and from the light absorption spectrum we obtain an indirect band gap energy of 1.62 eV and a direct band gap energy of 1.85 eV. This report shows the possibility for finding new structures of metal‐halides efficient in solar cells and points out new directions for further exploration of lead‐free metal‐halide solar cells.Here, a lead‐free silver bismuth iodide (AgI/BiI In 2009, CH+) included in the structure have been investigated, which also show promising optical properties.2I7 functions as a photovoltaic material, and solar cells with over 1 % PCE could be obtained.3 shows promising efficiency in solar cellsx1‐3Bix1+I4 (0<x<0.19) compounds has previously been performed, indicating that there exist several stable compositions and the structure is not trivial owing to the vacancies in the system.Bismuth is another low‐toxic alternative for replacement of lead and it was shown that it is possible to make functional solar cells with bismuth‐halide perovskites with a similar device structure as the lead perovskite solar cells.3) with molar ratio of AgI/BiI3=2:1 and AgI/BiI3=1:2 were used to synthesize materials with different crystal structures. These materials were used in a solar cell between TiO2, which acts as electron‐conducting layer, and poly (P3HT), which is used as hole‐transport layer. The PCE of the best devices was above 2 % under 1000 W m−2 (1 sun), AM 1.5 G simulated solar illumination. UV/Vis spectroscopy, X‐ray diffraction (XRD), and scanning electron microscopy (SEM) were used to investigate the properties of the material, and incident photon‐to‐current conversion efficiency (IPCE) and transient photovoltage decay measurements were used to try to understand the light‐induced processes in the solar cell. The results were also compared to results from devices with the previously reported material AgBi2I7.Here, solutions with a mixture of silver iodide (AgI) and bismuth iodide  measurements. Recently, Xiao et al.2I7 material and suggested that it cannot be formed in the ThZr2H7‐type structure, because the Bi−I bond length is too short and it may result in an unreasonably large mass density. Instead, it was suggested that the cubic AgBiI4 is formed with the lattice constant a=12.2223 Å. Moreover, the theoretically calculated AgBi2I7 has a much higher intensity of the (440) plane (d=2.16 Å), whereas the theoretically calculated AgBiI4 has a much higher intensity of the (400) plane, corresponding to d=3.05 Å. Herein, the (400) plane has the highest intensity, however, we cannot state which cubic structure is formed as the AgBiI4 and AgBi2I7 structures are very similar. A clear difference between the samples with space group Fd3‾m and R3‾m is observed at 2θ=42°, where Fd3‾m has only one diffraction peak from the (440) plane, and the R3‾m structure has two diffraction peaks from the (110) and (108) planes . We can therefore clearly distinguish the samples made from molar ratio solutions AgI/BiI3=1:2 and AgI/BiI3=2:1 by the space group difference. The previously reported structures for AgBiI4 and AgBi2I7 have the same Fd3‾m space group, which we also observe here for the material with molar ratio solution AgI/BiI3=1:2, whereas we observe a structure with space group R3‾m for the material from molar ratio solution AgI/BiI3=2:1. The space group R3‾m is in agreement with the crystal structure of Ag2BiI5, which we expect from the molar ratio in the solution. Here, we therefore hereafter use the space group to name the samples made from different molar ratio solutions. The diffraction peaks addressed with a star are in agreement with previously observed diffraction peaks in hexagonal Bi compositions.2 environment and they showed the same XRD patterns after 7 days .The crystal structures of the silver bismuth iodides made from solutions with different ratios of AgI and BiIA), A=100−R(λ)−T(λ) (R is the reflectance and T is the transmittance) in the 300–1600 nm range.The optical properties of the silver bismuth iodide samples were determined by measuring the transmittance and reflectance. Figure Fd3‾m has slightly higher absorption throughout the spectrum. Tauc plots were implemented in Figure α d E)r, α corresponds to the absorption coefficient and d the thickness of the thin film (here constant) and E is the photon energy. In the expression, r=1/2 for an indirect allowed band gap, and r=2 for a direct allowed band gap. From this expression the Tauc plots in Figure Eg) of the indirect optical transitions is estimated to be1.60 eV for the Fd3‾m sample and 1.62 eV for the R3‾m sample. The direct optical band gap energies are determined to be 1.78 eV for the Fd3‾m sample and 1.85 eV for the R3‾m sample.Both samples show similar character of spectra with absorption of 70–80 % above the band gap. The sample with space group Fd3‾m with grain size 400–800 nm and b) R3‾m with grain size 500–900 nm. The grains have grown densely together even if pinholes could be observed especially in Figure Fd3‾m sample in Figure R3‾m in Figure 2 can be observed at a few places most probably leading to short circuit in the solar cell at these points.The surface morphology of the crystallized films annealed at 150° C was characterized using SEM. Figure 2BiI5 and AgBi2I7 . Comparing to previously reported devices based on bismuth‐halide light absorbers, the sample with space group R3‾m shows significantly higher IPCE. The reason for the higher photoconversion efficiency is not clear at the moment, but it may be related to more efficient electron transport to TiO2, or longer lifetime of the photogenerated charges in the sample with space group R3‾m under different light intensity  of around 5.4 mA cm−2 is obtained, and the average PCE is around 1.5 % . The stability of the champion device was also investigated. The sample was kept under N2 environment in dark and the performance of the device shows almost the same efficiency after 40 days, with a slight loss (0.2 %) of efficiency, which is mainly owed to photocurrent loss . These promising results exemplify the possibility to tune the device and material performance of new lead‐free metal halides by changing the stoichiometry of the components, yielding different crystal structure and different optoelectronic properties.Figure R3‾m as light absorber in solar cells, and compared with the previously reported AgBi2I7 sample with space group on Fd3‾m. Both the materials with space group R3‾m and Fd3‾m have an indirect band gap and a direct band gap with slightly higher energy. The R3‾m‐based devices showed a maximum incident photon‐to‐current efficiency (IPCE) of 45 % and the Fd3‾m‐based devices showed a maximum IPCE of around 11 %. The device with the material with R3‾m space group structure shows a very promising power conversion efficiency (PCE) above 2 %. The results show the potential for finding new lead‐free metal‐halides and the possibility to tune the properties of bismuth halides adding different amounts of silver ions and different structures. In a future perspective, the large variety of possible metal‐halide materials, varying the elements, stoichiometry, and structure in metal‐halides, makes it therefore likely to find several materials with advantageous photovoltaic properties.In summary, we have characterized the lead‐free hexagonal metal‐halide with space group 3 (Sigma–Aldrich 99.99 %), AgI (Sigma–Aldrich 99 %), and P3HT (Sigma–Aldrich 99.995 %) were purchased and used as supplied. The solvent for precursors was butylamine (Sigma–Aldrich 99.5 %). The P3HT was dissolved in chlorobenzene (Sigma–Aldrich 99.8 %), the concentration of P3HT is 10 mg mL−1.BiI2 blocking layer and mesoporous TiO2 layer are the same was done by an etching method previously reported by our group.3 used were 1:2 and 2:1, the concentration of all solutions was 17 wt %. Precursor solutions were spin‐coated on the TiO2 substrate at 4000 rpm for 30 s under flow of N2. After heating at 150 °C for 30 min in a dry box (relative humidity is around 20 %), the light‐absorbing films were formed. Then, P3HT was spin‐coated on the top of light‐absorbing films under 3000 rpm for 30 s. Last, 80 nm‐thick gold electrode was evaporated on P3HT layers by thermal evaporation under the vacuum of about 10−5 mbar.The forming of TiOXRD: The structure of the metal‐iodide materials were determined by grazing incidence X‐ray diffraction (GIXRD), using a Siemens D5000 θ‐2 θ goniometer with CuKα (λ=1.54051 Å) radiation and 0.4° Soller‐slit collimator that had a resolution of 0.3° (2 θ) .PCE: The photovoltaic performance of cells were recorded by using a Keithley 2400 source meter with a scan rate of 50 mV s−1 under AM 1.5 G (1000 W⋅m−2) illumination with a solar simulator (Model: 91160), which was calibrated with a standard Si solar cell (Fraunhofer ISE), and the power supplier was Newport Oriel(Model: 69911). The solar cells were masked during the measurement and the active area was defined as 0.125 cm2.IPCE: The IPCE spectra were recorded using a Keithley multimeter (Model 2700) as a function of wavelength of the light from 350 to 900 nm. A monochromator  was used to obtain monochromatic light. The setup was calibrated with a standard Si solar cell (Fraunhofer ISE) prior to measurements. All solar cells were illuminated from the working electrode (glass substrate) side with an active area of 0.125 cm2 (circular shaped mask).Electron lifetime measurements: Electron lifetime was determined by transient photovoltage decay at different light intensities. The source was a white LED . The voltage was recorded with a 16‐bit resolution digital acquisition board  with a current amplifier .SEM measurements: The cross section of the solar cells was measured with SEM using a LEO 1550 FEG instrument  with in‐lens detector.UV/Vis: The optical reflectance and transmittance of the samples were measured with a PerkinElmer Lambda 900 double‐beam UV/Vis/NIR spectrophotometer equipped with an integrating sphere and a Spectralon reflectance standard. The absorbance A(λ) was measured by using the following equation: A(λ)=100−T(λ)−R(λ), T(λ) is the transmittance and R(λ) is the reflectance.2BiI5 solar cells; Jsc, Voc, fill factor (FF), and PCE for 20 solar cells, I–V measurement, IPCE measurement, and the photo of precursor solution, are provided in the Supporting Information.SEM images, EDX measurements, XRD patterns, histograms of parameters of AgThe 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.


Specifications TableValue of the data•Determination of the 3D microstructure of a new Laves phase intermetallic matrix composite through a focused ion beam (FIB) slice and view experiment and surface reconstruction.•STEM micrographs and STEM-EDX mapping identifying the elemental partitioning between C14 Laves matrix and bcc  precipitated laths.•Details of the determination and validation of the orientation relation between matrix and laths identified in 1Here we report data obtained from a Fe-30Ti-10Mo (at%) alloy 2Full details of the preparation of the alloy are provided in An FEI Helios NanoLab 600 dual beam FIB-scanning electron microscopy (SEM) system was used to perform a slice and view experiment through a ~15×15×15 µm volume by sequential FIB slicing followed by collection of secondary electron (SE) micrographs. The SE micrographs were then binarised and segmented using ImageJ to separate the C14 and A2 (bcc) phases, after which the images were stacked and an iso-surface produced using the Avizo software package.Electron transparent foils for transmission electron microscopy (TEM) were produced by electro-polishing using a solution of 10 vol% perchloric acid in methanol at −30 °C and 18 V, followed by final polishing using a GATAN precision ion polishing system operated at 5 kV, as in x–y, x–z and y–z slices of the reconstruction. From this, it was identified that the A2 phase does occur as laths and not as needles From SEM micrographs of the aged alloy characterised in In order to study the chemical segregation between the fine scale A2 plates and the C14 matrix STEM-EDX was employed, A2 and [30 20 3]C14 is shown in A TEM SADP and key of [805]


Here, we present experimental measurements of binding energies for water molecules occupying the second and, quite possibly, the third solvation shell surrounding a central Ca2+ ion in [Ca(H2O)n]2+ complexes. Results for these large, previously inaccessible, complexes have come from the application of finite heat bath theory to kinetic energy measurements following unimolecular decay. Even at n = 20, the results show water molecules to be more strongly bound to Ca2+ than would be expected just from the presence of an extended network of hydrogen bonds. For n > 10, there is very good agreement between the experimental binding energies and recently published density functional theory calculations. Comparisons are made with similar data recorded for [Ca(NH3)n]2+ and [Ca(CH3OH)n]2+ complexes.Further understanding of the biological role of the Ca For metal dications, such as Catransfer ; howevertransfer –20. In me et al.  succeedeh theory –25 can bh theory ,27. Usinolecules ,27. Pres2.−2 mbar) was generated by a Knudsen effusion cell  operating at approximately 600°C. Neutral calcium atoms collided with the molecular cluster beam to produce various neutral clusters including some with the composition Ca(H2O)n and Ca.Arm(H2O)n. Previous experiments have shown that argon atom evaporation is an essential part of the ‘pick-up’ process and facilitates the dispersion of energy on addition of a metal atom and after electron ionization [2O)n]2+ cluster dication was selected using the magnet and the electric sector field voltage was then scanned while the accelerating voltage and magnetic field remained constant. Equations given previously were then used to identify any fragment ions and determine their centre-of-mass kinetic energy 2+ complexes for n in the range 4–20. The assumptions made and the parameters specific for this particular series of experiments have been listed elsewhere 2+. In their measurements of hydration energies for alkaline earth metal ions, Rodriguez-Cruz et al. [2O)5(H2O)]2+, where the additional (H2O) denotes the presence of a water molecule that occupies an outer solvation shell and is hydrogen bonded to one or more molecules in the primary shell. This displacement of a water molecule was found to occur at an elevated temperature and was characterized by a lower than expected hydration enthalpy. Similar behaviour has been observed following kinetic energy release measurements on [Mg(NH3)n]2+ complexes [3)4(NH3)]2+ and [Mg(NH3)5(NH3)]2+ (or [Mg(NH3)4(NH3)2]2+) was attributed to the low binding energies calculated from finite heat bath theory. Unlike the experiments of Rodriguez-Cruz et al. [3)6]2+ [et al. [et al. [2O)5(H2O)]2+ complex approximately 22 kJ mol−1 higher in energy than a ground state structure where all the water molecules are coordinated to the central ion; an internal energy of this magnitude is easily achieved during electron ionization.Using finite heat bath theory –25, the lsewhere ,27. The z et al.  providedomplexes , and forz et al. , it is n [3)6]2+ , it is p 2+ complexes show binding energies that, even for n = 20, remain high and will probably not converge close to those recorded for H+(H2O)n clusters  until n is approximately 25 or more. In earlier discussions of the results derived from kinetic energy measurements, it has been argued that the nature of the experiment is such that only the lowest energy process available to a cluster will contribute to reaction (2.1) above 2+ complexes and included in their results are sequential binding energies out to n = 20. Similarly, Bai et al. 2+ and results presented previously for [Ca(NH3)n]2+ and [Ca(CH3OH)n]2+ that have also been derived from experimental data using finite heat bath theory 2+ and [Ca(CH3OH)n]2+ it was concluded that the first solvation shell consists of six molecules and so it is interesting to see that all three datasets converge to approximately 80 kJ mol−1 at that point.Finally, h theory ,27. The n = 7, the decline in binding energy is, in all cases, very much less rapid than is seen for the small clusters and would suggest that this pattern follows a transition to the second solvation shell where binding energy is now determined by hydrogen bond strength, but one that appears enhanced by the presence of the 2+ charge on the metal. It is instructive to compare 2O)nH+, (CH3OH)nH+ and (NH3)nH+ 2+ complexes, a similar conclusion on the long-range influence of charge was reached by Bush et al. 2+ complexes has made it possible to extract binding energies for up to 20 water molecules bound to Ca2+. The results suggest that the first solvation shell contains six water molecules; however, more significant is the observation that the 2+ charge on the metal cation has an influence on molecular interactions that extends far beyond the first solvation shell.The application of finite heat bath theory to kinetic energy release measurements recorded in the gas phase on [Ca(H


The composition of this phase was determined to be Na 2HfSi2O7, has been determined by laboratory powder X-ray diffraction and refined by the Rietveld refinement. The structure is a framework made of alternate layers of HfO6 octahedra and SiO4 tetrahedra linked by common O atoms. Sodium atoms are located in the voids of the framework, aligned into tunnels along the [010] direction. Na2HfSi2O7 is isostructural with the parakeldyshite Na2ZrSi2O7 phase.The structure of triclinic disodium hafnium disilicate, Na To overcome this problem, these radionuclides are often replaced by a stable element having similar properties as the radioactive element, for instance by using elements with a similar ionic radius or with the same oxidation state. Hence actinides are often replaced by neodymium, zirconium, europium, or hafnium  to monoclinic (Na2ZrSi4O11) or trigonal (Na8ZrSi6O18). If we focus on the chemistry of zirconosilicates with Si2O7 diortho groups and their analogs 2·H2O with zirconium totally substituted by hafnium. Adequate quantities of sodium silicate solution , sodium chloride, hafnium chloride, potassium chloride, sodium hydroxide and water were mixed thoroughly in a polytetrafluoroethylene (PTFE) vessel at room temperature for 30 minutes. A gel was obtained with a pH value around 13. The PTFE vessel was put in a Parr digestion apparatus for a hydrothermal synthesis over 10 days at 523 K. The resulting powder was washed, filtered, and dried overnight at 393 K. In spite of the drying process, the powder was still hydrated. Powder X-ray diffraction showed the compound to be isostructural to petarasite. The second step was the calcination of Hf–petarasite over 15 h at 1373 K under air which lead to a white powder. SEM observation of the powder showed large grains with Na, Hf, Si and O and smaller grains with supplementary K. The chemical composition of the major phase was determined by EDS to have the following stoichiometry Na1.7±0.2Hf1.0Si2.3±0.1O7.3±0.9 as compared to the theoretical stoichiometry of Na2HfSi2O7. Thus the major phase is very close to the expected one. The sample was analysed by differential thermal analysis to determine its melting point. There was no thermal event indicating a melting until 1623 K and the sample was still in powder form. The Na2HfSi2O7 phase therefore has a higher melting point than parakeldyshite which is below 1523 K  nahfsio, I. DOI: 1502957CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Agaricus bisporus in Escherichia coli. Applying this procedure a pure preparation of a single isoform of latent tyrosinase can be achieved at a yield of 140 mg per liter of autoinducing culture medium. This recombinant protein possesses the same fold as the enzyme purified from the natural source as evidenced by single crystal X-ray diffraction. The latent enzyme can be activated by limited proteolysis with proteinase K which cleaves the polypeptide chain after K382, only one The latent enzyme can amino acid before the main in-vivo activation site. Latent tyrosinase can be used as obtained and enzymatic activity may be induced in the reaction mixture by the addition of an ionic detergent (e.g. 2 mM SDS). The proteolytically activated mushroom tyrosinase shows >50% of its maximal activity in the range of pH 5 to 10 and accepts a wide range of substrates including mono- and diphenols, flavonols and chalcones.Tyrosinases are an ubiquitous group of copper containing metalloenzymes that hydroxylate and oxidize phenolic molecules. In an application context the term ‘tyrosinase’ usually refers to ‘mushroom tyrosinase’ consisting of a mixture of isoenzymes and containing a number of enzymatic side-activities. We describe a protocol for the efficient heterologous production of tyrosinase 4 from  They are of central importance for the pigmentation in vertebrates as the reactions catalyzed by tyrosinase provide the starting material for melanin biosynthesis2. Tyrosinases catalyze the ortho-hydroxylation of monophenols to o-diphenols  as well as the subsequent two-electron oxidation to the respective o-quinones , which is coupled with the reduction of molecular oxygen to water4. The active site of tyrosinase is composed of two copper ions which are coordinated by three histidine side chains each5 forming a type III copper center6. Activation of molecular oxygen is affected by binding of dioxygen to the type III copper center in a characteristic ‘side-on’ bridging mode (µ-η2:η2)9. Hydroxylation and oxidation of one monophenol to the corresponding o-quinone requires one molecule of O2, while two o-diphenols can be oxidized to o-quinones per molecule of O2 consumed 18. As tyrosinases can oxidize both small phenolic molecules and phenolic moieties of larger molecules (e.g. proteins) they have found a plethora of biotechnological applications in e.g. organic synthesis, determination of phenolic analytes, bioremediation as well as in medicine, food processing and engineering of (bio)materials20.The products of the reactions catalyzed by tyrosinase – Agaricus bisporus (‘mushroom tyrosinase’)20, supposedly mainly due to its ready commercial availability21. However, it should be noted that the purification protocols used to prepare the available commercial preparations do usually not yield homogenous tyrosinase and these are therefore likely to contain one or more unspecified ‘extras’ like laccase, β-glucosidase, β-xylosidase, cellulase, chitinase and xylanase activities23. The purity of these preparations is further compromised by the fact that Agaricus bisporus possesses genes coding for six different tyrosinases (AbPPO1 - AbPPO6)25. Of those six genes, at least two (AbPPO3 and AbPPO4)27 are expressed in significant amounts in the fruiting bodies which serve as the source material for the commercial preparations of ‘mushroom tyrosinase’. Considering these limitations of the widely used tyrosinase preparations in terms of both purity and batch-to-batch variability, an alternative enzyme source that manages to provide a single isoform of tyrosinase in a pure form and at a constant quality would promote both basic (protein-)biochemical research and biotechnological applications of tyrosinase. Herein, we describe such a protocol, yielding pure AbPPO4 in its latent form as well as providing access to the mature, active form of the enzyme.Most of these application utilize tyrosinase isolated from fruiting bodies of the common white mushroom A. bisporus cDNA with primers enframing the ORF for AbPPO4 (AbPPO4_fwd and AbPPO4_rev) yielded two distinct bands at 1.8 kbp (expected size of the gene) and 1.4 kbp  contained the complete gene encoding amino acids M1 to F611, the smaller band (1401 bp) was missing 435 bp corresponding to 145 amino acids. The loss of these bases occurred in such a manner that the reading frame was conserved and amino acids A436 to A580 were deleted from the translation product. This deletion starts in the middle of exon number 6 and spans the last intron as well as more than two-thirds of the last exon in the gene for AbPPO4. On the protein level the missing amino acids are all located in the C-terminal domain and contain the CXXC-motif as well as approximately half of the putative trans-membrane helix27. Expression of this construct was attempted but for all the conditions tested the heterologous protein was found exclusively in the insoluble fraction and no tyrosinase activity could be detected in the cell lysate.The initial PCR on the p Figure . By clonAbPPO4-gene28 the cloned full-length gene contains 23 mutations among which four are non-silent  are predicted to be non-detrimental to the enzyme’s function by SIFT29. Expression of the full-length construct (up to F611) confirms this prediction as the heterologous enzyme was found to be fully active.With respect to the published sequence for the 27 was expressed as a fusion protein with an N-terminal tag, namely glutathione-S-transferase from Schistosoma japonicum30. Initial expression attempts employing induction by addition of IPTG yielded a big amount of heterologous protein which was however almost exclusively in an insoluble form. Cultivation at lower temperatures and the use of autoinduction medium31 did produce a very small fraction of fusion protein in soluble form so that enzymatic activity could be detected in the cell lysate after 3 days of reaction. The addition of 500 mM NaCl to the cultivation medium did decrease the specific growth rate of the production strain by 30% causing a marked increase in total cultivation time but did also increase the fraction of soluble protein by several orders of magnitude. This expression protocol as described in Methods yields around 200 mg of fusion protein per liter of medium after capture and purification by affinity chromatography employing the affinity of GST to glutathione immobilized on cross-linked agarose. Removal of the fusion partner by the specific protease HRV 3 C was usually quite efficient with yields between 80% and 100% corresponding to approximately 110 to 140 mg of latent tyrosinase per liter of expression culture. The preparations were still active after 1 year of storage at 4 °C in the used 10 mM HEPES pH 7.5 buffer. For long-term storage freezing in liquid nitrogen32 is recommended as the preparations tend to darken after a few months of storage. This darkening, however, does not cause the loss of enzymatic activity.The latent form of the tyrosinase (up to T565)AbPPO4 in heterologous expression, the construct encoding only the main domain of the tyrosinase (up to S383)27 did not exhibit any tyrosinase activity.While the full-length construct did behave almost identical as the latent version of Proteolytic activation of the latent tyrosinase was tested with trypsin and proteinase K and both proteases induced tyrosinase activity in the latent enzyme. The activation by proteinase K was more efficient and did yield an essentially pure preparation of active tyrosinase while even after prolonged incubation with trypsin a second species of approximately 52 kDa remained in the reaction mixture Figure . The digAbPPO4 than the anionic sodium dodecyl sulfate (SDS). Maximal activation was achieved by applying 0.1 mM of CPC and reached a level which was 25% higher than that at the optimal SDS-concentration of 0.6 mM  was more effective in activating latent see Fig. . The act27 but otherwise intact protein . Since positive electrospray ionisation of free cysteine has been shown to generate mainly protonated cystine (which was attributed to oxidation processes occurring during positive mode electrospray)34 a similar mechanism may also be observed for the pseudo molecular ions of AbPPO4. The mass spectra of the activated tyrosinase indicated the presence of two protein variants with determined masses of 44181.5 ± 0.51 Da and 44449.6 ± 0.48 Da, respectively. These masses indicate proteolytic cleavage after lysine 382  and additionally for most of the tyrosinase molecules the removal of the first three N-terminal amino acids (Gly-Pro-Leu) from the vector-derived region .ESI-MS of the acidified (causes the loss of copper)see Fig.  yielded see Fig.  from thea = 287.30 Å, b = 52.09 Å, c = 152.66 Å, α = 90.00°, β = 98.03° and γ = 90.00°. The obtained crystals diffracted to a resolution of 3.25 Å (for further statistics see Table 33 and did only form in the presence of sodium hexatungstotellurate(VI) 36, the recombinant enzyme formed crystals containing exclusively the latent tyrosinase. Inspection of the electron density gave no indication for any deviation from the sequence shown in Fig. Latent tyrosinase crystallized in the monoclinic space group C 1 2 1 with 4 chains in the asymmetric unit giving rise to a unit cell of β-atoms. In the crystal containing TEW the interactions mediated by TEW are crucial for the stacking of the monomer chains while in the crystal of recombinant latent AbPPO4 this motif does not play a significant role for the formation of the lattice with the closest interchain contact being longer than 6 Å. The missing contribution from these strong interactions may be one decisive factor limiting the attainable crystallographic resolution37 which is 0.49 Å worse than in the crystal structure obtained using TEW.The amino acid side chains which form hydrogen bonds with the TEW polyoxyanion in the crystal of the enzyme purified from the natural source (HKKE starting at H116) are found in equivalent positions in the crystal of the recombinant enzyme but the lysine side chains are considerably more flexible as indicated by the lack of electron density beyond the CL-tyrosine was found to be 6.8 40.The optimal pH for the enzymatic conversion of 8 Figure . Enzymatase Fig. . The catase Fig. . Of the AbPPO4 was also tested with and does accept tyrosol, chlorogenic acid, p-coumaric acid, 4-tert-butlycatechol, octopamine, 4-methylcatechol, resorcinol, hydroquinone, protocatechuic acid, 3,4-dihydroxyphenylacetic acid, pyrogallol and 3-methoxyphenol as well as the flavonols fisetin, quercetin, its glycoside rutin, the flavanone naringenin and the chalcons isoliquiritigenin and butein .Besides the substrates chosen for kinetic measurements AbPPO4 discriminates between enantiomers of tyrosine showing pronounced differences in the rate of the tyrosinase reaction. For tyrosine 1 mM of the L-enantiomer is converted at a rate of 1.22 ± 0.0073 U mg−1, which is 2.58 ± 0.020 times faster than the rate on D-tyrosine. A slight increase in enantioselectivity is seen for the methyl ester of tyrosine for which the respective value is 14.3 ± 0.11 U mg−1 for L-tyrosine methyl ester representing a ratio of 3.70 ± 0.050 relative to the rate for the D-enantiomer.Activated 41 but for eukaryotic tyrosinases a yield of 4 to 6 mg per liter of culture is already considered large-scale42 and the same value was also reported for a related plant enzyme43. Expression strategies targeting fungal tyrosinases did usually rely on fungal hosts for the expression of soluble and active tyrosinase46. Substantial expression yields per liter of culture were reported for the secreted TYR2 from Trichoderma reesei overexpressed homologously (1 g l−1 in batch fermentation)45 or in Komagataella pastoris (24 mg l−1)46 and for a tyrosinase from Pycnoporus sanguineus produced heterologously in Aspergillus niger (20 mg l−1)44. As fungi do possess the necessary molecular tools to activate latent tyrosinases all the isolated enzymes were in their active form47. Bacterial expression of fungal tyrosinase does provide access to latent tyrosinases but was hampered by insufficient solubility of the expressed proteins which were also prone to enzymatic inactivity28. Enzymatically functional protyrosinase from Pholiota microspora was expressed in Escherichia coli48 but no value for the yield was reported. Recently, protyrosinase from Polyporus arcularius was produced in the same host with a yield of 54 mg latent tyrosinase per liter of culture medium49. Here, the latent form of AbPPO4 is produced by E. coli at a yield of 110 to 140 mg per liter of culture. The conversion of latent tyrosinase into the enzymatically active form is coupled to the proteolytic removal of the C-terminal domain which shields the active site of the tyrosinase50. The causal agents for this key-step in the maturation of fungal tyrosinases are still elusive and for only four fungal tyrosinases the exact location of this crucial event is known. Neurospora crassa TYR is activated in vivo by cleavage after F40851, active TYR2 of Trichoderma reesei extends up to G40045, TYR1 from Pholiota nameko is cleaved after F38752 and the C-terminal residue of active AbPPO4 is S38327. All those cleavage sites are found 31 or 30 (T. reesei TYR2) amino acids after the tyrosine motif (Y-X-Y/F or Y/F-X-Y) which is found close to the end of the central domain in all tyrosinases53. Preceding the cleavage site by 4 amino acids, the YG-motif, which is conserved in fungal tyrosinases20, is present. The activated AbPPO4 presented herein was cleaved by proteinase K after K382, which is only one amino acid away from the in vivo activation site S38327. This activated tyrosinase should therefore be an excellent model for the native enzyme. Digestion with proteinase K has also been used as a purification method for tyrosinase from mice54 and apple55. This stability against proteinase K digestion of tyrosinases from three different kingdoms of life suggests resistance against proteolysis by serine proteases as a general feature of tyrosinases.For bacterial tyrosinases productivities in the gram per liter range have been demonstrated with the application of an optimized fed-batch strategy56 or detergents like SDS57. Employing such a system, the enzymatic activity may be kept dormant in a preparation for a prolonged period of time until it is needed at which point it may be induced by the simple addition of a detergent. Latent AbPPO4 was activated by both the cationic detergent CPC and the anionic detergent SDS 58 and for MelB from Aspergillus oryzae produced in E. coli 59, making these enzymes both more specific towards L-tyrosine as well as faster on this substrate than activated AbPPO4. For TYR1 from P. nameko the kinetic parameters on tyrosine could not be determined due to insufficient solubility of the substrate52. For L-DOPA a slightly higher number of values (enzyme name: Km in µM | kcat in s−1) are reported for the recombinant tyrosinases from A. bisporus (AbPPO2: 1.22 | 141)58, P. nameko (TYR1: 1930 | 478)52, P. arcularius (Photo-regulated tyrosinase: 1040 | 223)49 and T. reesei (TYR2: 3000 | 22)45. In comparison to those enzymes activated AbPPO4 is less specific for L-DOPA and much faster on this substrate.The kinetic characterization of activated In conclusion, a protocol for the production of mushroom tyrosinase was established which is able to produce both latent and active tyrosinase in a pure form. Using this protocol it is possible to provide quantities of mushroom tyrosinase sufficient for providing even larger research projects with a defined tyrosinase preparation that does not suffer from the isoenzyme mixture and the side-activities frequently present in commercial preparations of tyrosinase isolated from mushrooms.L- and D-tyrosine were synthesized at the Department of Biophysical Chemistry, University of Vienna and are characterized by 1H-NMR and ESI-MS  or Carl Roth  and were at least of analytical grade. The methyl esters of 60 and are described in detail in the SI.RNA extraction and cDNA synthesis have been performed according to standard proceduresAbPPO4 was cloned out of frame into the expression vector pGEX-6P-1  and was brought in frame by PCR-based mutagenesis (for details see SI).The gene encoding 27, the cloned gene was adjusted accordingly. Towards that end the base triplets encoding the respective amino acids were deleted from the gene using the Q5® Site-Directed Mutagenesis Kit (NEB). Removal of the sequence corresponding to amino acids A566 to F611 was accomplished with the two primers lAbPPO4_fwd and lAbPPO4_rev, while E384 to F611 was removed using lAbPPO4_fwd and aAbPPO4_rev resulting in the bases encoding just the main domain of the tyrosinase33.As the enzyme isolated from the natural source did not contain the last 46 amino acids that are encoded by its geneAbPPO4 was expressed in E. coli BL21(DE3) in auto-inducing medium, namely ZYM-5052 without the trace element solution61 but with an additional 500 mM of NaCl. Cultures were grown at 20 °C in shaking flasks in media containing 100 mg l−1 Na-Ampicillin for 20 h. Then, 0.5 mM copper sulfate was added and expression was continued for 20 more hours (for details see SI).−1 NaCl in ddH2O, repelleted by centrifugation (8 min @ 3000 × g and 4 °C) and resuspended in lysis buffer  and 100 mg l−1 hen egg white lysozyme) at 100 g of wet cells per l. Cells were disrupted by 2 passages through a french press62 at a cell pressure of 850 bar. The samples were cooled on ice in between the passages through the french press and fresh PMSF was added to a final concentration of 2 mM in two portions at the end of each passage. The lysate was cleared by centrifugation (15 min @ 30800 × g and 4 °C), filtered (0.45 µm pore size PES membrane) and applied to a 5 ml GSTrap FF column at a flow rate of 0.5 ml min−1. The column was kept at 4 °C and 50 mM Tris-HCl, 150 mM NaCl pH 8.0 (@ 4 °C) was used as the mobile phase for elution of unbound material while the same solution with 20 mM reduced glutathione served as the elution buffer. The eluted fractions containing the fusion protein were concentrated by ultrafiltration  which was also applied for buffer exchange.The pelleted cells were washed with 10% of the original culture volume of 9 g lAbPPO4 fusion protein and the proteolysis reaction was allowed to proceed for at least 18 h at 4 °C in the running buffer of the affinity chromatography supplemented with 1 mM DTT in order to preserve the activity of the cysteine protease HRV 3C.GST was cleaved from the fusion protein by the action of picornain 3C  which was applied as a fusion-protein with GST (production protocol in the SI). 1 µg of protease was applied per 150 µg of GST-−1 and stored at 4 °C until use.After enzymatic cleavage, the tyrosinase was separated from the fusion partner as well as the protease by a second passage through the affinity column using identical conditions as for the first chromatographic step. The column flow-through containing the protein of interest was concentrated by ultrafiltration  and its buffer was exchanged to 10 mM HEPES pH 7.5 (@ 4 °C). This preparation was diluted to a concentration of 20 g lL-tyrosine in 50 mM sodium citrate buffer pH 6.8 at 25 °C. For activation of the latent enzyme 2 mM SDS were included in the assay mixture27. The monitored species was dopachrome at 475 nm (ε475 = 3600 M−1 cm−1)63, volumetric enzymatic activities were calculated from the linear part of the absorption-time curves (after the lag-phase but before the subsequent reactions towards melanin contribute significantly). One unit of enzymatic activity (U) was defined as the amount of enzyme that catalyzes the conversion of 1 µmol of substrate per minute of reaction.Tyrosinase activity was routinely assayed on 1 mM ® 500, 30 kDa molecular weight cut-off). For introduction into the nanoESI-QTOF mass spectrometer  by a syringe pump  @ 3 µl min−1 the protein solutions were diluted to approximately 1 µM in an aqueous solution containing 2% (v/v) acetonitrile and 1‰ (v/v) formic acid.For ESI-MS, which was done at the Mass Spectrometry Centre at the University of Vienna, the buffer of the protein solutions was exchanged to 5 mM ammonium hydrogen carbonate pH 7.8 by repeated ultrafiltration (VivaspinAbPPO4. Since the o-quinones generated by the enzymatic action of tyrosinase do not give rise to a stable and soluble product they were trapped by the potent nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH). MBTH couples to o-quinones via its amino group generating reasonably stable adducts that remain soluble and are easily detected photometrically64. Absorption curves and spectra were recorded on a Shimadzu UV-1800 spectrophotometer applying 1 cm cuvettes which were kept at 25 °C by a Julabo F25 MH thermostat in a circulating water-bath. Kinetic measurements were done in a total volume of 1 ml containing 50 mM sodium citrate buffer pH 6.8, 5 mM MBTH, 2% (v/v) N,N-dimethylformamide and different concentrations of the substrates to be tested as well as AbPPO4 (0.23 to 46 nM).A spectrophotometric assay detecting the appearance of the product in the reaction solution was applied for the determination of the kinetic parameters of m and kcat, the two parameters of the Michaelis-Menten model65, were calculated from the steady-state rate of product formation for the different substrate concentrations tested. Measurements were performed in triplicate and the reciprocals of the variances of the observed slopes were used as weights for the nonlinear regression applying the Levenberg-Marquardt algorithm66 as implemented in the program Dataplot (version 11/2010). Initial estimates for the two free parameters were generated by applying the Hanes-Woolf linearization of the Michaelis-Menten equation67.Molar absorption coefficients were determined from rapid oxidation of small concentrations of substrate under standard assay conditions applying tyrosinase concentrations in the µM range. KAbPPO4 was converted into its active form by treatment with proteinase K. The protease was used at a ratio of 1:10  in a reaction buffer containing 50 mM Tris-HCl pH 8 @ 25 °C, 100 mM sodium ascorbate and 20 g l−1 of latent AbPPO4 for a total reaction time of 90 min. The reaction was stopped by addition of 2 mM PMSF after which the solution was concentrated to less than 70 µl by ultrafiltration  and applied onto a size exclusion column  equilibrated with 50 mM sodium citrate pH 6.8 and run with the same buffer at 4 °C and with a flow rate of 0.5 ml min−1. The eluted fractions possessing tyrosinase activity were pooled and concentrated by ultrafiltration .Latent AbPPO4 crystals were refined based on an initial hit obtained with sodium cacodylate and PEG 4000 in a hanging drop vapour diffusion setup. Single crystals suitable for crystallography grew over the course of a few days in hanging drops initially made up of 1 µl of a 10 g l−1 solution of latent AbPPO4 in 10 mM HEPES pH 7.5 mixed with 1 µl of reservoir solution containing 50 mM sodium cacodylate pH 5.8 and 13% (w/w) PEG 4000 which was equilibrated via vapour diffusion with 1 ml of reservoir solution at 293 K. Crystals were harvested using Kapton® loops , soaked with cryo-protectant (50 mM sodium cacodylate pH 5.8 and 40% (w/w) PEG 4000) and plunged into liquid nitrogen where they remained until the diffraction experiment.Conditions for the growth of 68. Final model quality was evaluated by the MolProbity server69 and the model has been deposited in the PDB under entry number 5M6B.Data were collected at beamline ID-23 at the European Synchrotron Radiation Facility  at 100 K applying a wavelength of 0.873 Å (14.2 keV) and a PILATUS2 3 M detector. Data reduction (for details see SI) was carried out using XDSSI