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Low-temperature CO oxidation on multicomponent gold based catalysts

In this work the development of gold catalysts, essentially based on γ-alumina with small superficial fraction of Ce-Fe mixed oxides as support for the low temperature CO oxidation is proposed. Characterization results obtained by means of TEM, OSC, XPS, UV-Vis spectroscopy and H2-TPR are employed to correlate the activity data with the catalysts composition. The bare γ-alumina supported gold catalyst demonstrates the poorest activity within the series. The addition of CeO2 or FeOX improves the catalytic performance, especially observed for the CeO2-FeOx mixed oxide doped samples. This enhanced CO oxidation activity was related to the Ce-Fe interaction producing materials with promoted redox properties and therefore oxidation activity.

INTRODUCTION
Gold has been long discarded from catalytic applications due to the inert nature of massive gold. However, it is now about 30 years since the original breakthroughs of Hutchings (1985) and Haruta et al. (1987) demonstrating that gold can be an excellent catalyst when supported on metal oxides in a highly dispersed state. Since this discovery, intensive research has been devoted to the determination of the reactions types that can be efficiently catalyzed by gold nanoparticles, to the increase of the stability of the gold nanoparticles avoiding their sintering, to understand the role of the support in the catalytic activity and to demonstrate the similarities and distinctive properties of gold catalysis with respect to other noble metals (Corma and García, 2008) etc.
Within the properties that make gold unique as a catalyst, the ability of gold to catalyze reactions at ambient and sub-ambient temperature is one of the most important. The later opens up totally new application opportunities creating an entirely new field of low temperature catalysis with characteristics not previously found (Corti et al., 2007). For example gold catalysts are commonly deactivated by nanoparticles agglomeration, however, this sintering phenomena does not occur at sub ambient and room temperature. Furthermore, the poisoning of gold catalysts via carbonate formation due to the interaction of the support with atmospheric CO 2 can be avoided by storing the catalysts in a closed container at room temperature (Corti et al., 2007). The catalyst can preserve its activity under these conditions for 2 years (Cortie et al., 2006).
The oxidation of CO over gold catalysts is one of the most studied reactions in the last 20 years due to the high activity of gold catalysts for this reaction at ambient and sub-ambient temperature. Nowadays, a number of possible applications for catalysts able to perform CO oxidation at ambient temperature (as for example gold based catalysts) appear. Within the most extended application it should be mention: carbon dioxide lasers, gas sensors, respirators for protecting firefighters and miners from the CO poisoning, air-cleaning devices and many environmental issues.
The activity of gold-based catalysts toward CO oxidation is known to be sensitive to (1) the preparation method, (2) the nature of the supports, (3) the size of the gold particles, (4) the pre-treatment conditions, and (5) to the gold-support interactions (Trim, 2005). In particular, the choice of the support is of crucial relevance. Currently gold supported on a reducible transition metal oxide exhibit a significantly enhanced activity for CO oxidation compared to gold supported on non-reducible materials due to their ability to supply reactive oxygen. Au supported on CeO 2 and to lower extent on CeO 2 -MO x mixed oxides have been broadly studied (Gluhoi et al., 2003;Laguna et al., 2010). The relevance of these cerium based supports is mainly based on their high oxygen storage capacity (OSC), which is directly correlated to the creation, stabilization and diffusion of oxygen vacancies, especially on the oxide surface, due to the reversible redox behavior of the Ce 4+ /Ce 3+ couple (Veccheti et al., 2011).
The formation of oxygen vacancies in a CeO 2 -MO x supportis directly related to the formation of Ce-M solid solution. The dopant ions with electronegativity and ionic radius close to those of cerium cation are consider the most appropriate modifiers of structural and chemical properties of ceria. The presence of these dopants can provoke structural distortions such as ceria lattice contraction thus favoring the formation of oxygen vacancies. Among the transition metal cations, Fe 3+ has been successfully employed as a CeO 2 doping agent for CO oxidation reactions. The best oxidation activity usually is reported when Ce-Fe solid solution is formed and attributed to the increase of the ceria oxygen vacancies population (Laguna et al., 2011). Nevertheless, in some cases, above a certain amount, iron is not completely incorporated to the ceria network but segregates and it can play a role of an electronic modifier allowing a better dispersion of the added noble metal (Reddy et al., 2010).
Not at last place, the recently emerged new trend in the scientific community have to be taken into consideration, namely the need of lowering the rare earth metal compounds use and dependence, which leads us to consider the probability to substitute the bulk rare oxide support with a small superficial layer of it deposited on an "inert" oxide, such as alumina.
For all discussed above, the main goal of this work is the development of an efficient series of gold catalysts for low temperature CO oxidation using CeO 2 -FeO x mixed oxides supported on Al 2 O 3 as a support. The evaluation of the catalytic performance induced by the presence of certain amounts of iron in the catalyst formulation is another objective of this paper since the addition of iron accounts for the high performance of these systems and their viability in any possible environmental application.

Synthesis of the supports
In a typical preparation no matter ternary or quaternary systems, the desired amount of Ce(NO 3 ) 3 × 6H 2 O and Fe(NO 3 ) 3 × 9H 2 O (Aldrich) were impregnated on γ-alumina powder (Sasol). The impregnation was carried out in 50 mL of ethanol, evaporated till obtaining of a dry solid in rotary vapor at reduced pressure and temperature of 50 • C. The obtained solid was treated with NH 3 (Aldrich) solution 10 mol · L −1 during 30 min in order to assure the full conversion of the nitrates to hydroxides precursors. The support was then filtered, dried and calcined at 500 • C for 4 h.
The initial precursor quantities (CeO 2 or FeO x ) are calculated to be 15 wt.% of the final solid with FeO x to CeO 2 promotion varying from 0 to 3 wt.%. The samples are labeled as CeFeX/Al, where X indicates the theoretical FeO x (always calculated as Fe 2 O 3 ) loading.

Gold deposition
The gold deposition was performed by the direct anionic exchange method (DAE) (Ivanova et al., 2004). Aqueous solutions of the gold precursor HAuCl 4 (Johnson Matthey) 2 × 10 −4 M and support sieved in between 100 and 200 μm were used in order to obtain a final Au loading of 2 wt.%. The solution was heated to 70 • C and aged 20 min. After that the solution was cooled down to 40 • C and 50 mL of NH 3 (30% Aldrich) ware added. The slurry was then filtered, washed with water, dried in an oven at 100 • C overnight and calcined in air at 350 • C for 4 h.
Caution/safety note. The contact of ammonia with gold solution can result in the formation of gold ammonia complexes ("fulminating gold") which are explosive. Using this washing procedure may not be dangerous as the gold complexes are strongly attached to the support by DAE. Similar to the supports, in the adopted nomenclature the real gold loadings are omitted for simplification and the FeO x contents are expressed as the theoretical wt.% loading in the catalysts. For example, the Au/CeFe0.5/Al solid contains 2 wt.%. Au loading over 15 wt.% of Ce-Fe mixed oxide on Al 2 O 3 support in which the FeO x loading is 0.5 wt.% approximately.

CHARACTERIZATION TECHNIQUES
The chemical composition of the samples was determined by X-Ray microfluorescence spectrometry (XRMF) in an EDAX Eagle III spectrophotometer with a rhodium source of radiation.
High-Resolution Transmission Electron Microscopy (HRTEM) and High-Angle Annular Dark Field-Scanning Transmission Electron Microscopy (HAADF-STEM) images were recorded on a JEOL2010F instrument. The spatial resolution at Scherzer defocus conditions in HRTEM mode is 0.19 nm, while the HAADF-STEM studies were performed using an electron probe of 0.5 nm of diameter and a diffraction camera length of 10 cm. The average size of Au particles and its distributions were estimated by counting about 70 Au particles.
The Temperature Programmed Reduction (TPR) experiments were carried out in a conventional quartz reactor connected to a thermal conductivity detector (TCD). The reactive gas stream, 5% H 2 in Ar (Air Liquide) was passed through a 50 mg of sample with a flow rate of 50 ml.min −1 and the temperature rose at 10 • C · min −1 from room temperature to 900 • C. A molecular sieve 13× was used to retain the H 2 O produced during the reduction and/or CO 2 which could be desorbed from the solid surface.
For the Oxygen Storage Complete Capacity (OSCC) 100 mg of catalyst was loaded into a U-shaped quartz reactor and the temperature was raised in a He flow (50 mL/min) until 350 • C. Then, 10 O 2 pulses of 1 mL were injected every 2 min. After that, the sample is submitted to 10 CO pulses of 1 mL each (every 2 min). The sample is then degassed during 10 min in a He flow and subjected to a new series of oxidizing pulses (10 O 2 pulses) and then exposed to five alternating pulses (CO-O 2 -CO-O 2 -CO-O 2 ). The OSC is determined by the amount of CO 2 formed after the first CO pulse of the alternated ones. This method is based on the one presented by Duprez et al. (Kacimi et al., 1993;Royer and Duprez, 2011). The gas composition at the exit of the reactor was analyzed by a mass spectrometer PFEIFFER Vacuum PrismaPlus controlled by the program Quadera®.
The UV-Vis spectra were recorded on a Varian® spectrometer model Cary 100, equipped with an integrating sphere and using BaSO 4 as reference. All the spectra were collected in a diffuse reflectance mode and transformed to a magnitude proportional to the extinction coefficient through the Kubelka-Munk function F(α).
X-ray photoelectron spectroscopy (XPS) analyses were performed on a Leybold-Hereus LHS-10/20 photoelectron spectrometer equipped with multichannel analyzer EA200. The sample powders pressed in small stainless steel troughs of 4 mm diameter were placed on a ceramic carousel. The pressure in the analysis chamber was around 5 × 10 −9 Torr. The analyzed area was ∼1.4 mm 2 and the pass energy was set at 150 eV. Spectra were obtained by using Mg Kα 1,2 radiation (1253.6 eV). The binding energies were calculated with respect to the C(C, H) component of the C 1 s peak fixed at 284.8 eV. Data treatment was performed with the CasaXPS program (Casa Software Ltd., UK). The atomic fractions were calculated using peak areas normalized on the basis of acquisition parameters and sensitivity factors provided by the manufacturer.

CATALYTIC ACTIVITY SET-UP
The activity measurements were carried out in a U-shape glass flow reactor at atmospheric pressure. The catalysts were pretreated in a 30 mL min −1 activation flow of 21% O 2 balanced in He (from room temperature to 350 • C, 5 • C min −1 ). After the activation, a reactive flow 3.4% CO (Air Liquide, 99.997%), and 21% O 2 (Air Liquide, 99.999%) balanced by helium, was passed through the reactor at room temperature. The gas total flow was 42 mL min −1 and the quantitative analysis was carried out with a Blazers OmnistarBentchop mass spectrometer. The catalysts were tested in the reaction flow at room temperature until reached the steady state. Then, the systems were heated to 350 • C at 5 • C · min −1 .
When necessary, the same reaction was carried out at temperatures below 0 • C. The reactor, loaded with a fresh and activated sample, was immersed into a cooling bath composed by liquid N 2 and acetone. Once the temperature stabilized, the reactive flow described above was flushed through the reactor. The temperature was increased slowly to the room temperature and the quantitative analysis of the reactives and products was carried out on a Blazers OmnistarBentchop mass spectrometer calibrated using gas mixtures of CO and CO 2 in helium.

CHEMICAL COMPOSITION: BULK AND SURFACE
The chemical composition of the prepared solids is presented in Table 1. Bulk composition of the supports seems to be close to the intended values. All the Ce-Fe mixed systems presented similar amounts of cerium oxide (∼15 wt.%) and iron oxide increased from 0.62 wt.% in the CeFe0.5 sample till 2.42 wt.% in the CeFe3 sample. Gold deposition provokes a slight decrease in both cerium and iron oxide loadings. This phenomena could be related to the highly basic media (NH 3 , 30% v/v) employed during the gold deposition. It seems that a part of the support oxides is dissolved during the process affecting the catalysts composition. Regarding gold loadings, the experimental values neighboring 2 wt.% are obtained, excepting the Au/Al 2 O 3 catalyst for which a 40% of gold loss is observed. It was reported elsewhere  that always 30-32% of gold loss is observed when the DAE method assisted by NH 3 is applied for the preparation of Au supported on alumina.
For the surface composition the atomic ratios of all present components from the XPS analysis have been used to calculate the corresponding mass ratios for sake of comparison in between the two methods of characterization. However, the Ce/Fe and Au/Ce atomic ratios were also included in the Table 1. Analyzing the results several points should be considered; the surface layer composition and its relation to the bulk composition. Concerning the surface layer composition, the XPS results show that approximately 20-25 wt.% of the mixed oxide is presented on the catalysts surface on which the gold particles have been deposited. Nevertheless, the surface and bulk compositions significantly differ. An enrichment of cerium and iron oxides in the solids surface is observed compared to the bulk composition, confirming the successful deposition of the Ce-Fe mixed oxide on the alumina surface. All the solids present between 19 and 25 wt.% of superficial ceria loading except the Au/CeFe1.5/Al sample where the cerium oxide content on the surface was remarkably higher (34.5 wt.%). The increase of the iron oxide layer is proportional to that of ceria one excepting the Au/Fe0.5/Al sample, where the iron oxide surface concentration is higher than expected, as confirmed also by the lowest Ce/Fe atomic ratio. As for the gold compositions, a general decrease of gold amount was obtained compared to the bulk values. As a detail, this decrease on the available gold on the surface seems to be less important within the Ce-Fe mixed oxides. The formation of the Ce/Fe solid solution within this series of samples have been already discussed in details in reference (Reina et al., 2013) and confirmed for the majority of the samples, being the CeFe2/Al the limiting value above which the FeOx phase segregation occurs. Considering this solid solution formation an increase of the amount of ceria oxygen vacancies is expected with the increase of the extent of solid solution formation. As proposed by Laguna et al. (2010) these structural defects may act as nucleation centers for gold deposition thus improving the gold dispersion linearly with the concentration of defects. The later agrees with the fact that Au/CeFe1.5/Al is the system that admits the higher amount of gold on the surface since this system presents the highest concentration of defects on its surface and a strong gold-support contact (Reina et al., 2013). Concerning the textural properties, the catalysts are mesoporous materials with specific surface areas of around 200 m 2 · g −1 governed by the presence of the primary γ-Al 2 O 3 support. A slight increase of the specific surface area of the final solids after gold deposition is observed. This effect has been reported earlier (Konya et al., 2003) and attributed to the increase of the pore volume caused by the presence of gold nanoparticles inside the pore structure.

X-RAY PHOTOELECTRON SPECTROSCOPY SPECTRA (XPS)
Selected XPS spectra of the prepared supports are presented in Figure 1. Ce3d spectra are shown in Figure 1A. Six peaks corresponding to three pairs of spin-orbit doublets can be identified. The complex shape of the cerium 3d spectra arises from the multiplet effect such as hybridization of Ce 4f orbitals with O 2p levels and final state (Le Normand et al., 1988) and is characteristic of CeO 2 . However, the presence of Ce 3+ must not be discarded. Slight but important differences can be extracted from the spectra, if the attention is focused on Ce 3+ . Currently, the main Ce 3+ peaks appear at about 885 and 905 eV. In our mixed systems, especially from CeFe1/Al to CeFe2/Al, these peaks can be noticed overlapped with the Ce 4+ ones. The presence of Ce 3+ could be linked to the existence of oxygen vacancies which appear along the support series due to the formation of Ce-Fe solid solution.
The presence of Ce 3+ in the CeFe3/Al sample is less notorious according to the shape of the spectra. This system marks the limit where Ce-Fe solid solution no longer occurs in good agreement with the discussion published in reference Reina et al. (2013). Iron oxide, Fe 2p spectra is presented in Figure 1B. Fe/Al exhibited two XPS peaks attributed to Fe 2p 3/2 and Fe 2p 1/2. The Fe 2p 3/2 is narrower and stronger and with greater area than Fe 2p 1/2 caused by the spin-orbit (j-j) coupling effect; Fe 2p 3/2 has degeneracy of four states whilst Fe 2p 1/2 has only two states. The obtained binding energies of Fe 2p 3/2 and Fe 2p 1/2 for the Fe/Al sample are 711.4 ± 0.2 and 724.96 ± 0.2 eV, respectively, in good agreement with the data obtained by Yamashita and Hayes (2008) for a Fe 2 O 3 standard. In addition, the Fe 2p 3/2 peak in Fe 2 O 3 must have a satellite located at ∼8 eV higher than the main Fe 2p 3/2 peak. In our sample, this satellite can be intended at 719.2 ± 0.2 eV. The small amount of iron in the rest of the solids limits the resolution of the spectra, however, for the CeFe3/Al and CeFe2/Al the same profile that the one observed for Fe/Al was obtained with a slight shift that may account to the Ce-Fe interaction. XPS spectra of the prepared gold catalysts are presented in Figure 2. Not remarkable changes in the Ce 3d spectra were found after gold deposition (Figure 2A) indicating that there is no change in the electronic density of ceria atoms which remains mainly as Ce 4+ . Nevertheless, it should be pointed out that Au/CeFe1.5/Al sample did modify its profile in some extension toward a better defined Ce 4+ which agrees with the increased of the superficial CeO 2 presented in Table 1. The later could also be related to the fact that gold tend to nucleate on the ceria oxygen vacancies (associated with Ce 3+ ) thus covering the small portion of Ce 3+ on the catalyst surface. As for the Fe 2p spectra (Figure 2B), the inclusion of gold did not significantly modify the profiles, except from the Au/CeFe0.5/Al sample where gold deposition seems to unearth Fe but in a different oxidation state since the peaks position are far from the rest of the series. In this sample the binding energies of Fe 2p 3/2 and Fe 2p 1/2 are 706.3 ± 0.2 and 719.6 ± 0.2 eV, respectively, close to the energies obtained for a Fe 0.94 O standard in reference Yamashita and Hayes (2008) confirming the change of the iron oxidation state in this particular case, which also results in some incertitude in the calculation of the Ce/Fe atomic ratio. Finally Au 4f spectra are reported in Figure 2C. In this region each gold species shows two peaks Au 4f 7/2 at 83.4 ± 0.2 eV and Au 4f 5/2 at 87.2 ± 0.2 eV characteristic of metallic gold (Haruta et al., 1993). A weak shift on the peak positions was observed. This shift may indicate differences on the Au-Ce interaction as proposed by Rodriguez et al. (Ma et al., 2007) and in good agreement with the properties derived from the support composition in terms of Ce-Fe interaction and the presence of ceria electronic defects that could alter gold electronic density. Each peak has been successfully fitted with a Guassian-Lorenztian curve with one component supporting the metallic character of gold in this series of catalysts. However, this interaction is not the only factor to consider for explaining the shift since a change in the gold particle size should be also taken into account. Furthermore, the assumption that gold is present on its metallic form is quite reasonable considering that samples have been calcined at 350 • C. It was in fact published that at calcination temperatures higher than 300 • C gold is present just as Au 0 (Park and Lee, 1999).

TEM
Selected TEM images of the gold catalysts are shown in Figure 3.
The white spots correspond to the presence of heavier elements such as Ce, Ce-Fe, and/or Au supported on alumina. No clear differences in dispersion, size and shape are evidenced among the catalysts. However, we must take into account that the high atomic weight of cerium atoms in the samples makes the detection of gold particles difficult due to the low mass and diffraction contrast. Although, it should be pointed out that in all samples the gold particles are regularly distributed on the whole support and the particle size ranges from 3 to 5 nm as reported in (Reina et al., 2013). As for example, TEM measurements for Au/Ce/Al found 3.8 ± 0.5 nm gold average size and 4 ± 0.2 nm for the Au/CeFe2/Al sample which make us to consider 4 nm average gold particle size for all calculation, e.g., dispersion, reaction rate etc. It should be pointed, however, that even if this slight differences do not influence the normalization of the catalytic activity can significantly contribute to the shift in the XPS gold peaks as pointed above.

UV-VIS SPECTROSCOPY
UV-vis spectra of the prepared gold catalysts are presented in features. This band is due to the charge transfer from 2p valence band of O 2− to 4f band of Ce 4+ (Centeno et al., 2002). As a relevant phenomena, a widening and shifting of the ceria absorption edge is observed evidencing Ce-Fe interaction. It should be mentioned that the shift of the ceria absorption edge occurs till 2 wt.% of Fe content since the Au/CeFe3/Al sample did not show this band. A weaker Ce-Fe interaction could be expected for this solid. In addition this sample and the Au/Fe/Al sample exhibited a broad absorption at 393 nm. This band was attributed by Reddy and co-workers to O → Fe 3+ ligand-to-metal charge transfer of isolated Fe ions in tetrahedral and octahedral coordination (Reddy et al., 2010). Furthermore, a broad absorption band was found in the range 500-570 nm for most of the samples. This contribution is assigned to the well-known plasmon resonance of gold nanoparticles. For the Au/Fe/Al sample the gold contribution is masked by the extended absorption of iron oxide in the visible region ascribed to d-d transitions 6 A 1g → 4 T 1g and 6 A 1g → 4 T 2g in α-Fe 2 O 3 (Reddy et al., 2010). This contribution could be envisaged also for the Au/CeFe3/Al system for which the higher iron concentration suggests oxide segregation (Reina et al., 2013).

TPR
Redox properties are of especial relevance when oxidation reactions are considered. An approximation to the redox behavior of the gold catalysts was obtained by the TPR-H 2 experiments. Figure 4 shows the TPR-H 2 profiles of the prepared solids.
The Fe 2 O 3 -Al 2 O 3 support ( Figure 5A) presents 3 overlapping zones of reduction corresponding to the two steps of the reduction from Fe 3+ to Fe 2+ and to Fe 0 of the surface and bulk iron oxide. Wimmers et al. (1986) studied the reduction of Fe 2 O 3 and proposed a reduction in two steps Fe 2 O 3 → Fe 3 O 4 → Fe, with no formation of FeO. For the same oxide, other authors proposed a three steps reduction process considering FeO formation dealing with: Fe 2 O 3 →Fe 3 O 4 at about 400 • C, Fe 3 O 4 → FeO at about 600 • C and finally FeO → Fe at higher temperatures (Boccuzzi et al., 1999). The later is in good agreement with our profile. The CeO 2 -Al 2 O 3 ( Figure 5A) presents only one reduction zone at around 495 • C attributed to the Ce 4+ to Ce 3+ reduction of the superficial ceria. As for the Ce-Fe mixed supports ( Figure 5B) two zone of reduction can be clearly seen in the temperature range (300-600 • C). The two reduction peaks involve both iron oxide and cerium oxide reduction process that are not possible to separate. The main different was observed in the first reduction zone that becomes more intense and shifts to lower temperatures (except in the CeFe1.5 sample) with the iron load in the sample pointing the Ce-Fe synergetic effect. This peak could be due to the first reduction step of Fe 3+ species (Laguna et al., 2011). However, the simultaneous reduction of Ce 4+ and Fe 3+ cannot be discarded. Finally the CeFe3/Al presented a shoulder at around 700 • C as was observed in the Fe/Al sample reinforcing that iron segregation occurs in this solid as it was mentioned in UV-Vis data discussion.
Concerning to the gold catalysts (Figures 5A ternary system and 5B quaternary system), as expected the presence of gold decreases the temperature of reduction by facilitating the mobility of the H 2 molecule on the surface of the solid. It was reported earlier (Jacobs et al., 2004) a shift in the reduction peaks related to the surface oxygen of CeO 2 in the presence of gold at much lower temperatures Because of the later, Au/Ce/Al sample shows two reduction zones while the Ce/Al solid shows only one. The low temperature reduction (centered at 150 • C) zone is assigned to the noble metal promoted ceria surface reduction and the high temperature reduction process is ascribed to the ceria bulk reduction. An overlapping of the latest reduction steps in the Au/Fe/Al system was observed. In the quaternary Au/CeFe/Al systems, the addition of gold provoked the apparition of three peaks instead of the two original ones. In fact the last reduction peak must be attributed once again to the bulk ceria reduction. Currently the amount of consumed hydrogen was higher for the Ce-Fe mixed systems pointing that this quaternary systems possess enhanced redox properties which generally leads to a better catalytic behavior in CO oxidation reactions.

OSCC AND OSC
For further understanding of the promoter effect of iron addition to the ceria redox properties, OSCC and OSC measurements of Ce/Al and CeFe2/Al samples and their corresponding gold analogs were carried out. The OSCC provides information about the maximum reducibility of the samples while the OSC informs about the most reactive and most available oxygen atoms. The OSCC measurements expressed as the sum of the μmolCO 2 formed for all the CO pulses contacted with the solid, as a function of the temperature is presented in Figure 6. As a general trend the increase of the temperature increases the OSCC value more pronounced for the supports than for the catalysts. The Fe containing samples exhibited superior OSCC in the whole studied temperature range. This result indicates that the inclusion of iron in the catalyst formula enhanced the oxygen mobility.
In order to find out the number of oxygen layers involved in the process the measurements of the OSC was carried out. For the OSC theoretical calculations, the number of surface oxygen atoms and the BET area of each sample ( Table 1) were considered. More precisely it was considered that (1) only oxygen atoms bonded to the cerium ones participate in the oxygen storage process; (2) the surface is considered homogeneous (3) only one of the four oxygen atoms is involved in the storage (CeO 2 → Ce 2 O 3 + "O") (4) null gold metal contribution to the reduction, e.g., the gold metal could not be reoxidized after the calcinations of the samples.
The results are presented in Table 2. For the supports, similar to the OSCC higher the temperature higher the number of oxygen layers involved in the reduction of the surface-effect enhanced by the presence of iron. However, almost no accessible oxygen for the immediate exchange was found at 150 • C. The addition of gold changes dramatically the oxygen mobility in the whole temperature range, especially at the lowest temperature, where the oxygen availability increases around 30 times. Nevertheless, for the rest of the temperatures a maximum seems to be reached indicating that above 250 • C the number of oxygen layers remains constant. Figure 7A shows the light-off CO oxidation curves for the prepared supports. All the ternary systems based on Ce-Fe mixed oxide supported on Al 2 O 3 exhibited a superior catalytic activity compared to the binary systems Fe/Al or Ce/Al evidencing the beneficial effect of introducing iron oxide to ceria. It has been recently reported that a noticeable increase of the catalytic performance in CO oxidation for a ceria-alumina commercial support can be achieved by employing iron oxide as a doping agent (Reina et al., 2012). It was demonstrated that the incorporation of iron species into ceria lattice creates structural sites with high electronic density acting as preferential place where CO molecules oxidized. The activity of the supports corresponds to the following order: CeFe2/Al ›CeFe1.5/Al ›CeFe1/Al ›CeFe0.5/Al ›CeFe3/Al ›Fe/Al ›Ce/Al ›Al. The later agrees with the enhanced electronic properties exhibited for the CeFe2/Al sample as demonstrated by the OSC experiments.

CATALYTIC ACTIVITY CO OXIDATION
Concerning gold catalysts, all of them (except Au/Al and Au/Fe/Al) reached total conversion at room temperature ( Figure 7B). These results evidences the different behavior between gold supported on reducible oxides such as iron and cerium oxide and gold supported on non-reducible oxides such as alumina. The high activity of gold-ceria catalysts may be attributed to the presence of reactive oxygen as observed by OSC measurements for the ceria containing samples which suggests a support assisted CO oxidation via Mars van Krevelen mechanism. Higher the OSC higher the oxygen mobility and better the catalytic behavior toward CO oxidation.
In order to discern better the catalytic behavior, a sub-ambient CO oxidation study was carried out. Figure 8 shows the activity test at low temperature for the gold catalysts. The specific reaction rates at 0 • C and 25 • C and turnover frequencies (TOF), using the dispersion of Au atom exposure to reactants flow, are calculated and presented in Table 3. For the dispersion a mathematical model consisting of estimating the average size of the gold nanoparticles as a function of the selected geometry and gold atomic radii was employed and directly used . The TOF then is the normalization of the reaction rate to the dispersion values expressed in s −1 .
In the case of the ternary solids, very good activity has been observed being the Au/Ce/Al system better than the Au/Fe/Al. At 25 • C the results show that the Au/Fe/Al sample exhibits superior rate than a similar Au/Fe 2 O 3 catalysts measured in comparable conditions reported in reference Kung et al. (2007) (5.08 × 10 −4 vs. 3.4 × 10 −4 ). In addition, the TOFs values of practically all the samples are higher than that calculated TOFs for similar Au/FeO x /CeO 2 -Al 2 O 3 catalysts measured in the same conditions reported in Reina et al. (2012).
For the quaternary systems, an improvement of the activity is observed. However, a clear trend with the iron oxide loading cannot be established, indicating that the rate of the CO oxidation is governed mainly by the presence of gold and that a similar gold particle size results in a similar reaction rates. Nevertheless, the quaternary solids, exhibited better ability to abate CO compared to the ternary solids (Au/Ce/Al or Au/Fe/Al), being Au/CeFe2/Al the system that totally eliminate CO at lowest temperature (−32 • C). It was reported, that the support could influence the activity indirectly either by increasing the number of low coordinated Au atoms (Janssens et al., 2007)   the presence of the contact Ce-O-Fe surface due to the solid solution formation, at which interface a lowering of the energy barrier for the oxygen migration occurs (Trovarelli, 1999). Ce-Fe contact evidenced by our XPS and UV-Vis data resulted to be critical for a high performance in the CO oxidation. In this sense, Au/CeFe3/Al was the less active within the Ce-Fe mixed systems, meanwhile Au/CeFe2/Al was the most active. The later agrees with the information extracted on the UV-Vis data where Ce-Fe strongest interaction was achieved for the Au/CeFe2/Al whilst for the Au/CeFe3/Al solid iron oxide segregation was intended. Furthermore, the higher capacity of the Ce-Fe mixed catalysts to abate CO is also related to the improved redox properties demonstrated by the TPR and OSC experiments. Gold supported on iron doped-cerium oxide has a larger amount of labile reducible oxygen, which generally leads to more active catalyst in the oxidation reactions.

CONCLUSIONS
A series of highly effective CO oxidation catalysts has been developed in the present paper. The materials are able to completely eliminate CO even at sub-ambient temperatures reaching full conversion of CO at (−32 • C) in the best situation. XPS data points that the catalyst surface is enriched in cerium and iron oxide compared to the bulk and also evidences that the interaction between metallic gold particles and the support depends on the support composition. The modification of the ceria electronic properties by the inclusion of iron oxide has been demonstrated by the UV-vis experiments. Enhanced redox properties and oxygen mobility are achieved when iron is employed as a ceria dopant leading to promising materials for CO oxidation reactions with potential environmental applications.
As a final remark it should be underlined the viability of the systems based on inert support as a matrix for the homogeneous rare earth doped metal oxide catalytically active layer.


extfieldsofstudy: Medicine Chemistry

text:
1-Ethyl-4-{2-[1-(4-methylphenyl)ethylidene]hydrazinylidene}-3,4-dihydro-1H-2λ6,1-benzothiazine-2,2-dione

In the title compound, C19H21N3O2S, the dihedral angle between the aromatic rings is 6.7 (2)° and the C=N—N=C torsion angle is 178.0 (2)°. The conformation of the thiazine ring is an envelope, with the S atom displaced by 0.802 (2) Å from the mean plane of the other five atoms (r.m.s. deviation = 0.022 Å). In the crystal, molecules are linked by C—H⋯O interactions, generating C(5) chains propagating in [010]. A weak C—H⋯π interaction is also observed.

In the title compound, C 19 H 21 N 3 O 2 S, the dihedral angle between the aromatic rings is 6.7 (2) and the C N-N C torsion angle is 178.0 (2) . The conformation of the thiazine ring is an envelope, with the S atom displaced by 0.802 (2) Å from the mean plane of the other five atoms (r.m.s. deviation = 0.022 Å ). In the crystal, molecules are linked by C-HÁ Á ÁO interactions, generating C(5) chains propagating in [010]. A weak C-HÁ Á Á interaction is also observed.   Table 1 Hydrogen-bond geometry (Å , ).
MS acknowledges the HEC Pakistan for providing a PhD fellowship and the University of Sargodha for the X-ray diffraction facility. The dihedral angle between the C1-C6 and C13-C18 aromatic rings is 6.68 (15)° and the C10=N2-N3=C11 torsion angle is 177.98 (19)°. The conformation of the C1/C6/C9/C10/N1/S1 thiazine ring is an envelope, with the S atom displaced by 0.802 (2) Å from the mean plane of the other five atoms (r.m.s. deviation = 0.022 Å). A very similar conformation was observed in a related structure (Shafiq et al., 2013). Atoms C7 and C8 in (I) are displaced from the mean plane of the thiazine ring by -0.416 (5) and 0.704 (6) Å, respectively.
In the crystal, the moelcules are linked by C-H···O interactions (Table 1) to generate C(5) chains propagating in the b axis direction. A weak C-H···π interaction to the C13-C18 ring also occurs (Table 1).

Refinement
The H atoms were placed in calculated positions (C-H = 0.93-0.97 Å) and refined as riding. The methyl group was allowed to rotate, but not to tip, to best fit the electron density. The constraint U iso (H) = 1.2U eq (C) or 1.5U eq (methyl C) was applied.  The molecular structure of (I), showing displacement ellipsoids at the 50% probability level. where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max = 0.001 Δρ max = 0.45 e Å −3 Δρ min = −0.40 e Å −3

Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.


extfieldsofstudy: Biology Medicine

text:
Structure of LdtMt2, an l,d-transpeptidase from Mycobacterium tuberculosis

The crystal structures of two fragments of the l,d-transpeptidase from M. tuberculosis have been determined at 1.45 and 1.86 Å resolution. The extramembrane part of this enzyme consists of three domains: two domains related to the immunoglobulin fold and a catalytic domain belonging to the ErfK/YbiS/YhnG family at the C-terminus.

Introduction
The cell wall of Mycobacterium tuberculosis is a complex multilayered structure with a unique architecture that protects the organism against both physical and chemical stress (Hett & Rubin, 2008). This barrier supports the survival of this pathogen in host macrophages and confers resistance to many antibiotics (Hett & Rubin, 2008;Gengenbacher & Kaufmann, 2012;Russell, 2011). The increasing number of multidrugresistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of M. tuberculosis makes treatment using current strategies increasingly difficult (Cole & Riccardi, 2011). Since M. tuberculosis expresses a significant number of class A -lactamases (Flores et al., 2005;Hugonnet & Blanchard, 2007), -lactams are not considered to be a promising frontline medication against tuberculosis. However, interest in the treatment of tuberculosis by targeting the mycobacterial cell wall using a combination therapy of potent lactamase inhibitors of the clavulanate family and carbapenem-type -lactams has been revived. For instance, the clavulanate-carbapenem combination has shown a bactericidal effect against dormant mycobacteria and drug-resistant strains and in a mouse tuberculosis model (Hugonnet et al., 2009;England et al., 2012).
In the complex mycobacterial cell wall, the peptidoglycan (PG) layer is responsible for rigidity and osmotic stability and provides the foundation of the cell envelope (Hett & Rubin, 2008). The mycolic acids and glycolipids that form a wax-like coat in the outer layer are covalently linked to arabinogalactan, which in turn is coupled to N-acetylmuramic acid units in the PG (Brennan & Crick, 2007). PG is a complex polymer built up of a glycan chain of alternating units of N-acetylglucosamine and N-acetylmuramic acid and short oligopeptide stems coupled to the N-acetylmuramic acid units via a lactate moiety. The peptide stems are cross-linked, connecting different glycan strands into a meshwork that builds up the PG layer surrounding the cell membrane (Vollmer et al., 2008;Meroueh et al., 2006). The integrity of the peptide cross-links is vital for bacteria and the enzymes involved in the formation of these linkages are targeted by -lactam antibiotics. The amino-acid composition and crosslink architecture show large variations when comparing different bacterial species. M. tuberculosis displays a common peptide-stem sequence (l-Ala-d-Glu-Dap-d-Ala) which is also found in Escherichia coli and Bacillus subtilis, but with characteristic modifications. These are the high level of amidation on the d-glutamate (d-Glu) and diaminopimelate (Dap) moieties and a high proportion (80%) of Dap-Dap (3-3) cross-links (Kumar et al., 2012;Lavollay et al., 2008). The peptide linkages are produced by periplasmic transpeptidases that are active in PG polymerization (Lovering et al., 2012). The common Dap-d-Ala (3-4) cross-links are formed by d,d-transpeptidases, which are also called penicillin-binding proteins (PBPs). In contrast, the Dap-Dap (3-3) cross-links are created by l,d-transpeptidases (Mainardi et al., 2005), which represent a different class of enzymes that are not related to the d,d-transpeptidases (Biarotte-Sorin et al., 2006;Bielnicki et al., 2006). l,d-Transpeptidases are also responsible for the coupling of nonconventional d-amino acids to PG peptide stems, which has been suggested to be a stress-induced cell-wall adaptation in Vibrio cholerae (Cava et al., 2011).
The M. tuberculosis H37Rv genome encodes five proteins that all contain a characteristic l,d-transpeptidase domain (Rv0116c, Rv0192, Rv0483, Rv1433 and Rv2518c). Ldt Mt2 (Rv2518c) has been reported to be expressed at the highest level and has been found to be essential for virulence. Inactivation of this gene led to an attenuated phenotype and to increased susceptibility to clavulanate/amoxicillin treatment in vitro and in a mouse tuberculosis model (Gupta et al., 2010). The importance of Ldt Mt2 in virulence makes it an attractive target for the development of inhibitors and novel antibiotics. Structurally characterized l,d-transpeptidases from B. subtilis (Bielnicki et al., 2006) and from Enterococcus faecium (Biarrotte-Sorin et al., 2006) define the architecture of the catalytic domain. However, both display an additional domain unrelated to the approximately 200-amino-acid long segment preceding the catalytic domain in Ldt Mt2 that may govern the positioning of the catalytic module in the PG layer of the cell wall.
Here, we report the three-dimensional structure of Ldt Mt2 (l,d-transpeptidase 2 from M. tuberculosis) based on the X-ray crystallographic structures of two fragments of Ldt Mt2 representing the extramembrane part of the protein (residue range 55-408). The structure analysis reveals that Ldt Mt2 folds into three domains: two domains in the N-terminal part, both of which show an immunoglobulin-related fold, and a C-terminal transpeptidase domain. This domain composition is different from the two-domain structure of the extramembrane part of Ldt Mt2 proposed recently (Erdemli et al., 2012). The crystal structures of the Ldt Mt2 constructs allow modelling of the full-length extramembrane part of the enzyme (residues 55-408), providing an estimate of the maximal distance of the catalytic site from the membrane and thereby the approximate distance at which 3-3 cross-links are formed in the PG layer. Mass-spectrometric analysis provides further evidence that Ldt Mt2 forms covalent adducts with the -lactam antibiotics imipenem and ampicillin.

Gene expression and protein purification
The expression construct for the complete extramembrane segment of Ldt Mt2 (UniProt code O53223; Rv2518c) included amino acids 34-408, lacking only the predicted transmembrane domain (residues 18-34) and the intracellular part of the polypeptide chain (residues 1-17). Additional constructs were based on the domain arrangement obtained from a bioinformatics analysis of the protein sequence (see x3). The construct used in structure determination comprising the two N-terminal domains A and B (the AB module) included residues 55-250, while the construct comprising the B and C domains (the BC module) encoded residues 149-408 (Fig. 1a).
The coding sequences for these constructs were amplified by PCR using Pfu Turbo polymerase (Stratagene) with M. tuberculosis H37Rv genomic DNA as the template. Each construct was expressed in the pNIC28Bsa4 vector (GenBank accession No. EF198106) with a cleavable N-terminal His 6 tag (H 2 N-MHHHHHHSSGVDLGTENLYFQ*SM). Proteolytic tag removal by TEV protease (the cleavage position is indicated by an asterisk) resulted in recombinant proteins with two additional amino acids (Ser-Met) at the N-terminus. All proteins were expressed in E. coli BL21 (DE3) cells at 294 K in LB medium supplemented with 30 mg ml À1 kanamycin. Gene expression was induced by the addition of 0.1 mM IPTG at an OD 600 of 0.5-0.6 and the cells were harvested 20 h postinduction. The pellets were washed in 0.3 M NaCl, 10 mM Tris-HCl pH 8.0 and the cell walls were disrupted by flashfreezing and treatment with lysozyme (40 mg ml À1 ) and DNase I (2 mg ml À1 ) followed by sonication. The recombinant proteins were purified by affinity chromatography using His-Pur Ni-NTA resin (Thermo Scientific) and were eluted with an increasing imidazole gradient. Subsequently, the buffer was exchanged to 0.15 M NaCl, 25 mM Tris-HCl pH 8.0 using a desalting column (HiPrep 26/10; Pharmacia Biotech). The His 6 tag was removed by TEV protease cleavage with 5 mg TEV per milligram of recombinant protein at 293 K in the presence of 2 mM DTT. Cleaved proteins were separated from residual uncleaved proteins and TEV protease by a second Ni-NTA affinity step. Finally, a size-exclusion chromatography step was performed using a Superdex 200 column (Pharmacia Biotech) equilibrated with a buffer consisting of 0.15 M NaCl, 25 mM Tris-HCl pH 8.0. Pure protein samples were concentrated using a Vivaspin 20 centrifugal device (Sartorius, Gö ttingen, Germany) and the protein concentration was determined according to the Bradford assay using reagents from Thermo Scientific (Rockford, Illinois, USA) and a BSA standard. The samples were analyzed by SDS-PAGE and native PAGE (PHAST system, Pharmacia Biotech), aliquoted in the final buffer (0.15 M NaCl, 25 mM Tris-HCl pH 8.0), flash-frozen in liquid nitrogen and stored at 193 K. The selenomethionine-substituted BC-module construct was produced via the metabolic inhibition method (Doublié, 1997) and purification was carried out using the same steps as for the native protein.

Protein crystallization
Protein crystallization screening was carried out at protein concentrations of 17.5 mg ml À1 for the construct comprising the B and C domains and 23.2 mg ml À1 for the construct comprising the A and B domains by the vapour-diffusion method using a Mosquito Crystallization Robot (TTP LabTech Ltd, Melbourn, England) and the commercially available crystallization screens PACT, JCSG+, NeXtal Classics II Suite (Qiagen) and SaltRX (Hampton Research). Crystals of the AB module were obtained in sitting drops at 293 K by mixing 0.15 ml protein solution and 0.15 ml well solution [0.2 M ammonium sulfate, 0.1 M bis-tris-HCl pH 6.5, 25%(w/v) PEG 3350] and equilibrating against 50 ml well solution. The crystal used for data collection was flash-cooled in liquid nitrogen without using a cryoprotectant. The crystals of the BC module were grown in a hanging-drop format at 293 K by mixing 1 ml protein solution and 2 ml well solution (3 M sodium acetate pH 6.0, 0.1 M bis-tris propane-HCl pH 7.0) and equilibrating against 1.0 ml well solution. Crystals of the selenomethionine-substituted BC module were obtained in conditions identical to those for the native protein. The crystals of the BC module (native and selenomethionine-substituted) were cryoprotected by a short soak (20 s) in mother liquor supplemented with 30%(v/v) ethylene glycol prior to freezing. The crystals were frozen in nylon loops and stored in liquid nitrogen until data collection.

Data collection
X-ray data sets were collected to 1.45 Å resolution for the AB module and to 1.86 Å resolution for the BC module on beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France. In addition, a data set was collected to 2.7 Å resolution from a crystal of the SeMetsubstituted BC module on beamline ID29 at the ESRF at the Se edge at a wavelength of 0.97677 Å . All data sets were collected at 100 K. The X-ray data were processed and scaled using XDS (Kabsch, 2010; AB module) and MOSFLM (Leslie, 2006;BC module) and scaled with SCALA from the CCP4 suite .
The crystals of the AB module belonged to space group C2, with unit-cell parameters a = 114.4, b = 27.6, c = 68.0 Å , =114.1 , and contained one molecule in the asymmetric unit with a solvent content of 47.8%. Crystals of the BC module (native and selenomethionine-substituted) belonged to space group I2 1 2 1 2 1 , with unit-cell parameters a = 116.0, b = 121.4, c = 123.3 Å , and contained two molecules in the asymmetric unit with a solvent content of 68%. The statistics for all data sets are summarized in Table 1.

Structure determination and refinement
The structure of the BC module was solved by Se-SAD phasing using the data sets obtained from crystals of the SeMet-substituted protein. The Se sites were identified and refined from the peak data set using SHELX (Sheldrick, 2008). Of the 12 selenium sites in the asymmetric unit, 11 were found by SHELX, with CC values of 47.2% (CC all ) and 30.1% (CC weak ). An almost complete model was built by ARP/wARP (Mooij et al., 2009) using the SeMet peak data set. From this point, the 1.86 Å resolution native data set was used to complete the model by iterative rounds of manual model building using Coot (Emsley et al., 2010) and crystallographic refinement with REFMAC5 (Murshudov et al., 2011) applying local NCS symmetry and TLS. The final model contained two chains of the BC module comprising protein residues 149-408, 12 acetate ions, two Na + ions and 480 water molecules. The final crystallographic R and R free values were 16.5% and 18.7%, respectively (Table 1).
The structure of the AB module was solved by molecular replacement using Phaser (McCoy et al., 2007) with the B domain (149-250) derived from the BC module as the search model. Density modification using Parrot (Cowtan, 2010) was employed to improve the electron-density map, in particular for the unknown domain A at the N-terminus. The A domain was built manually using Coot (Emsley et al., 2010) and refined to 1.45 Å resolution by cycles of restrained refinement using REFMAC5 (Murshudov et al., 2011). The final structure comprised amino-acid residues 57-250, two sulfate ions and 309 water molecules; the crystallographic R and R free values were 16.2% and 21.8%, respectively (Table 1).
Both protein models were validated using Coot (Emsley et al., 2010) and MolProbity (Chen et al., 2010) to monitor the stereochemistry and model quality. A summary of the refinement statistics and model parameters is given in Table 1.
Structural comparisons were carried out using the DALI algorithm (Holm & Rosenströ m, 2010;Holm & Park, 2000). Molecular contacts and interacting surfaces were analyzed using the PISA server (Krissinel & Henrick, 2007). Figures were produced using the program PyMOL (http://www. pymol.org). The coordinates of the AB module and BC module have been deposited in the Protein Data Bank under accession codes 4hu2 and 4huc, respectively.

Mass-spectrometric analysis of antibiotic binding
Samples of Ldt Mt2 (5 mg) were dispensed in water without antibiotics and with 2 mM imipenem (Sigma) or ampicillin (Duchefa Biochemie, Haarlem, The Netherlands) and incubated for 45 min at 295 K. Subsequently, the samples were diluted in 0.5 ml denaturing buffer [5%(v/v) acetonitrile, 0.1%(v/v) formic acid, 0.5 mM TCEP] and loaded onto a CapLC system and an ESI-Q-TOF quadrupole/orthogonal acceleration time-of-flight mass spectrometer (Waters Corp.) using the method described by Sundqvist et al. (2007). The spectra were combined and deconvoluted with the Maximum Entropy 1 algorithm from the MassLynx software suite (Waters Corp.) to obtain the molecular weights of the protein molecules and the protein-antibiotic adducts.

Results and discussion
3.1. Domain organization of Ldt Mt2 , construct design and protein production The lppS gene (Rv2518c) encodes an enzyme consisting of 408 amino acids denoted Ldt Mt2 . The polypeptide chain contains a short intracellular segment (residues 1-17), a predicted transmembrane domain (residues 18-34) and a larger extramembrane part (residues 35-408). The amino-acid sequence of Ldt Mt2 was analyzed with respect to potential domain organization using transmembrane-domain prediction (Cserzö et al., 1997) and sequence comparisons by BLAST (Altschul et al., 1990) and ClustalW (Thompson et al., 1994). This resulted in a well defined prediction of the catalytic domain (residues 250-408). The sequence similarity of residues 150-250 (30-40% identity) to three other M. tuberculosis proteins (Rv0116c, Rv0483 and Rv1433) and several proteins derived from different corynebacteria genomes that also contain a predicted l,d-transpeptidase domain suggested that this fragment may be a separate domain. Furthermore, the sequence comprising residues 34-250 was subjected to domain-border analysis/prediction (http://www.tuat.ac.jp/ domserv/cgi-bin/DLP-SVM.cgi#Wahtis; Ebina et al., 2009) as well as to secondary-structure prediction by JPred (Cole et al., 2008) to define the domain borders. Finally, two likely starting points at the N-terminus were the end of the predicted transmembrane helix coinciding with a predicted lipoproteinattachment site (Rahman et al., 2008) at position 34/35 and the beginning of the first predicted secondary-structure element at position 55. Taken together, these analyses suggested that the extramembrane part of the enzyme consists of three domains: two smaller domains (domain A, residues 34/55-146; domain B, residues 149-250) and a catalytic domain (domain C, residues 250-408) belonging to the ErfK/YbiS/YhnG family (Bielnicki et al., 2006;Fig. 1a). Based on this analysis, three constructs were designed: a full-length construct (residues 34-408), the AB module (residue 55-146) and the BC module (residues 149-408).
The full-length extramembrane fragment was expressed in E. coli and purified to homogeneity. Based on size-exclusion chromatography, the protein is a monomer in solution. However, extensive crystallization screens did not result in crystal formation. The AB-and BC-module constructs also resulted in soluble protein and each construct behaved as a monomer in solution.

The structure of the BC module
The crystal structure of the BC module was solved to 1.86 Å resolution by Se-SAD phasing from crystals of selenomethionine-substituted protein. The electron density for the two polypeptide chains (residues 149-408) in the asymmetric unit is well defined (Fig. 2a). Six surface residues were modelled in double conformations (Arg297 and Ile301 in chain A and Met237, His246, Arg297 and Lys370 in chain B). The model also contains 480 water molecules and 12 acetate ions, which mostly interact with surface residues. Two of the acetate ions are located in the active-site cleft of domain C. Finally, each polypeptide chain contains a bound metal ion. We modelled the bound metal as an Na + ion based on the electron density (i.e. no remaining difference density after refinement), typical metal-ligand distances in the range 2.2-2.5 Å and the composition of the ligand sphere. The Na + ion is located at the C-terminus of a surface helix and is coordinated by the carbonyl O atoms of residues 342 and 345 and by three water molecules.
The BC module consists of the catalytic domain of the typical ErfK/YbiS/ YhnG fold and a smaller domain with an immunoglobulin-related fold (Fig. 1b). The crystal asymmetric unit contains two molecules with a monomer-monomer interface of approximately 800 Å 2 , suggesting crystal-packing contacts rather than dimer formation. The structures of the two molecules in the asymmetric unit in the crystals of the BC module are essentially identical, displaying a C r.m.s.d. of 0.3 Å for 259 aligned residues. The overall structure of this module and the interactions of the two polypeptide chains in the asymmetric unit are very similar to a recently determined structure of a corresponding module of Ldt Mt2 , albeit obtained from a different expression construct (residues 120-408 as judged from Fig. 1b in Erdemli et al., 2012; the atomic coordinates were not available in the PDB).
The core of the transpeptidase domain consists of a -sandwich formed by two -sheets packed together. Comparison of this domain with a related protein from E. faecium (PDB entry 1zat; Biarrotte-Sorin et al., 2006) using DALI results in a Z-score of 19.3 and a C r.m.s.d. of 1.9 Å for 120 aligned residues. A similar comparison with the B. subtilis homologue (PDB entry 1y7m; Bielnicki et al., 2006) gives a Z-score of 17.7 and a C r.m.s.d. of 1.6 Å for 109 aligned residues. The key residues of the catalytic site, Cys354 and His336, correspond to Cys442 and His421 in the E. faecium homologue. In Ldt Mt2 the active site is located under a lid formed by a pair of antiparallel -strands and their connecting loop (residues 298-324) that protrude from the core of the catalytic domain (Fig. 3). This feature is not present in the B.subtilis enzyme and the tip of the lid is disordered in the E. faecium protein, making the active site solvent-accessible, while in Ldt Mt2 access is limited by the lid. The large hydro-  phobic residues of the lid (Tyr298, Tyr308 and Tyr318) and the residues surrounding the entrance to the active site (Tyr330, Phe334 and Trp340) contribute to the closure and to the exclusion of solvent, which may support efficient catalysis.
Residues 382-408 form a C-terminal extension which is not present in the two characterized structural homologues. This sequence stretch runs along the C domain and contributes to the domain interface by forming additional contacts with the B domain (Fig. 1b). The three tryptophan residues (Trp394, Trp398 and Trp401) in this segment form extensive stacking interactions at the domain interface and are likely to stabilize the relative orientation of the two domains. The pattern of tryptophan residues in this sequence stretch is retained in Rv0483 (LprQ), one of the homologues from the M. tuberculosis H37Rv genome.

The structure of the AB module of Ldt Mt2
The structure of the segment comprising the A and B domains was solved to 1.45 Å resolution by molecular replacement using the B domain (residues 149-250) as the search model. The structure of the AB module includes residues 57-250, approximately half of the extramembrane part of Ldt Mt2 , and 309 water molecules. Apart from the first two residues of the construct, the electron density for the polypeptide chain is well defined (Fig. 2b). Seven residues, Val59, Val66, Asn121, Arg123, Thr138, Glu162 and His214, were modelled in double conformations. Each domain in the model contains a bound sulfate ion located on the protein surface. In domain A the sulfate is anchored to the protein through interactions with Asn95, Asn97, Arg99 and Arg122, whereas in the B domain the ligand forms hydrogen bonds to the side chains of Tyr201, Arg209 and Arg211.
The two domains are arranged in a V-shaped manner and both display a variant of the immunoglobulin fold built up by asandwich of two antiparallel -sheets (Figs.  1c and 4). A structural classification of immunoglobulin-like -sandwich domains defined four subclasses, s-type, h-type, ctype and v-type, based on strand connectivity and topology (Bork et al., 1994). Both immunoglobulin-like domains of Ldt Mt2 belong to the c-type Ig fold, with strand orders a-b-e-d and c-f-g in the two antiparallel -sheets (Fig. 4). The active site of Ldt Mt2 . The lid covering the active site formed by a pair of antiparallelstrands and their connecting loop (residues 298-324) is shown in red. The active-site residues Cys354 and His336 are depicted as green sticks. The bulky hydrophobic residues of the lid (Tyr298, Tyr308 and Tyr318) and its surroundings (Tyr330, Phe334 and Trp340), shown as red and orange sticks, respectively, limit access to and contribute to the closure of the active site. The similarity between the A and B domains is reflected in the C r.m.s.d. of 2.7 Å based on 85 aligned residues. The main difference in their overall structure is the short -helix (residues 180-187) found between -strands 2 and 3, which is not present in domain A, and the loop connecting 6 and 7, which is substantially longer in the B domain (Figs. 4 and 5a). The sequence identity derived from structural alignment of the two domains is 12% which does not provide strong evidence for gene duplication.
The domain interface accounts for 350 Å 2 of buried surface area including 13 and ten buried residues from the A and B domains, respectively. The domain interface is formed by the loops connecting -strands 1 and 2 and -strands 5 and 6 in domain A and the loop connecting -strands 6 and 7 in domain B. The two domains are connected by a short linker Ala149-His150 forming hydrogen bonds to the main chain of the B-domain residues involved in the interface (Gly236 and Phe237). The size of the ABdomain interface, the short linker and the fact that B factors of the linker residues are in the range 18-22 Å 2 as in the remaining part of the structure suggest that the domain linker is not flexible and the orientation of the two domains is kept in this V-shaped form (Fig. 1c). The structures of the B domain in the AB and BC modules are essentially identical and superposition gives an r.m.s.d. of 0.4 Å for 101 aligned residues, although the B-factor profiles for these domains are different in the two crystal forms, most likely owing to different crystalpacking interactions.
The two constructs used in the crystal structure analysis cover approximately the whole length of the extramembrane part of the polypeptide chain of Ldt Mt2 and the results presented here redefine the domain composition of this enzyme. A recent structure analysis described the extramembrane part of the protein as a twodomain structure extending from the membrane (Erdemli et al., 2012). The structure of the AB module, which covers a larger proportion of the N-terminal residues of the extramembrane part of the enzyme (residues 55-250) than in the previous analysis (residues 120-408), shows that this segment of Ldt Mt2 contains an additional domain, domain A, at the N-terminus (Fig. 1c)    previously. The extramembrane part of the polypeptide chain of Ldt Mt2 is thus a three-domain entity, with two immunoglobulin-like domains rather than one and a C-terminal transpeptidase domain.
A structural comparison using DALI identified the closest structural homologues of the two immunoglobulin-like domains as the periplasmic copper-resistance protein from Pseudomonas syringae (PDB entry 2c9r; Zhang et al., 2006;Z-score of 10.2 and C r.m.s.d. of 2.4 Å ) and the N-terminal domain of Halothermothrix orenii amylase (PDB entry 3bc9; Tan et al., 2008) and a similar domain of the endo--1,4mannanase from Cellulomonas fimi (PDB entry 2x2y; Hekmat et al., 2010) with similar Z-scores (6.0-10.1) and C r.m.s.d. values (2.6-3.1 Å ). Finally, the cell-wall-located S-layer protein from Geobacillus stearothermophilus (PDB entry 2ra1; Pavkov et al., 2008) aligned with a Z-score of 7.8 and a C r.m.s.d. value of 3.2 Å . Structure-based sequence alignments show that in the case of the B domain the sequence identities to these structural homologues are all below 10%, thus indicating no significant relationship at the amino-acid sequence level. However, the A domain shows a higher degree of amino-acid sequence conservation when compared with the copper-resistance protein from P. syringae (18.5%) and with the homologous domains of the mannanase (18.5%) and a glucodextranase from Arthrobacter globiformis (15.2%) (Mizuno et al., 2004). Functional analogy can be excluded in the case of the periplasmic copper-resistance protein, as the copper-binding residues are not present in Ldt Mt2 . However, the similarity to the two domains that are present in the enzymes processing high-molecular-weight polysaccharides (mannanase and glucodextranase) could potentially be relevant as the PG backbone is a duplex of carbohydrate chains.
In the multi-domain amylase enzyme from H. orenii it has been experimentally demonstrated that the small noncatalytic immunoglobulin-like domain mediates the interaction of the enzyme with the high-molecular-weight substrate (Tan et al., 2008). The residues identified as conserved between domain A and the corresponding domains from the polysaccharideprocessing enzymes mannanase or glucodextranase are either part of the hydrophobic core or are located on the -sheet surface (Fig. 5b).

The extramembrane Ldt Mt2
As the two constructs include essentially the whole sequence of the extramembrane part of Ldt Mt2 and contain an overlapping domain (domain B), the structure of the fulllength protein excluding the short intracellular and membrane-stretch parts can be reconstructed by superposition of the AB and the BC modules based on the B domains (Fig. 6). The three-domain structure of this transpeptidase can extend from the plasma membrane to a distance of at least 80 Å (plus the stretch of residues 34-54  Model of the complete extramembrane part of Ldt Mt2 from M. tuberculosis. The model was derived by superposition of the B domains common to the structures of the AB and BC modules. The maximal distance between the membrane and the catalytic site, where cross-link formation in the cell-wall peptidoglycan by Ldt Mt2 can occur, can be approximated to at least 80-100 Å . bacterial cell wall. These domains define the distance of the catalytic site and thus the site of formation of the 3-3 linkages in the PG relative to the membrane. Cross-link formation appears to occur at two levels carried out by the BC modulelike l,d-transpeptidases (Rv0116c and Rv1433) and by the one domain longer Ldt Mt2 -type (ABC module; Fig. 6) enzymes (Rv2518c, Rv0483 and Rv0192).

Binding of b-lactam antibiotics to Ldt Mt2
The inhibitory effect of -lactam-type antibiotics towards transpeptidases is based on the formation of a covalent adduct with the active-site serine residues in d,d-transpeptidases or cysteine residues in l,d-transpeptidases, as has been demonstrated for the l,d-transpeptidase from B. subtilis (Lecoq et al., 2012) and for Ldt Mt1 (Rv0116c), a homologue of Ldt Mt2 (Dubé e et al., 2012). Incubation of the BC module or of the full-length extramembrane fragment (residues 34-408) of Ldt Mt2 from M. tuberculosis with the -lactam-type antibiotics imipenem (299.3 Da, carbapenem class) and ampicillin (349.4 Da, penicillin class) led to the formation of covalent enzyme-antibiotic adducts as shown by ESI-MS (Table 2 and Supplementary Material 1 ). The observed molecular masses of the extramembrane fragment (residues 34-408; 40 074.0 Da) and the BC module (residues 149-408; 28 482.0 Da) were very close to the calculated masses from the sequence: 40 073.8 and 28 481.8 Da, respectively (Supplementary Material). The mass difference between the samples before and after incubation with 2 mM ampicillin or 2 mM imipenem corresponds to the exact mass of the respective antibiotic (Table 2). Together with the previous demonstration of binding of imipenem and meropenem to Ldt Mt2 using ITC (Erdemli et al., 2012), these data indicate that the enzyme can recognize and bind a variety of -lactam antibiotics, which also suggests that there is potential for the design of more specific inhibitors of Ldt Mt2 .


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Studying the oxidation of water to molecular oxygen in photosynthetic and artificial systems by time-resolved membrane-inlet mass spectrometry

Monitoring isotopic compositions of gaseous products (e.g., H2, O2, and CO2) by time-resolved isotope-ratio membrane-inlet mass spectrometry (TR-IR-MIMS) is widely used for kinetic and functional analyses in photosynthesis research. In particular, in combination with isotopic labeling, TR-MIMS became an essential and powerful research tool for the study of the mechanism of photosynthetic water-oxidation to molecular oxygen catalyzed by the water-oxidizing complex of photosystem II. Moreover, recently, the TR-MIMS and 18O-labeling approach was successfully applied for testing newly developed catalysts for artificial water-splitting and provided important insight about the mechanism and pathways of O2 formation. In this mini-review we summarize these results and provide a brief introduction into key aspects of the TR-MIMS technique and its perspectives for future studies of the enigmatic water-splitting chemistry.

INTRODUCTION
In nature, the splitting of water to molecular oxygen (O 2 ) is catalyzed by the membrane-bound pigment-protein photosystem II (PSII) of plants, algae, and cyanobacteria (Vinyard et al., 2013). The catalytic site of the water-splitting reaction is an inorganic tetra-manganese mono-calcium penta-oxygen (Mn 4 CaO 5 ) cluster (Figure 1) that forms, together with its protein ligands, the water-oxidizing complex (WOC) of PSII (Yano et al., 2006;Umena et al., 2011). Water-splitting by the Mn 4 CaO 5 cluster is energetically driven by the strongest biological oxidant, P680 + (with a midpoint potential of ∼1.25 V), generated by the light-induced charge separation within the Chl-containing reaction center (RC) of PSII (Diner and Rappaport, 2002;Ishikita et al., 2005). A redox-active tyrosine residue (Y Z ) is the essential electron transfer intermediate between the photoactive RC and the Mn 4 CaO 5 cluster of PSII. Following light absorption, the Mn 4 CaO 5 cluster is oxidized step-wise (one electron at a time) and thereby cycles through five redox states, known as S i states (where i reflects the number of oxidizing equivalents stored by the cluster) (Figure 1). The four-electron four-proton oxidation chemistry of two water molecules is completed when the four oxidizing equivalents are accumulated within the WOC, and the highly reactive S 4 state relaxes into the most reduced S 0 state with the concomitant O-O bond formation and release of O 2 (Messinger et al., 2012;Cox and Messinger, 2013). This reaction cycle of water oxidation is also known as the Kok cycle (Kok et al., 1970).
During the last few decades an enormous progress in elucidation of the WOC structure and in understanding the mechanism of the water-splitting became possible due to employment of numerous biophysical techniques (summarized in Aartsma and Matysik, 2008;Messinger et al., 2009a,b; also see refs therein). Among them, time-resolved isotope-ratio membrane-inlet mass spectrometry (TR-IR-MIMS) in combination with isotope labeling (Konermann et al., 2008;Beckmann et al., 2009) provided the most direct information on the S i state dependent substrate water binding to the WOC (Messinger et al., 1995;Wydrzynski et al., 1996). These findings were recently reviewed in detail by Hillier and Wydrzynski (2008), Messinger et al. (2012), and Cox and Messinger (2013) and are, therefore, only briefly discussed here. However, TR-MIMS has also been successfully employed and yielded important data on other structural and mechanistic aspects of the water-splitting chemistry in both natural PSII and in variously designed artificial O 2 -evolving catalysts. In this minireview, we summarize these recent investigations and also provide some comments on perspectives of the TR-MIMS technique for future studies of water-splitting and O 2 evolution.

KEY CONCEPTS OF TR-MIMS
The concept of TR-MIMS was first applied in 1963, when Georg Hoch and Bessel Kok began to use mass spectrometer with a semipermeable membrane as inlet system (Hoch and Kok, 1963). This allowed to separate the liquid sample from the high vacuum space of the mass spectrometer, while at the same time it was permeable to the gaseous analytes. This excellent solution allowed continuous on-line measurements of dissolved gaseous analytes (either dissolved in solution or directly from the gas phase) with a temporal resolution of a few seconds. Therefore, FIGURE 1 | Cyanobacterial PSII structure and Kok cycle of photosynthetic water oxidation by the Mn 4 CaO 5 cluster. The arrows within PSII indicate the direction of electron transfer which comprises the following redox-active cofactors: inorganic Mn 4 CaO 5 cluster, redox-active tyrosine Z (Y Z ), the primary electron donor P680 that includes a pair of Chls a (P D1 and P D2 ) and two accessory Chls (Chl D1 and Chl D2 ), the primary pheophytin (Pheo D1 ) acceptor, the primary (Q A ) and the secondary (Q B ) quinone acceptors. The phytyl tails of the Chl's and Pheo's, and the isoprenyl chains of the quinones have been cut for clarity. The light-induced S state transitions of the Mn 4 CaO 5 cluster are indicated by arrows with "hν" labels. The PSII structure and the zoomed structural model of the Mn 4 CaO 5 cluster in the center of the Kok cycle are based on the recent PSII crystal structure at a resolution of 1.9 Å (PDB entry 3ARC; Umena et al., 2011). the TR-MIMS technique is ideally suited for investigations of photosynthetic and artificial water-oxidation/O 2 evolution (for instance, see Konermann et al., 2008;Beckmann et al., 2009). For an outline of other TR-MIMS applications in biological and in industrial systems, see reviews by Lauritsen and Kotiaho (1996) and Johnson et al. (2000). Recent technological advances in MIMS instrumentation are summarized in Davey et al. (2011).
A schematic view of a TR-MIMS set-up employing an isotope ratio mass spectrometer is shown in Figure 2. This type of mass spectrometers is normally equipped with an electronimpact ion source, magnetic sector field analyzer, and individual detectors (Faraday cups) that provide simultaneous detection of several masses (ions) with high sensitivity and signal stability. For its ability to monitor and to selectively analyze all isotopologues (molecules that differ only in their isotopic composition) of gaseous products with one instrument, the TR-MIMS approach in combination with isotope enrichments became indispensable tool for kinetic and functional analyses of photosynthetic enzymes (Konermann et al., 2008;Beckmann et al., 2009). The key part of the TR-MIMS instrument is a gas inlet system that is integrated within a MIMS cell. The design of MIMS cells may vary depending on the measuring purposes (Konermann et al., 2008;Beckmann et al., 2009), but all of them contain a gaspermeable membrane functioning as analyte inlet system into the vacuum of the mass spectrometer. The coupling of such a cell to various light sources (e.g., Xenon lamps or lasers) allows carrying out the measurements of light-induced O 2 evolution in photosynthetic samples or light-driven O 2 -evolving artificial catalysts. Before entering the ion source of the mass spectrometer the analytes pass through a cryogenic trap (Figure 2), which freezes out water vapor that inadvertently pervaporate through the membrane in trace amounts.
Enrichment of aqueous sample suspension with oxygen's heavy isotope ( 18 O) for isotope ratio measurements of O 2 (and/or CO 2 ) isotopologues is a powerful and commonly used tool in studies of water-splitting chemistry and/or related reactions. Therefore, most of the experiments are carried out in H 18 2 Olabeled sample suspensions/solutions.

IS WATER THE IMMEDIATE SUBSTRATE OF PHOTOSYNTHETIC O 2 EVOLUTION?
It is widely accepted that water is the immediate substrate for photosynthetic O 2 production. However, Metzner (1978) suggested that instead hydrogen carbonate (bicarbonate; HCO − 3 ) is the immediate substrate for O 2 formation that is subsequently replenished by the reaction of CO 2 with H 2 O. This hypothesis was discounted for long because the isotopic equilibration between 18 O-water and HCO − 3 is too slow to account for early isotope labeling studies (Ruben et al., 1941;Stemler and Radmer, 1975;Stevens et al., 1975;Radmer and Ollinger, 1980). Due to the discovery that a carbonic anhydrase (CA) activity is associated with PSII (Lu and Stemler, 2002;Villarejo et al., 2002;Moskvin et al., 2004) the "bicarbonate-as-substrate hypothesis" needed to be re-investigated with refined expriments. Indeed, due to rapid exchange of HCO − 3 and CO 2 species by CA, the 18 O-label could "escape" from HCO − 3 to water (which has a several orders higher concentration than the added 18 O-labeled HCO − 3 ), and, thus, lead to the lack of O 2 yield from HCO − 3 (Stemler and Radmer, 1975;Radmer and Ollinger, 1980). Two different TR-MIMS approaches were taken recently and both exclude that HCO − 3 is a physiologically significant substrate (Clausen et al., 2005a;Hillier et al., 2006). Clausen et al. (2005a) reported that under H 18 2 O-labeled and CO 2 /HCO − 3 -depleted conditions the typical oscillation pattern of 18 O-enriched O 2 evolution is obtained in response to single light flashes, but didn't find any evidence for CO 2 release. The latter would be expected in case the Metzner's hypothesis would be correct. Hillier et al. (2006), in their TR-MIMS study, employed 18 O/ 13 C-labeled HCO − 3 to probe the capability of PSII (from higher plants and cyanobacteria) to oxidize HCO − 3 . The authors were able to detect an extremely small (and, thus, non-physiological) flux of 18 O from HCO − 3 into O 2 similar to that observed in an early TR-MIMS study of Radmer and Ollinger (1980). Moreover, no relationship between O 2 evolution and PSII-associated CA activity was found by McConnell et al. (2007) in their TR-MIMS examination of PSII preparations from higher plants.

IS HYDROGEN CARBONATE A LIGAND TO THE WOC?
Hydrogen carbonate had been proposed to bind as integral cofactor to the Mn 4 CaO 5 cluster after accumulation of many experimental results indicating: (i) the requirement of HCO − 3 ions for optimal stability and functionality of the WOC (Van Rensen and Klimov, 2005), and (ii) it's important role for the photoassembly reaction of the Mn 4 CaO 5 cluster (Dasgupta et al., 2008). Moreover, in the PSII crystal structure from Thermosynechococcus elongatus at 3.5 Å resolution, HCO − 3 was modeled as a nonprotein ligand bridging Mn and Ca ions within the WOC (Ferreira et al., 2004).
Earlier, interesting TR-MIMS experiments were performed by Stemler (1989) and Govindjee et al. (1991), in which formate FIGURE 2 | Representation of a TR-IR-MIMS set-up. Gaseous products, produced by sample suspension (for instance, by PSII samples) in the cell, penetrate through a gas-permeable membrane into a high-vacuum space, pass through a cryogenic trap (which removes water vapor from a flow of gaseous analytes), and enter the isotope ratio mass spectrometer. Here, gaseous analytes are first ionized in the ion source by electron impact, and are then separated according to their m/z ratios by a magnetic field in the sector analyzer that allows simultaneous online detection by individual collector cups (e.g., a 7-cup Faraday detector array). MS signals are monitored and analyzed using a personal computer. See text for further details.

FIGURE 3 | TR-MIMS experiments demonstrating that HCO −
3 is not a tightly bound ligand to the Mn 4 CaO 5 cluster in spinach PSII membrane fragments. (A) Amount of released CO 2 upon formate addition (black arrows) to intact PSII membranes is the same as in the case of PSII membranes without the Mn 4 CaO 5 cluster (due to 75-min pre-incubation with 80 mM N 2 H 4 ). Due to enrichment of sample suspension with H 18 2 O (3%) CO 2 was detected as C 16 O 18 O at m/z 46. (B) Addition of the strong reductant NH 2 OH (white arrows) at concentrations known to cause rapid reduction of the Mn 4 CaO 5 cluster and release of Mn ions as Mn II into the solution didn't lead to a release of CO 2 /HCO − 3 above background. In order to avoid the overlay of CO 2 and N 2 O signals (the latter is known to be produced during interaction of NH 2 OH with the Mn 4 CaO 4 cluster), the N 2 O signal was shifted from m/z 44 to m/z 46 by employing the 15 N-labeled NH 2 OH for these experiments. To facilitate equilibration between CO 2 and HCO − 3 all measurements were performed in the presence of externally added CA (to a final concentration of 3 μg ml −1 ). Modified from Shevela et al. (2008b).

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November 2013 | Volume 4 | Article 473 | 3 was tested to induce the release of HCO − 3 (that can be detected by TR-MIMS as CO 2 ) from PSII. Although Govindjee et al. (1991) provided clear evidence for the formate-induced release of CO 2 /HCO − 3 , the HCO − 3 binding site was not specified in this study. Based on numerous previous data indicating that HCO − 3 is a ligand to the non-heme iron (NHI) at the electron acceptor side of PSII, the released CO 2 was suggested to derive from this binding site. However, later formate was shown to bind both at the acceptor and the donor (water-splitting) side of PSII (Feyziev et al., 2000), and therefore, the released CO 2 /HCO − 3 could also originate from the water-splitting side.
In order to specifically probe the possible binding of HCO − 3 to the Mn 4 CaO 5 cluster at the donor side of PSII, Shevela et al. (2008a,b) re-examined and extended the earlier TR-MIMS investigations. Thus, a comparison of the formate-induced C 16 O 18 O yields (Figure 3A), under H 18 2 O-enriched conditions, in intact PSII and Mn-depleted PSII was performed. This was complemented by experiments in which the gaseous products produced by a quick reductive destruction of the of the Mn 4 CaO 5 cluster by 15 N-labeleld NH 2 OH ( Figure 3B) were studied. Both approaches clearly demonstrated that the detected CO 2 /HCO − 3 does not originate from the inorganic core of the water-splitting site of PSII (Shevela et al., 2008a,b). Independent evidence for absence of HCO − 3 bound to the WOC was provided by FTIR and GS-MS experiments (Aoyama et al., 2008;Ulas et al., 2008). Moreover, in recent x-ray crystallography studies at resolutions of 1.9-3.0 Å no HCO − 3 was found in the vicinity of the Mn 4 CaO 5 cluster, while they all clearly show HCO − 3 bound to the NHI on the acceptor side of PSII (Guskov et al., 2010;Umena et al., 2011) (also, see Figure 1). Thus, it can be excluded that HCO − 3 is a tightly bound ligand of the Mn 4 CaO 5 cluster.
However, none of the mentioned studies negates the option that a mobile, weakly bound, and rapidly exchanging HCO − 3 affects the activity of the WOC. Thus, in case of the TR-MIMS measurements, weakly bound HCO − 3 may have been removed during the required degassing of the MIMS cell prior to formate or NH 2 OH additions to PSII samples. Therefore, the possible loss of weakly bound HCO − 3 and the amount of HCO − 3 associated with PSII under air-saturated condition remain to be established in future TR-MIMS experiments.

WHEN AND HOW DOES SUBSTRATE WATER BIND TO THE WOC?
Indisputably, the most significant and unique contribution of the TR-MIMS instrumentation in understanding of water-oxidation mechanism in photosynthesis was its application for studying substrate binding in the different S i states of the WOC. In these experiments the binding of water to the WOC is probed by the rapid injection of H 18 2 O into the PSII samples which were preset into the desired S i state by pre-illumination with 0, 1, 2, or 3 flashes. Then, after desired incubation times, O 2 evolution is induced by a sequence of additional flashes. The exchange rates of the two substrate molecules are then calculated from the change of the 16 O 18 O and 18 O 18 O yields as a function of incubation time (see Figure 4A for protocol of the TR-MIMS measurements of substrate water exchange in the S 3 state

state of PSII (A) and experimentally obtained substrate water exchange rates in spinach thylakoids (B). (A)
The S 3 state is populated by two pre-flashes given at 2 Hz (shown by the two first black vertical arrows). This is followed by the rapid injection of H 18 2 O into the PSII sample (shown by blue vertical arrow) and subsequent fast mixing of the injected H 18 2 O with the sample. Evolution of O 2 isotopologues is then induced by a 3rd flash, given at varying delay times (from 0 to 10 s) after the H 18 2 O injection (signified as incubation time). Finally, a series of four flashes is given at 2 Hz to induce O 2 yield used for normalization. state in spinach thylakoids at 10 • C and pH 6.8. Symbols in both plots are experimental data, and the lines in the top and bottom plots are biexponential and monoexponential fits, respectively. The biexponential fit yields rate constants of ∼40 s −1 for the fast phase and ∼2 s −1 for the slow phase. The slow phase in the 16 O 18 O data is matching the rate found in the monoexponential fit of the 18 O 18 O data (Messinger et al., 1995;Hillier et al., 1998;Wydrzynski, 2000, 2004). Adapted from Cox and Messinger (2013).
since they determine the time resolution of the TR-MIMS measurements. In the first H 16 2 O/H 18 2 O-exchange TR-MIMS experiments the water exchange kinetics could not be resolved (Radmer and Ollinger, 1986;Bader et al., 1993). The development of the MIMS cell by Messinger et al. (1995), which allowed for fast mixing of H 18 2 O with the sample and also implemented O 2 removal from the labeled water by the glucose-glucose oxidase-catalase method, greatly improved the time resolution down to the milliseconds scale and allowed measurements of substrate water exchange in all semistable S i states (Messinger et al., 1995;Hillier et al., 1998;Hillier and Wydrzynski, 2000). Figure 4B illustrates characteristic water exchange kinetics in the S 3 state as measured in spinach thylakoids with the time resolution of 8 ms. In this figure, the yields of the singly-labeled ( 16 O 18 O) and doubly-labeled ( 18 O 18 O) isotopologues of molecular oxygen are plotted as a function of H 18 2 O incubation time in the S 3 state. While the former plot reflects the result when only one of the two possible 18 O-water substrates is exchanged, the latter one is for the case when both 18 O-waters are exchanged. The biphasic behavior of the 16 O 18 O rise (detected at m/z 34) (see Figure 4B) is known to represent the exchange rates of two independent slowly and fast exchanging substrate water molecules bound at separate sites within the WOC. In contrast, the 18 O 18 O product (monitored at m/z 36) exhibits a monoexponential rise with a rate equal to that of the slow phase kinetic of the 16 O 18 O data, thus-reflecting the exchange of the same "slowly" exchanging substrate water as observed at m/z 34. This finding clearly confirms that the two phases of the 16 O 18 O data are an intrinsic feature of the WOC and do not originate from PSII heterogeneity (Messinger et al., 1995;Hillier et al., 1998).
Further TR-MIMS experiments also revealed that the "slowly" exchanging water is bound to the WOC in all semi-stable S i states, while the "fast" exchanging water was detected only in the S 2 and S 3 states (Hillier et al., 1998;Wydrzynski, 2000, 2004;Hendry and Wydrzynski, 2002). Thus, the TR-MIMS technique provides not only the most direct evidence for independent substrate water binding within the WOC, but also allows to monitor the change in their binding affinities throughout the reaction cycle. For a complete overview of the TR-MIMS findings in this field, we refer the readers to reviews by Hillier and Messinger (2005), Hillier and Wydrzynski (2008), Beckmann et al. (2009), Messinger et al. (2012, and Cox and Messinger (2013).

THE 16 O/ 18 O ISOTOPE EFFECT AND PHOTOSYNTHETIC WATER-SPLITTING
Up to now there is no final agreement on whether isotopic discrimination during O 2 production by photosynthetic watersplitting in PSII contributes to the so-called Dole effect, which describes the finding that the percentage of the 18 O isotope in atmosphere is higher (by 23‰) than in oceanic waters (Dole, 1936;Tcherkez and Farquhar, 2007). While many gas isotope ratio studies clearly showed that oxygen produced by O 2 -evolving organisms is isotopically identical to the water they are suspended in (Dole and Jenks, 1944;Stevens et al., 1975;Guy et al., 1993;Helman et al., 2005), recent 18 O-enriched TR-MIMS experiments indicated that the 18 O isotope is favored by the WOC for O 2 production, thus-suggesting a significant 16 O/ 18 O isotope effect in the photosynthetic water-splitting (Burda et al., 2001(Burda et al., , 2003. This finding was challenged by recent theoretical estimations that suggest a very small isotope effect (Tcherkez and Farquhar, 2007). Undoubtedly, a resolution of these conflicting results can be provided by revisiting TR-MIMS studies. These future studies should be designed to account for: (i) technical limitations/drawbacks of the previous TR-MIMS experiments (for instance, the absence of fast H 18 2 O mixing upon its addition to sample suspension inside the MIMS cuvette Bader et al., 1987;Burda et al., 2003), (ii) possible contribution of isotopic fractionation due to transfer of O 2 isotopologues through the membrane inlet toward the high vacuum of mass spectrometer recently reported by Hillier et al. (2006), and (iii) current knowledge of the S-state dependent substrate water binding and exchange rates as derived from TR-MIMS measurements (for reviews, see Hillier and Wydrzynski, 2008;Cox and Messinger, 2013). However, we note here, that without specific investigations of the 18 O isotope effect in photosynthetic water-oxidation, our TR-MIMS studies do not reveal any oxygen isotope discrimination in photosynthetically produced O 2 (for instance, see Figure 6C and text below for explanations), indicating that any such effect must be small at best.

IN SEARCH FOR INTERMEDIATES OF WATER SPLITTING BY TR-MIMS APPROACH
While most states of the Kok cycle (S 0 , S 1 , S 2 , S 3 ) are semistable, the S 3 Y • Z and S 4 state are known to be a highly reactive intermediates that until very recently were not characterized. Clausen and Junge (2004) attempted to stabilize and identify putative intermediate(s) of the S 4 state by applying a high partial O 2 pressure in order to shift the equilibrium of the terminal S 4

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November 2013 | Volume 4 | Article 473 | 5 → S 0 + O 2 + nH + reaction backwards. Based on their UVabsorption transients the authors observed half suppression of Mn oxidation under only 10-fold increase of ambient O 2 pressure (2.3 bar). These results were considered to be the first indication for an intermediate in the S 3 → S 4 → S 0 transition and as a possible route for stabilizing it (Clausen and Junge, 2004). Although a further delayed Chl fluorescence study corroborated these results (Clausen et al., 2005b), experiments by time-resolved X-ray absorption spectroscopy (TR-XAS) (Haumann et al., 2008) and by visible fluorescence study (Kolling et al., 2009) (Shevela et al., 2011a). In these experiments direct monitoring of 18 O 2 evolution from 18 O-labeled water against a high level of 16 O 2 in a suspension of PSII complexes became possible due to a specially designed high pressure MIMS cell (for details, see Figure 5A). This study demonstrated that neither an inhibition nor altered flash-induced pattern of O 2 evolution take place under up to 50-fold increased concentration of dissolved O 2 around PSII ( Figure 5B). These findings show that the terminal water-splitting reaction/O 2 release in PSII is highly exothermic, and are in line with the results obtained by TR-XAS (Haumann et al., 2008) and variable fluorescence (Kolling et al., 2009) studies.

APPLICATIONS IN ARTIFICIAL PHOTOSYNTHESIS
One of the central goals of artificial photosynthesis is the development of bio-inspired, efficient and robust catalysts that are able to split water employing the energy of sunlight in a fashion similar to the water-oxidizing Mn 4 CaO 5 cluster in PSII (Concepcion et al., 2012;Nocera, 2012;Wiechen et al., 2012). Therefore, data concerning catalytic rates and turnover numbers (stability) of newly synthetized O 2 -evolving catalysts are highly important for their further development. In this regard, in addition to traditionally used amperometric methods for O 2 detection (Renger and Hanssum, 2009), TR-MIMS can be applied as a highly sensitive method for studying the O 2 -evolving capability of these complexes. However, a major advantage and uniqueness of the TR-MIMS technique in this field is that, in combination with 18 O-labeling experiments, it can be employed for studying the pathways of O 2 formation in reactions catalyzed by the 'potential' solar water-oxidation catalysts (Poulsen et al., 2005;Beckmann et al., 2008;Sala et al., 2010;Shevela et al., 2011b;Najafpour et al., 2012;Vigara et al., 2012 originates from the bulk water as it has been monitored by TR-MIMS in the reactions of O 2 -evolving catalysts with oxygentransferring oxidizing agent, oxone (HSO − 5 ) (Poulsen et al., 2005;Beckmann et al., 2008;Shevela et al., 2011b) (see Figure 6A). In the case of "true" water-splitting, 18 O-fractions in bulk water and in evolved O 2 are expected to be same (i.e., 18 α theor = 18 α exp ) as depicted in Figure 6B for the reaction of a synthetic catalyst (CaMn 2 O 4 · H 2 O oxide) with photogenerated oxidizing agent [Ru III (bipy) 3 ] 3+ (Ru III photo ), and in Figure 6C for natural lightinduced water-splitting reaction performed by PSII. It's worth mentioning here, that the initial phase of the presented traces until stable 18 α values (Figure 6) is a technical artefact, merely caused by the response time of the membrane-inlet system of the mass spectrometer which seems to be related to the overall O 2 concentration. However, the difference in time needed to reach final 18 α value in two water-splitting reactions shown in Figure 6 also reflects a much slower reaction rate for the reaction of the oxide with Ru III photo . Thus, O 2 evolution for this reaction was detected only after 1 min of illumination since this time is required to build up a sufficient concentration of photosensitizer Ru III photo (data not shown here; for details, see Shevela et al., 2011b). We note that one of the attractive extensions to the described TR-MIMS approach for the characterization of watersplitting catalysts is the coupling of the TR-MIMS instrument to an electrochemical cell (for further details, see Konermann et al., 2008 and refs therein).

CONCLUSIONS AND FUTURE PERSPECTIVES
Application of the TR-MIMS technique and isotope labeling for studies of various biophysical aspects of photosynthetic watersplitting and O 2 production is continuously growing. It provides not only insightful and unique information (which is sometimes not accessible by other methods) about this fundamental biological process, but also becomes an essential and highly precise tool for testing artificial water-oxidizing catalysts. Future applications of TR-MIMS for studies of watersplitting chemistry and O 2 production could follow from advances in membrane materials, different designs of the membrane-inlet systems, coupling with electrochemistry and spectroscopy, and technological developments of the mass spectrometers.