Fast Detection Allowing Analysis of Metalloprotein ElectronicStructure by X-ray Emission Spectroscopy at Room TemperatureKatherine M. Davis,† Brian A. Mattern,‡ Joseph I. Pacold,‡ Taisiya Zakharova,† Dale Brewe,§

Irina Kosheleva,⊥ Robert W. Henning,⊥ Timothy J. Graber,⊥ Steve M. Heald,§ Gerald T. Seidler,‡

and Yulia Pushkar*,†

†Department of Physics, Purdue University, West Lafayette, Indiana 47907, United States‡Department of Physics, University of Washington, Seattle, Washington 98195, United States§Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, United States⊥Center for Advanced Radiation Sources, The University of Chicago, Chicago, Illinois 60637, United States

*S Supporting Information

ABSTRACT: The paradigm of “detection-before-destruction” was tested for ametalloprotein complex exposed at room temperature to the high X-ray flux typical ofthird-generation synchrotron sources. Following the progression of the X-ray-induceddamage by Mn Kβ X-ray emission spectroscopy, we demonstrated the feasibility ofcollecting room-temperature data on the electronic structure of native Photosystem II, atrans-membrane metalloprotein complex containing a Mn4Ca cluster. The determinednondamaging observation time frame (about 100 ms using a continuous monochromaticbeam, with a deposited dose of 1 × 107 photons/μm2 or 1.3 × 104 Gy, and about 66 μsin pulsed mode using a pink beam, with a deposited dose of 4 × 107 photons/μm2 or 4.2× 104 Gy) is sufficient for the analysis of this protein’s electron dynamics and catalyticmechanism at room-temperature. Reported time frames are expected to be representative for other metalloproteins. Thedescribed instrumentation, based on the short working distance dispersive spectrometer, and experimental methodology arebroadly applicable to time-resolved X-ray emission analysis at synchrotron and X-ray free-electron laser light sources.

SECTION: Spectroscopy, Photochemistry, and Excited States

Recently developed third-generation synchrotron sourcesand free-electron lasers provide unparalleled bright X-ray

beams with defined time structure.1,2 These are indispensabletools for time-resolved structural and functional analysis inchemistry and biology (via crystallography,3 X-ray scattering,4

and X-ray spectroscopy5,6) as well as for cell and tissueimaging.7 The main limitation in the application of these X-raysources and techniques is X-ray-induced damage to thematerials or biological molecules that can affect their structureand chemical composition.8,9 This includes changes in theredox states of metal cofactors and their coordinationenvironment, which are thought to occur particularlyquickly.10,11 One possible way to counteract this damage is tocool biological samples to cryogenic (preferably 10−20 K)temperatures.12 However, this currently routine approachprevents the analysis of dynamics such as electron transfer,conformational changes, and bond formations that occur inbiological samples at room temperature (RT). In recent years,and, in particular, with LCLS coming online, it was proposedthat the collection of data with sufficiently high time resolutionwould overcome X-ray damage.13 The concept for sensitivebiological samples is that by utilizing setups with high timeresolution, we can always define some time window for datacollection before X-ray-induced damage occurs. While plausiblefrom both a physical and chemical standpoint, it needs

experimental validation, in particular, when a biological sampleis exposed to the high (full) flux achievable with third-generation synchrotron sources.We chose Photosystem II (PS II) as our biological system

due to its high sensitivity to X-ray-induced damage.10,14,15 PS IIis a central constituent for the natural photosynthetic processthat allows for dioxygen formation during photosynthesisoccurring in plants, green algae, and cyanobacteria. The processof splitting water (2H2O → O2 + 4e− + 4H+) in the trans-membrane protein complex of PS II requires a catalyst, theMn4Ca cluster, embedded in the unique protein environment.This assembly is termed the oxygen-evolving complex(OEC).8,12,16 There is a substantial scientific interest inuncovering the mechanism of this water splitting catalysisthat occurs at room temperature and follows the Kok cycle.17

This cycle describes five visible-light-induced transitionsbetween the so-called S states (S0−S4) corresponding to fiveredox states of the OEC. In this cycle, oxygen evolves in the S3→ S0 transition, and the S4 state is proposed to represent atransient intermediate.18,19

Received: May 16, 2012Accepted: June 26, 2012Published: June 26, 2012

Letter

pubs.acs.org/JPCL

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The mechanism of PS II action can be studied by Mn Kβ X-ray emission spectroscopy (XES) (also known as photon-in−photon-out spectroscopy), Figure 1A, which is highly sensitiveto the oxidation state of the Mn centers and the spin state ofthe Mn4Ca cluster.20−23 This technique was used here todemonstrate the feasibility of RT XES analysis of the nativeprotein prior to the onset of X-ray-induced damage. The MnKβ spectra of PS II in several S states were reported in 2001from cryogenic (20 K) measurements utilizing an X-rayemission spectrometer designed with spherically bent crystalanalyzers (SBCAs).24 Conventional SBCA-based setups recordemission spectra point-by-point (unless the SBCA is positionedoff-Rowland circle),25 and mechanical movement of thespectrometer/detector assembly is needed to record the nextpoint in the spectrum. This makes such a setup particularlyimpractical for time-resolved analysis. While cryogenic XESanalysis of PS II was available in 2001,24 no transition of thisapproach to RT has been reported so far, illustrating associatedexperimental challenges. To the best of our knowledge, noother metalloprotein has been characterized to date by XES atRT.To achieve maximal time resolution, we chose to record X-

ray emission spectra using dispersive optics that allow forinstantaneous detection (Figure 1B).26−29 In this implementa-tion, the time resolution of the spectrometer is determined bythe time structure of the X-ray beam (X-ray pulses of about 100ps at third-generation synchrotron sources and femtosecondpulses at free-electron laser sources). The crystal type andgeometry of the spectrometer determine the energy range and

resolution, Figures 1 and S1−S3 (Supporting Informa-tion).26−29 While a number of spectrometer designs havebeen reported previously, no time-resolved experiments havebeen realized with such a spectrometer, and no feasibilityanalysis for measurements with dilute/biological samples (inthis case, PS II samples have a 0.5 mM concentration of Mnions) has been reported.This study reports two different miniature X-ray emission

spectrometer (miniXES) configurations suitable for the analysisof Mn Kβ emission. The spectrometers use flat dispersiveoptics, and their design is based on the observation that amicrofocused incident beam permits one to obtain good energyresolution from an analyzer crystal located a few centimetersfrom the sample.26−29 The first instrument uses the Ge 440reflection in a Johansson arrangement30 and allows for a 100 eVcollection range containing the Kβ′, Kβ1,3 (emission from the3p level), and Kβ″ peaks and demonstrates an instrumentalenergy resolution of ∼1.5 eV, Figures 1D and S2 and S3(Supporting Information). This instrument allows sufficientspace to fit a microfridge assembly into the sample position,allowing for low-temperature measurements (see the Support-ing Information Materials and Methods section and Figure 3B).To improve the energy resolution, the second design insteaduses the GaP 440 reflection in a von Hamos configuration31

(Figure S1, Supporting Information). This von Hamos designhas a decreased collection range of 50 eV, including only theKβ main lines. However, it demonstrates a higher energyresolution of ∼0.3 eV, Figures 1C, D and S1 and S3(Supporting Information), which is comparable to that of

Figure 1. (A) Absorption of the X-ray photon results in the ejection of a 1s core−shell electron and the subsequent repopulation of the created holewith an electron from the 3p level (Kβ emission lines). (B) Resulting X-ray fluorescence is reflected by a flat crystal onto the position-sensitivedetector (PSD). Data from the PSD are read out in defined time intervals, Δt. Pixel-to-energy calibration of PSD allows reconstruction of the XESspectrum. (C) Mn Kβ main lines in MnO are recorded on a 2D-PSD. (D) Comparison of MnO Kβ spectra obtained with two reportedspectrometers. The GaP 440 von Hamos miniXES demonstrates an energy resolution comparable to that of spectrometers using spherically bentcrystal analyzers.24

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spectrometers utilizing spherically bent crystal analyzers, theinstruments heretofore employed for the analysis of PS II.24

Good energy resolution is evident in the well-resolved shoulderon the left side of the MnO Kβ1,3 peak, as previously observedusing traditional instruments, Figure 1D.20 The majority ofroom-temperature data were collected using the von Hamosspectrometer, Figures 2 and 3.

RT XES measurements on the dark-adapted S1 state of PS II(see the Supporting Information Material and Methodssection) were first carried out using a monochromatic X-raybeam; see Table 1 for experimental details. During themeasurements, a fresh portion of PS II (protected by asynchronized shutter) was exposed to a full-intensitymonochromatic microfocused X-ray beam. In order to resolvethe onset of X-ray-induced damage in time, the acquired imageswere read from the position sensitive detector (PSD) in definedtime intervals, the shortest at ∼20 ms. The time required fordata read out from the Pilatus detector used in this study is ∼3ms. Images corresponding to defined exposure times collectedfrom multiple points on the sample were added together.Spectra from different samples were added together after theenergy calibration. The summation of PSD images was carried

Figure 2. (A) Mn Kβ X-ray emission spectra for the undamaged OECof PS II recorded at room temperature using continuousmonochromatic (green) and pulsed pink (red) X-ray beams. Thetotal amount of PS II protein used to obtain displayed spectra is ∼80(green) and ∼40 mg (red). The linear background (about 30%(green)/50% (red) of the signal intensity) was subtracted; see theMaterials and Methods section in the Supporting Information. (B,C)Effect of the progression of X-ray-induced damage on the Mn Kβ XESspectra of PS II, obtained using the rotating sample holder. Data areshown for consecutive 100 ms exposures with continuous X-rayillumination (B) and in intervals of three pulses of the X-ray beam (C).The shift of the XES spectrum to higher energy is due to thephotoreduction of the OEC containing MnIII and MnIV ions to MnII.

Figure 3. X-ray-induced damage to the OEC expressed as a percentageof MnII plotted versus the photon dose for RT (A) and 80 K (B) data;note the difference in the x-axis. (A) RT data were collected from theJohansson (red squares, excitation energy of 7.5 keV, static sampleholder) and von Hamos miniXES (blue striped squares and greencircles, corresponding to dynamic and static sample holders, excitationenergy of 7.09 keV) spectrometers, respectively. (B) The purpletriangular data were recorded with the Johansson spectrometer at 80 Kand an excitation energy of 7.5 keV. Energy scales for all data arerelative to the MnO reference compound (first moment of 6490.82eV). Also note that the first moments are sensitive to procedures of thebackground removal and noise in the spectra.

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out either with software, when images from single-pointexposures were collected and stored individually, or by usingthe spinning sample holder that allowed for simultaneousdetection/addition of data from multiple points on the sample.No differences were observed in spectral shape or time ofdamage progression between these two methods of dataaveraging. In Figure 2A, we plot the spectrum obtained in thetime interval less than the damage threshold (determined to be60−100 ms while using monochromatic beam, correspondingto 1.3 × 104 Gy).Additionally, RT measurements were done with a pulsed

pink beam (BioCARS, Advanced Photon Source),32 Figures 2Aand 2C and Table 1. Detector images from points on thesample exposed to one, two, or more consecutive 22 μs X-raypulses were collected and added up. Spectra from differentsamples were added together after calibration. In Figure 2A, weplot the spectrum obtained using three consecutive X-ray pulses(corresponding to a dose of 4.2 × 104 Gy) that did not showindications of damage.The time-resolved nature of the XES experiment allowed us

to record emission spectra of undamaged PS II at RT andanalyze the dose-dependent progression of X-ray-induceddamage (see below as well as Figures 2B and 3A). Thisanalysis ensured that data for undamaged PS II were obtained;thus, detection with higher (such as ns or ps) time resolution,technically possible with the described setup, was not necessaryfor this particular experiment.To additionally verify that undamaged data were indeed

recorded, we used several approaches. (1) Data were visuallycompared to previously published cryogenic S1 PS II spectra24

and found to be identical in terms of spectral shape.Unfortunately, we could not compare the first moment ofour PS II spectrum to that reported previously for the S1 stateas no data were provided for a reference compound. (2) Werecorded PS II, S1 state XES at 80 K using the Johanssonspectrometer design (Figure S2, Supporting Information) thatallows sufficient space to fit a microfridge assembly into thesample position (see the Supporting Information Materials and

Methods section). Obtained RT data were comparable withthose taken at 80 K. The value of the first moment for RT PS IIS1 state XES agreed well with the value obtained for the low-temperature spectrum recorded with the same spectrometer,Figure 3. Overall, the X-ray emission spectral shape obtained atRT and low T are similar, indicating that no significant chargeredistribution is happening within the OEC upon PS IIfreezing. (3) We did additional analysis of RT Mn K-edgeXANES (Figure S4, Supporting Information) with equivalentX-ray exposure. Many other studies have documented XANESof the S1 state and the subsequent X-ray-induced damage,making it an ideal test to validate our RT damage thresh-old.14,16,33 Rough XANES scans were taken over short timescales (∼23s scans, equivalent to a 4.5 ms exposure at theintensity used for emission measurements with a monochro-matic X-ray beam) with a seven-times-reduced incident flux toobserve the main edge shift. No shifts were detected withexposures equivalent to 100 ms of the full flux utilized in theXES experiment.After confirming the time window for the collection of

undamaged data, we proceeded to analyze the time course of X-ray-induced damage. The sensitivity of the Kβ1,3 line position tothe Mn redox state is used to monitor the photoreduction ofthe S1 state (containing two MnIII and two MnIV ions), resultingin the formation of MnII.14,16,33 The accumulation of MnII ionsis reflected in a shift to higher energies in the X-ray emissionspectra. In Mn Kβ emission spectra, the splitting of the Kβ′ andKβ1,3 lines is due to the exchange interaction between the 3phole and the valence electrons on the Mn 3d level. Thus, thissplitting is sensitive to the oxidation state of Mn.20,32 With thedecrease in oxidation state from MnIV,III to MnII, the Kβ1,3 peakshifts toward higher energies as the number of unpairedelectrons increases on the 3d level, resulting in larger splittingof the Kβ′ and Kβ1,3 lines, Figure 2B, C.12,20,24,32 Increases inthe MnII concentration as a function of X-ray exposure aredetermined by comparing the experimental spectra withcalibration spectra. Calibration spectra contain different ratiosof MnII in solution to undamaged S1 state data. Note that theaddition of 10% of MnII into undamaged data results in a verysmall shift; thus, error bars are included in Figure 3. Typically,for damage analysis, ∼60−100 ms (RT) and ∼60 s (lowtemperature, LT) worth of 2D-PSD exposures are averagedtogether to improve S/N. Assuming a linear response of thepeak shift to the content of MnII in the sample over small timescales (∼ms) as a first approximation, we can argue that thedamage observed in an averaged set of exposures (100 ms forRT and 60 s at 80 K) actually reflects the damage caused by halfof the dose deposited during that time frame plus the dosedeposited in the time interval(s) preceding. Note that we donot assume this linearity across the entire damage profile, onlyin “short” averaged exposures. This approximation therefore ismuch less accurate for the LT data due to the longer exposuretimes and causes some shape distortion of the damage profile.The main purpose however, is to accurately depict the dosagesover which the damage occurs. Without applying thisapproximation, the damage threshold would be skewed towardmuch higher dosages. The ±10% error bars account for thepossible uncertainty resulting from a visual comparison of theexperimental and calibration spectra, particularly whencomparing data having different energy resolution and thereforedifferent spectral shape (note that 80 K measurements wereonly possible with the Ge440 Johansson spectrometer design).The dose dependence of the MnII content (80 K) obtained

Table 1. Comparison of the Experimental Parameters forContinuous Beam and Pulsed Mode

experimental characteristics 20-ID:monochromaticbeam

BioCARS: pinkbeam

excitation energy (keV) 7.09 or 7.5(indicated in thetext)

peak energycentered at 7.85keV

photon flux ∼1012 photons persecond

∼7 × 1010 photonsin 22 μs pulse

spot size on the sample (μm2 −vert. × horiz.)

∼106 × 85 ∼43 × 120

shortest exposure 20 ms 22 μsdose delivered in the shortestexposure, photons/μm2

2.2 × 106 1.36 × 107

dose delivered in the shortestexposure, Gya

0.26 × 104 1.4 × 104

pink beam repetition rate n/a 41.1 HzaDosages in grays were calculated assuming a sample thicknessequivalent to one penetration length (distance at which the X-rayintensity is attenuated by a factor of e, approximately equal to 2.7times) at the Mn Kβ emission energy (6490 eV). For the PS IIsolution approximated as water, one penetration depth is equal to 520μm. For the measurements at BioCARS, the incident energy wasassumed to be 7.85 keV, while the real incident beam had a spread ofenergies centered at 7.85 keV.

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here is similar to that previously reported by the analysis of MnXANES at 100 K (Figure S6, Supporting Information).14

Figure 3 clearly demonstrates the dramatic increase in therate of X-ray damage at RT. Previously, the progression of X-ray-induced damage to PS II at RT was accessed by Mn K-edgeXANES.15,34−36 The RT dependence of damage on the photondose obtained here is also similar to that previously reported(Figure S5, Supporting Information). However, one mustdistinguish these two analyses (XANES and XES) by theirdifferent rate of dose deposition. Previous XANES analysis useda flux of 1−5 × 1012 photons/s/mm2 15,18 as compared to 1.1 ×1014 photons/s/mm2 of monochromatic beam in this XESstudy. The comparison indicates that even with a considerablyincreased (20 times) rate of dose deposition, the damageremains proportional to the accumulated dose and does notdisplay a dependence on the rate of dose deposition. InGrabolle et al.,15 a lag phase (about 10 s with 1 × 1012 photons/s/mm2

flux resulting in 1 × 1013 photons/mm2 of totalexposure) was reported to precede the onset of X-ray damage.This exposure converted to units of μm−2 corresponds to 1 ×107 photons/μm2, which is comparable to the flux depositedduring the first 80 ms of our XES experiment with themonochromatic beam, Table 1. As we could not visualize anydamage in the 60−100 ms exposure time, our results do notcontradict earlier observations of a lag phase. To further probethe effect of dose deposition rate on the progression of X-ray-induced damage to the metal center in the protein, weperformed measurements with a pink X-ray beam of highintensity, delivered to the sample in the form of 22 μs pulses.By doing so, we increased the rate of dose depositionapproximately 3000 times. We have not observed an increasein the rate of X-ray-induced damage under these conditions.Rather, some decrease in the damage was noted, Figure 2C.The statistics of the data set obtained with the pink beam werenot sufficient for a detailed analysis of damage progression.With a maximum of 20 pulses, we did not achieve full proteindamage. This experiment will be repeated in the future forfurther investigation of damage progression under theseconditions.The obtained damage threshold for RT X-ray studies of PS II

confirms that X-ray-induced damage will not prevent reliableRT analysis of the S states of the Kok cycle. This includes theproposed S4 state (lifetime of a few milliseconds) and itsassociated electron transfer and bond formation dynamics. Weexpect other metalloproteins to be similar11 or less sensitive toX-ray-induced damage, and thus, our technical demonstrationopens the area of RT XES analysis of metalloproteins withsynchrotron sources. In addition, serial femtosecond crystallog-raphy, which is currently under development at free-electronlasers,8,13,37,38 can be augmented with the described XES setupto monitor the redox state on metal cofactors in the conditionof crystallographic experiments. The present experimentalmethodology is readily modified to conduct time-resolvedanalysis of other metalloproteins and to perform laser pump/X-ray probe experiments on proteins and diluted molecular orsolid-state systems. Its simplicity should allow for a broadadaptation, and its time resolution is limited only by the timestructure of the X-ray source.

■ EXPERIMENTAL METHODSPS II-enriched thylakoid-membrane particles were preparedfrom spinach.39,40 The oxygen evolution of 300 μmol O2/(mgChl·hr) was measured by a Clark-type electrode. Sample

preparation, handling, and storage environments were com-pletely dark, save for dim green light. Samples were maintainedon ice during preparation.XES spectra were collected at the Advanced Photon Source

(APS) at Argonne National Laboratory on beamlines 20-IDand BioCARS.32 At 20-ID, a Si(111) monochromator was usedto prepare the monochromatic beam (Table 1), which wasfocused using Rh-coated KB mirrors, and a He-filled ionchamber was used to monitor the intensity of the X-rays, I0.The pulsed pink beam was focused using a KB mirror systemwith the beam reflecting off of the Si stripe to reject higherundulator harmonics. We typically refer to the X-ray beam afterpassing through the KB mirror system as a “pink beam” becauseonly the low-energy component, below the mirror cutoff, isreflected; see the Supporting Information. A high-heat-loadwhite-beam X-ray chopper was used to produce a pulse traincomposed of 22 μs long X-ray pulses at a rate of 41.1 Hz. Thisconfiguration was used for the radiation damage studies. ASi(111) channel-cut monochromator was periodically insertedinto the pulsed pink beam ∼0.5 m upstream of the fluorescencespectrometer (described below) to enhance the energyresolution for calibration purposes. The monochromator(s)was (were) calibrated via the KMnO4 pre-edge, and MnO XESand Fe foil XANES were taken periodically to correct for anyshift in energy calibration. XES emission spectra were recordedusing a Pilatus (Dectris) 2D-PSD.The two miniature XES spectrometer (miniXES) config-

urations employed have very different characteristics. The firstinstrument uses the Ge 440 reflection in a Johanssonarrangement13,29 and allows for a 100 eV collection rangecontaining the Kβ′, Kβ1,3, and Kβ″ peaks and demonstrates aninstrumental energy resolution of ∼1.5 eV, Figures 1D and S2and S3 (Supporting Information). The second instrument usesthe GaP 440 reflection in a von Hamos configuration28,31

(Figure S1, Supporting Information). This von Hamos designhas a decreased collection range of 50 eV, including only theKβ main lines and higher-energy resolution of ∼0.3 eV, Figures1C, D, and S3 (Supporting Information).24 The Mn Kβ spectraobtained for various Mn oxides corresponding to the differentoxidation states of Mn were in agreement with previouslypublished results20,24 and are not shown here.An in situ calibration of the detector pixels is achieved by

measuring the positions of the elastic scattering peaks whilescanning the monochromator through the desired X-rayemission energy range, Figure S3, (Supporting Information).This is done after the completion of the emission measure-ments. The calibration methodology has been described indetail elsewhere.26,28,29 The 2D-PSD exposures were equivalentto either 7 or 20 ms of irradiation per sample point.Approximately 25 images were taken per point, resulting in0.5 s of total exposure. To resolve the damage in time, wetypically added together ∼60−100 ms (RT), ∼60 s (LT), andthree pulses worth of 2D-PSD exposures to improve S/N; seeFigures 2, 3 and S5 and S6 (Supporting Information).

■ ASSOCIATED CONTENT

*S Supporting InformationSpectrometer, sample, and experimental details, Figures S1−S6.This material is available free of charge via the Internet athttp://pubs.acs.org.

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■ AUTHOR INFORMATIONCorresponding Author*E-mail: ypushkar@purdue.edu.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe research at Purdue was supported by the DOE, Office ofBasic Energy Sciences DE-FG02-10ER16184 (Y.P.) and theNSF Graduate Research Fellowship under Grant No. 0833366(K.D.). Research at the University of Washington is supportedby the DOE, Office of Basic Energy Sciences DE-SC0002194.PNC/XSD facilities at the Advanced Photon Source andresearch at these facilities are supported by the U.S.Department of Energy, Basic Energy Sciences, a MajorResources Support grant from NSERC, the University ofWashington, Simon Fraser University, and the AdvancedPhoton Source. Use of the Advanced Photon Source, an Officeof Science User Facility operated for the U.S. Department ofEnergy (DOE) Office of Science by Argonne NationalLaboratory, was supported by the U.S. DOE under ContractNo. DE-AC02-06CH11357. Use of the BioCARS wassupported by the National Institutes of Health, NationalCenter for Research Resources, under Grant NumberRR007707. The time-resolved setup at BioCARS was fundedin part through a collaboration with Philip Anfinrud (NIH/NIDDK).

■ ABBREVIATIONSOEC, oxygen-evolving complex; XES, X-ray emission spectros-copy; 2D-PSD, two-dimensional position-sensitive detector;RT, room temperature; LT, low temperature; PS II, photo-system II; XANES, X-ray absorption near-edge structure

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