Real-time capture of domain movements during copper amine oxidase catalysis by mix-and-inject serial crystallography

Domain: physics
Published: 2025-12-18
Editorial outcome: minor_revision

Abstract
Proteindynamicsplayacrucialroleinvariousphysiologicalfunctions,includingenzymecatalysis.Toexploreconformationalchangesduringenzymecatalysis,weconductmix-and-injectserialcrystallography,anadvancedtechniquetocapturetime-resolvedproteinstructuresinrealtime,usingthemicrocrystalsofbacterialcopperamineoxidasecontainingaprotein-derivedquinonecofactor.Within50msofmixingthemicrocrystals(<4μm)withapreferredsubstrate(2-phenylethylamine)underanaerobicconditions(reductivehalf-reaction),weobservedomainmovementsassociatedwithsubstratebindingandformationofametastablereactionintermediate,aproductSchiff-baseofthequinonecofactor.At100–1000msaftermixing,conformationaltransitionfromaminoresorcinoltothesemiquinoneradicalformsofthereducedcofactorprogressesgradually,likelydependingonthereplacementoftheproductaldehydebythenext-cycleaminesubstratethattriggersthecofactorconformationalchange.Overall,thisstudyprovidesstructuralinsightintoenzymecatalysisaccompanyingtheactive-siteconformationalchangesthatarehardlyscrutinizedbystudiesinsolution.

Introduction
Proteins exhibit various motion modes over a wide range of time scales, including the thermal vibration of individual bonds in the femtosecond range, rotation or conformational changes of local structuresinthepico–microsecondrange,anddomain/wholeprotein movementinthemillisecond(ms)range1,2.Suchdynamicsplayfundamental roles for proteins in exhibiting various cellular functions1. Forreceptorsandenzymes,themotionsinducedbybindingofligands andsubstrates/effectorsareessentialforspecificrecognition.Thermal fluctuation of the proton/hydrogen acceptor/donor is linked to enzymecatalysis viahydrogen tunneling3.Adjustmentofthe proto- nationstatesoftheactivesiteviaconformationalchangesinthesur- roundingresidues is crucial to rearrange the hydrogen bonds or modulatethereactionefficiency.Enzymesoptimizetheenvironment oftheiractivesitesandacceleratespecificreactionsbymodulatingthe proteindynamics.

Development of mix-and-inject serial crystallography (MISC), which is an application of serial femtosecond X-ray crystallography (SFX)usinganX-rayfree-electronlaser(XFEL)4–9,hasbeenatechnical breakthrough to investigate the millisecond changes in the atomic structures associated with enzyme catalysis10–14. In MISC, a micro- crystal suspension of an enzyme is mixed with the substrate and ejectedtotheXFELinteractionpoint,enablingthedirectobservation of the time-resolved structural changes during the catalytic reaction10–14.Namely,MISCisacrystallographicversionofaclassical continuous-flow or stopped-flow technique used for spectro- photometric detection of time-resolved reaction intermediates formedduringenzymecatalysisinsolution15,16.Inaddition,laserexci- tation of chromophores contained in the enzyme system, which is typically used for initiation of the reaction in most pump-probe experiments,isnotrequiredinMISC.Theseaspectsunderscorethe versatilityofMISC,makingitapplicabletovariousenzymereactions.

We focus on copper amine oxidase, a ubiquitous enzyme that occurswidelyinprokaryotesandeukaryotesandparticipatesinvar- iousphysiologicalfunctionssuchasmicrobialaminemetabolismand plant germination17. Its peptidyl cofactor, 2,4,5-trihydrox- yphenylalaninequinone(TPQ),isgeneratedviaametalion-dependent post-translational modification17–19. TPQ cofactor plays the central redoxroleintheoxidativedeaminationofprimaryaminesviaaping- pongmechanismconsistingofreductiveandoxidativehalf-reactions (SupplementaryFig.1).Intheinitialreductivehalf-reaction,oxidized formofthecofactor(TPQox)reactswiththesubstratetoformasub- strateSchiff-base(TPQssb),whichisconvertedintoaproductSchiff- base(TPQpsb)andhydrolyzedtoformthefirstreactionproduct(a corresponding aldehyde) and 2e−-reduced form of the cofactor (TPQamr).TPQamrremainsinequilibriumwiththeradicalformofTPQ (TPQsq) by transferring 1e− to Cu(II). Subsequently, oxidative half- reactionstartswiththedioxygenaccepting2e−/2H+fromtheTPQamr/ Cu(II)orTPQsq/Cu(I)coupledependingontheenzymesource,forming thesecondproduct(HO)andaniminoquinoneform(TPQimq),which 2 2 finallyrevertstoinitialTPQoxviahydrolysisorTPQssbviatransimination, releasingammonia.Weextensivelystudythemechanismofreductive half-reaction ofthe enzyme from Arthrobacter globiformis (AGAO) by conductingin-solutionkineticstudiesviastopped-flowmeasurements anddeterminethekineticconstantateachreactionstep.Inaddition,the structures of reaction intermediates that form in the TPQamr/TPQsq equilibriumstatearedeterminedbyX-rayandneutroncrystallography at high resolution16,20–27 (Fig. 1). Taking advantage of these previous findings,wehereshowanMISCstudyofAGAOmicrocrystalsconducted forfurtherunderstandingoftheinterplaybetweentheproteindynamics andcatalyticmechanisms.

Results
MISCmeasurementanddataprocessing
ToobtainsufficientdiffractiondataviaXFELirradiation,weprepared AGAOmicrocrystalswithauniformsizeofapproximately2.5×4μm (parallelogrammicplates;thickness<1μm)andsuspendedthemina crystallization buffer (approximately 108–109mL−1), as previously described28.Thecrystalsuspensionandsubstratesolutioncontaining 2-phenylethylamine (2-PEA; the most preferred substrate29 with K =2.5μM,k =76s−1determinedatpH6.8,30°C16)wereprepared m cat under anaerobic conditions at pH 9.0 so that the pH-dependent equilibrium between TPQox and TPQsq shifts toward TPQsq 24, and mixed inside the injector capillary to give the final substrate con- centrationof4mM.Thereactiontimeelapsedaftermixingtheenzyme crystalswiththesubstrate(hereafterdefinedasthedelaytime[Δtm]) was controlled using injector capillaries with different distances betweenthemixingandirradiationpointsandchangingthetotalflow rateofthereactionmixture(seetheMethodssectionfordetails).XFEL diffractiondatawereobtainedatΔtm=22,25,50,100,200,300,400, 500,and1000ms.Assumingthatthereductivehalf-reactionincrys- talsproceedsrapidlyasinsolutionwiththedecreasingorderofrate constants(seeSupplementaryFig.1;k1>k2>k3>k4)23,theΔtmwasset atincreasingtimeintervalstocapturerapidstructuralchangesinan early stage of the reaction. To obtain the initial enzyme structure (containingTPQox)beforestartingthereaction,single-flowSFX30,31of thecrystalsuspensioninabufferwithout2-PEAwasconductedandthe resultantdatasetwasdefinedasΔtm=0ms.Subsequently,diffraction imagescollectedatallΔtmpointsweresubjectedtodataprocessing.

Duringtheinitialintegrationofdiffractiondata,wenotedbimodal distributionsofthelengthsofaunitcellparameter,a-axis,amongthe indexedimagesatΔtm=22,25,and50ms(Fig.2,middle),unlikethe normaldistributionsofotherparameters(b,c,α,β,andγ)withsingle peaks (SupplementaryFig. 2). Histograms of the number ofimages countedfora-axisvalueswerebestfittedtothesumoftwonormal distributionpeaks.Thea-axislengthsatthetwopeakswereestimated to differ by 0.68–0.69Å in the curve fitting of the three data sets, indicatingthepresenceoftwomicrocrystalregionswithunitlatticesof differenta-axislengths.AstheAGAOmicrocrystalsizeconsiderably exceeded the XFEL beam diameter (1.5μm), various microcrystal regionswereirradiatedbythepulsedbeam(SupplementaryFig.3). Therefore, for subsequent structure refinement, we divided the dif- fractiondatasetsatthethreeΔtmpointsintotwogroupscontaining less than10% of the counterpartdistribution peaks(Fig. 2, bottom) designatedas22–50ms(L)and(S)forlongandshorta-axes,respec- tively,and calculated them separately,together with the diffraction data sets at other Δtm points (0, 100, 200, 300, 400, 500, and 1000ms), which exhibited single distribution peaks in all unit cell parameters.Structuremodelswererefinedbyapplyingthestructures of TPQ intermediates produced during the reductive half-reaction with 2-PEA (Fig. 3a). All data collection and structure refinement detailsarepresentedinSupplementaryTables1and2.Differencesina- axis lengths between the (L) and (S) data sets at Δtm=22, 25, and 50ms were 0.81, 0.87, and 0.75Å, respectively, after final data processing.

Structurescapturedin50ms
Structuresdeterminedforthe22–50ms(L)datasetswereidenticalto theinitialenzymestructurebeforestartingthereaction(0ms),con- taining TPQox and four water molecules (W1–W4) in the substrate- binding pocket (Fig. 3b, above the line; Supplementary Fig. 4). Population of the TPQ structure without a bound substrate decreasedininverseproportiontoΔtm(Fig.3c).Theseresultssuggest thatthedatasetswithalonga-axisarederivedfromtheinnercore regionofthemicrocrystals(SupplementaryFig.3)andthatthediffu- sionoftheaminesubstratetakes50–100msuntilreachingthecore region that remains in the initial substrate-free TPQ structure. In contrast,structuresdeterminedforthe22–50ms(S)datasets,which possibly diffracted from the peripheral region of the microcrystals, showed the substrate-bound cofactor assignable to TPQpsb (Fig. 3b, below the line)16,21, as determined previously by conventional cryo- geniccrystallography32.PopulationoftheTPQpsbstructureincreased inproportiontoΔtm(Fig.3c).Hence,time-resolvedcrystalstructure containingTPQpsbwascapturedasametastablesynchronizedcatalytic intermediateinthisstudy.However,thepredictedfirstintermediate containingTPQssb(Fig.3a)wasnotcaptured,possiblyduetothemuch fasterratesofitsformationanddecaycomparedtotherateofsub- strate diffusion into the microcrystals. The a-axis shortening asso- ciated with substrate binding is discussed later in relation to unidirectional proteinshrinkage,includingdomainmovement.

Structurescapturedin100–1000ms
Diffraction data sets at Δtm=100–1000ms had the a-axis lengths correspondingtothoseofthedatasetsat22–50ms(S)(Supplemen- taryTable1),confirmingthatallAGAOmoleculesinthemicrocrystals reactedwith2-PEA.TherefinedstructuresdeterminedfortheseΔtm points, except for Δtm=1000ms, were essentially identical to pre- viouslyreportedstructures24,26,containinganequilibriummixtureof TPQamr andTPQsq (Figs.3band4a).TPQpsbhydrolysistoformTPQamr (Fig. 3a) proceeded slowly during 50–100ms. The slower decay of TPQpsb comparedtoits formation,even atΔtm=22ms,is likelythe reasonforitscaptureasametastableintermediate16,21.Occupancyof TPQamr relative to TPQsq, calculated after structural refinement, decreased gradually with the increase in Δtm and reached 0% at 1000ms(Fig.3c).Meanwhile,occupancyofconformeraofTyr296, which stabilizes the TPQsq radical by forming a strong hydrogen bond27,concomitantlyincreasedduring100–1000ms(Fig.4b).Hence, transitionfromTPQamr toTPQsqwasvisualizedinreal-timeduringthe laterstage(Δtm>100ms)ofthereductivehalf-reaction.Theorderof magnitudeofapparentrateconstantsofthesesteps(TPQpsb→TPQamr, 20s−1(1/[50ms]);TPQamr→TPQsq,1s−1(1/[1000ms]))wascompatible withthatoftherateconstants(102and39s−1,respectively)determined in solution at 4°C and pH 6.8 via stopped-flow measurements23, althoughtheratesincrystalswereslowerby5–40foldevenatahigher temperature(26°C).

Notably, substrate-derived electron densities in all structures refined forthedatasets atΔtm=100–1000mswere disconnected fromthoseofthereducedcofactors(Fig.3b)andcouldbeassigned asthereactionproduct, phenylacetaldehyde(PAA), oraminesub- strate,2-PEA(Fig.4a).Thetwoligandsarealmostindistinguishable byX-raycrystallographybecauseofverysimilarelectrondensities derivedfromthealdehydeoxygenandaminenitrogenatoms,but ourrecent neutron crystallographic study27unequivocally demon- stratedthebindingofanadditionalaminesubstratereplacingPAA, 2-PEA,tothereducedenzymecontainingTPQsq.Thisnewlybound 2-PEApossiblytriggerstheconformationalchangefromTPQamr to TPQsqviaelectrostaticinteractions27.Althoughthetimingatwhich PAA is replaced by 2-PEA during Δtm=100–1000ms remains unknown, the substrate-derived electron densities possibly repre- sentthemixtureofPAAand2-PEA.Atomictemperaturefactors(B- factors) of bound PAA/2-PEA also decreased gradually during Δtm=100–400ms and reached a constant level after 500ms in inverse proportion to the relative occupancy of TPQsq (Fig. 4b), indicatingthecompletereplacementofPAAby2-PEAstablybound inthehydrophobicpocket.

Methods
Samplepreparationandcrystallization
RecombinantAGAOwaspurifiedintheinactiveprecursorformand converted into the Cu(II)/TPQ-containing active form, as previously described16. Protein concentrations were spectrophotometrically determinedusingtheextinctioncoefficientat280nm(13.2)fora1% solutionoftheactiveform16.AGAOmicrocrystalswerepreparedusing acombinationofmicro-seedingandbatchcrystallizationmethodsin 1.05Mpotassium-sodiumtartratecontaining25mMHEPESbuffer(pH 7.4),aspreviouslydescribed28.ThepHofthemicrocrystalsuspension was adjusted to 9.0 by repeatedly replacing the upper buffer with 1.05Mpotassium-sodiumtartratecontaining25mMTAPSbuffer(pH 9.0)aftereachmicrocrystalsedimentation.ThefinalpHwasmeasured using a pH meter. Microcrystal size and concentration were deter- minedusingtheBZ-X710all-in-onefluorescencemicroscope(Keyence, Osaka, Japan). Subsequently, microcrystal suspension was filtered through a nylon mesh (pore size: 20μm; Sysmex, Kobe, Japan) via unforcedgravitationalsedimentationunderatmosphericpressure.To establish fully anaerobic conditions, the microcrystal suspension (approximately 108–109 crystals mL−1; protein concentration, 30mgmL−1) and substrate solution in the crystallization buffer (pH 9.0)wereplacedinaglovebox(AS-600PC;ASONE,Osaka,Japan)filled with N2 gas for at least 24h. Finally, plastic vessels containing the microcrystalsuspensionandsubstratesolutionweredoublyenclosed inanO2-impermeableplasticbaginsidethegloveboxandtransported to the SPring-8 Angstrom Compact Linear Accelerator (SACLA) beamline,BL2(Hyogo,Japan).

Datacollectionandprocessing
DiffractiondatawerecollectedattheSACLAbeamline39viaMISC10–14. Two types of injectors were used for the measurements: A double- focusingmixingjet40andgasdynamicvirtualnozzleconnectedviaa PEEKtubetoaT-junctionmixer11.Briefly,microcrystalsuspensionand substrate solution were fully loaded into 15-mL and 50-mL sample reservoirs, respectively, in an anaerobic glovebox (Coy Laboratory Products, MI, USA) at the beamline. The reservoir containing the microcrystalsuspension wasreciprocally and gentlyrotated to pre- ventmicrocrystal sedimentation. For MISC,the tworeservoirswere connectedtoahigh-performanceliquidchromatographypump(LC- 20AD; Shimadzu, Kyoto, Japan) with a water-filled PEEK tube. The microcrystalsuspensionandsubstratesolutionwerepushedoutby pumpingwaterviaapistoninthereservoirs,avoidingdirectcontactof thesampleswithwater,anddeliveredthroughthePEEKtubetothe liquid injectors with an inner diameter of 75 μm. Flow rates of the microcrystal suspension and substrate solution were 0.02 and 0.23mLmin−1, respectively, with the double-focusing mixer (Δtm: 50–400ms)and0.125mLmin−1(forboth)withtheT-junctionmixer (Δtm:500and1000ms).Themicrocrystalsuspensionmixedwiththe substratesolutioninsidetheinjectorwasejectedtotheXFELirradia- tionpointonadiverseapplicationplatformforhardX-raydiffraction at SACLA (DAPHNIS)31,41. Δtm was controlled by changing the flow distances inside the injector or T-junction system for Δtm=50–1000msataconstanttotalflowrate(0.25mLmin−1)orset- tingthetotalflowrateat0.57and0.50mLmin−1forΔtm=22and25ms, respectively,usingtheinjectorwiththeshortestflowdistance.Δtm was calculated basedonthedistancebetweenthemixingandXFELirra- diationpoints,capillaryorPEEKtubewidth,andflowrate.Forsingle- flowSFXexperiments(thedatasetwasdefinedasΔtm=0ms),5-fold dilutedmicrocrystalsuspensionalonewasdeliveredataflowrateof 0.25mLmin−1 with the gas dynamic virtual nozzle26. Data sets for a single-flowSFXofthesubstrate-freeinitialenzyme(0ms)andMISCat Δtm=500and1000mswerecollectedfirst(proposalnumberofthe Japan Synchrotron Radiation Research Institute: 2020A8013), then those at Δtm=22–50ms (2021A8013), and lastly those at Δtm=100–400ms(2021B8029).AsanaverageoftheMISCmeasure- ments,operationtimeofabout8hincludingtheadjustmenttimeand atotalof300mgproteinwererequiredforcollectingthediffraction imagessufficientforasingledataset.

XFELdiffractionpatternswereobservedatanambienttempera- ture (approximately 26°C). A MultiPort Charged Coupled Device detector was set up with a short-working-distance octal sensor arrangement42atasample-to-detectordistanceof50mm.XFELbeam was focused at a beam diameter of 1.5μm in full-width at half- maximumusingacoupleofellipticalmirrorsintheKirkpatrick–Baez geometry43. The pulse beam had a wavelength of 1.24Å (10.0keV), repetition rate of 30Hz, temporal width within a 10-femtosecond duration,andpulseenergyofapproximately640μJatthelightsource. Datacollectionwasguidedviareal-timeanalysisusingtheSACLAdata- processingpipeline44.DatawereretrievedusingtheSACLAapplication programminginterface45andfilteredusingCheetah46,witheachimage over 20 spots accepted asa hit. Hitimages wereindexed and integrated using CrystFEL v 0.9.1 package47. Detector background estimatedbyaveragingthedarkimageswassubtractedfromthedif- fractionpatterns.DetectormetrologywasrefinedusingGeoptimiser48. IndexingwasperformedusingXGANDALF49.Datacollectionandpro- cessingdetailsarepresentedinSupplementaryTable1.

Structuresolutionandrefinement
Initial phase was determined via molecular replacement using Phaser50.ThesearchmodelwasbasedonthecoordinatesoftheAGAO monomer(ProteinDataBankaccessioncode:3wa2)afterremovingall ligands,includingwatermoleculesandmetalions.Refinements,elec- trondensitymapcalculations,andsolventmoleculeassignmentswere performedusingPhenixv1.19.2package51.Occupanciesofalternate conformersfoundinseveralresidues,includingTPQandTyr296,were refinedusingoccupancyrefinementinPhenix,withtheinitialvalues settoequaloccupancy(=0.5)foreachconformer.Manualrebuilding wasperformedusingCootv0.9.852.Duringrefinement,watermole- culesandotherligands,suchasCuandTPQ,wereaddedstepwiseto themodel.Specifically,TPQox,TPQpsb,TPQamr,andTPQsqmodelswere constructed based on the Fo–Fc polder omit map. PyMOL (Schrö- dinger, New York, USA) was used to construct figures. Statistical information related to the refinement process are provided in Sup- plementaryTable2.

X-raydiffractionanalysisoflatticeorientation
TodeterminethelatticeorientationinalargeAGAOcrystal,theoxi- dized form of enzyme was crystallized initially at pH 7.4 in dialysis buttons16.Thecrystalsgrowntoamillimetersizewerefurtherdialyzed against 1.05M potassium-sodium tartrate containing 25mM TAPS buffer (pH 9.0). The buttons were then transferred to the crystal- lizationbuffer atpH 9.0 containing 45 % (w/v) glycerol ascryopro- tectantandkeptovernight.Thecrystalswerefrozeninliquidnitrogen. X-ray diffraction images of the crystals were measured under cryogenic conditions at a beamline station BL44XU in SPring-8 and were processed with DIALS53. The lattice orientation was determinedwiththedirectionsoftherespectiveunitvectors(a/|a|,b/| b|,andc/|c|)ofa-,b-,andc-axesofthereallattice,whichwerecalcu- latedwitha*-,b*-,andc*-axesofthereciprocallatticeobtainedfroma crystalsettingmatrixAinaDIALSlogusingthefollowingequations (alsorefertothesourcefile):a=(b*×c*)/V*,b=(c*×a*)/V*,c=(a*×b*)/ V*, and a·a* = b·b* = c·c* = 1, where V* (volume of reciprocal lattice)=a*·(b*×c*).

Dataavailability
All crystallographic models (refined against the raw structure factors described in the article) and associated reflection data have been depositedintotheProteinDataBank(PDB)underaccessioncodes9LZD (structure of the TPQox form determined by single-flow SFX); 9LZ8 (structure at Δtm=22ms with long a-axis); 9LZ7 (structure at Δtm=22mswithshorta-axis);9LZA(structureatΔtm=25mswithlong a-axis);9LZ9(structureatΔtm=25mswithshorta-axis);9LZC(structure at Δtm=50ms with long a-axis); 9LZB (structure at Δtm=50ms with short a-axis); 9LZ3 (structure at Δtm=100ms); 9LZ4 (structure at Δtm=200ms); 9LZ5 (structure at Δtm=300ms); 9LZ6 (structure at Δtm=400ms);9LZE(structureatΔtm=500ms);and9LZF(structureat Δtm=1000ms).PDB3WA2wasinitiallyusedasthestartingmodelfor structuredetermination.ThesourcedataunderlyingFigs.2,3c,4b,6a and 7a, and Supplementary Figs. 2, 6, and 7c are provided with this paperasaSourceDatafile.Sourcedataareprovidedwiththispaper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Murakawa et al focuses on domain movements during copper amine oxidase catalysis.\nMix-and-inject serial crystallography, an advanced technique to capture time-resolved protein structures in real time provides\nstructural insight into enzyme catalysis accompanying the active-site conformational changes that are unobservable by\nstudies in solution.\nThis study is very interesting not only to fellow specialists working in related areas but also to the general readership of\nNature Commun., attracting attention, and therefore is recommended for publication in this journal.\nHowever, the followings can be considered before the publication.\nIn Fig.3a, is the position where Phenylethylamine(2-PEA) enters during the reaction from TPQpsb to TPQamr between\nTPQamr and TPQsq?\nIn Fig.2 and Fig.3, the observed difference of 3mS from 22mS to 25mS seems to be larger than others. Are there any reason\nof that in the measurement?\nIn Fig.3, how do you measure the average occupancy of TPQamr and TPQsq? Is the way different form that used in Fig.4?\nLines 142-143\n\"The orders of the observed rates of these steps (TPQpsb → TPQamr [50–100 ms] and TPQamr → TPQsq [100–1000 ms])\nwere compatible with the rate constants (102 and 39 s−1, respectively) determined in solution at 4°C and pH 6.8 via\nstopped-flow measurements23 but slower by 10–50 fold.\"\nIs the expression of \"orders ---- were compatible with the rate constants\" correct?\nLines 239-241\n\"Collectively, these results suggest that the gradual transition from TPQamr to TPQsq during 100–1000 ms (Fig. 3c) is due to\nthe slow replacement of PAA by 2-PEA depending on the diffusion-controlled rates of desorption of PAA from the substrate-\nbinding pocket and re-binding of 2-PEA.\"\nAt \"Line 87\", \"under anaerobic conditions at pH 9.0 so that the pH-dependent equilibrium between TPQamr and TPQsq\nshifts toward TPQsq24\" is written, while the gradual transition from TPQamr to TPQsq during 100–1000 ms is due to the\nslow replacement of PAA by 2-PEA.\nAt least in crystals, this equilibrium reaction does not occur depending on pH?\nIn Extended Fig.2, only data of 0 ms was measured in solutions without 2-PEA.\nIs it not appropriate to compare with the data from 22 minutes onwards?\nIn Extended Fig.2, actually, the length of a-axis in data of 0 ms is different from each of those of (L) and (S).\nIt is better to show data of 0mS and that of 22mS-1000mS, respectively.\n\"Method section\" (Line 356-424\" is described between \"References section\": (Lines 263-354) and (Lines 426-460).\nIs it OK?", "reviewer_2": "Review Manuscript “Real time capture of domain movements during copper amine oxidase catalysis by mix-and-inject serial\ncrystallography”\nThe authors Murakawa and collaborators present a very comprehensive time -resolved serial crystallography study on a\ncopper amine oxidase with time point ranging from 22 ms to 1 s. This work follows the reaction cycle of AGAO mixed with 2-\nPEA in anaerobic conditions using the sample delivery method MISC (mix-and-inject serial crystallography). All the\nexperimental details (apart from the stop flow experiments) are provided either in the methods or the supplementary\ninformation. We have only minor remarks on this paper, and we strongly support the publication in Nature Communications.\nSI\n• Additional information on the pH/ pKa and rates of the reactions in solution in the scheme in Extended Data Fig. 1 would\nbe helpful.\n• Additionally, the stopped flow data that has been used to calculate these rates and is referred to several times in the text\nshould really be in the SI, and the details should be in the methods. Or if this is already published data then it needs a\nreference.\n• Could you add a figure giving the the chemical structures of PAA and 2-PEA\n• Extended figure 5 & 6: 2-PEA diffusion in solvent channel: there is a program that allows the user to visualize the solvent\nchannel: MAP_CHANNELS by Juers and Ruffin (J. Appl. Cryst 2014). This program or maybe a newer program could help\nto measure the solvent channel, and it could help to support or explain why 2D diffusion model is preferred.\nManuscript\n• Abstract\no Line 38-39: \"active-site conformational changes that are unobservable by studies in solution\" is quite a broad statement\nand may not be true, maybe NMR in solution could help. Could you please comment.\n• Results / MISC measurement and data processing section\no Line 86: A reference is missing for \"(2-PEA; the most preferred substrate)\"\n• Discussion section:\no Since there are 2 different redox states of Cu between TPQamr and TPQsp, could X-ray Emission Spectroscopy help to\ncorroborate some hypothesis about the slow replacement of PAA by 2-PEA? If you had access to this technique, would you\nuse it, and would it help?\no The temperature differences between the stopped flow experiments (15 C) and MISC experiments (26 C) could also be a\nreason for differences in the rates.\nMethods\n• We would like a bit more details on the sample consumption. On average how many ml / ul (or mg of protein) have been\nused for each time point?\n• The MISC sample delivery method has been used quite extensively in the US with Schmidt and collaborators. We were not\naware that SACLA is offering this sample delivery method. Could you please give more details on MISC: is it provided by the\nfacility? Or by collaboration? Is it the same nozzle design used by Schmidt et al?\nAdditional comments from the team review\nLine 28 (within the abstract)\nPlease state somewhere that the time-revolved studies are for the reductive half reaction.\nLine 32:\nPlease state the average crystal size used for the tr-SFX work\n(see line 84 for values; 2.5 × 4 μm parallelogram plate × < 1 μm thick).\nLine 33: “amine substrate”\nambiguous, please state what substrate was used for this study (2-phenylethylamine).\nLine 54\na microcrystal suspension of an enzyme is mixed with the substrate and ejected to the XFEL emission point,\n“emission” should probably be “interaction”\nLines 56 – 57\nNamely, MISC is a crystallographic version of a classical continuous-flow or stopped-flow technique used for detection of\ntime-resolved reaction intermediates formed during enzyme catalysis in solution15,16.\nReads better as:\nNamely, MISC is a crystallographic version of a classical transient kinetic, spectroscopic analysis of continuous-flow or\nstopped-flow technique used for detection of time-resolved reaction intermediates formed during enzyme catalysis in\nsolution15,16.\nLine 86 “(2-PEA; the most preferred substrate)”\nPlease provide KM, and kcat values for relevant pH values here and provide appropriate references.\nLine 96: “we noted unusual distributions of”\nAs you state a few lines later, this is fit to a simple bimodal distribution; therefore, please replace “unusual” with “bimodal”.\nLine 101 and following sentences: “indicating the presence of two microcrystal regions with unit lattices of different a-axis\nlengths...”\nThis sentence implies two different microdomains within the microcrystals, which may be true and could be consistent with\nthe partially bleached crystals shown with macrocrystals in Extended Figure 3. However, the bimodal distribution only\ndemonstrates that successful indexing of the tr-SFX images reveals that there are two, non-isomorphic populations in the\nwhole slurry. The illustration in Extended Data Fig. 3 suggests that the two species result from X-ray a narrow X-ray beam\npassing through the flat face of the crystal plate. The X-ray beam shape is Gaussian, not a top-hat profile, and so there is not\nsuch a big difference between the crystal and X-ray beam sizes. Indeed, the thin plate dimension is smaller than X-ray\nbeam. Moreover, the X-ray beam passed through randomly oriented crystals; it is possible that indexing may fail if/when the\nX-ray beam passes through the bleached and non-bleached regions of the microcrystals. Given that the unit cell changes by\nsuch a small amount (~0.3%), it is likely that indexing probably still succeeds in these cases too.\nAnalysis of the data from Extended Data Table 1 indicates that the a-axis unit cell edge for all of the timepoint datasets is\ndifferent compared to the t = 0, ground state. The % change for the “Long” cell datasets are all small and positive 0.295,\n0.348, and 0.191 for the 22, 25, and 50 ms datasets, respectively. However, the % change for the “Short” cell species and all\nof the longer timepoints across the series are also all small but negative (-0.124, -0.202, -0.197, -0.191, -0.217, -0.238,\n-0.243, -0.321, -0.295). The distribution for first three timpoints are clearly bimodal, but more homogeneous for the later six\ntimepoint data.\nFigure 2 and Extended Data Table 1:\nPlease annotate the image (or indicate in the legend) the unit cell dimension for the peak of each species. Are these the\nsame values reported in Extended Data Table 1?\nExtended Data Fig. 3\nPlease comment on why the image of the large macrocrystal indicates that the crystal had not fully reacted even after 30\nminutes. The tr-SFX data assumes many thousands of random orientations to sample the whole Ewald sphere and thereby\nbuild up the complete dataset. So, the tr-SFX results with microcrystals indicates that only about 1 second is needed for a\ncomplete reductive half reaction?\nPlease support the statement, “strict directionality of substrate diffusion” with an analysis and appropriate figure(s) of the\ncrystal packing and solvent channels with respect to the observable crystal parallelogram plate morphology. If this analysis\nhas been done in another paper, then it should be referenced. For instance, examination of the lattice packing in PDB code\n3WA2 (with different unit cell edge lengths in C121 space group) clearly reveals two ~ orthogonal dimensions with rather\nlarge solvent channels and a third orientation with much smaller channels.\nExtended Data Fig. 6\nThis figure is a good place to show the crystal lattice packing and how it impacts access to the active sites within the lattice.\nLine 464 – 466: “(proposal 2017B8038, 2020A6507, 2020A8013, 2021A6606, 2021A8613, 2021B6606, 2021B8029,\n2022A6705, 2022A8020, 2022B6705, 2022B8013, 2023A6805, 2023A8009, 2023B6805, 2023B8013, 2024A8021, and\n2024B8016)”\nThis suggests that the team accessed SACLA during 17 different beamtimes spanning about 7 years to collect the data,\nwhich is fine and it is appropriate to indicate which SACLA beamtimes contributed to the publication. However, it is very\nlikely that some of the time was used for testing and development, while some of the time was used for the actual tr-SFX\ndata collection reported in this manuscript. Please indicate which of the proposals were used to collect the reaction timepoint\ndatasets and how long it took to collect each timepoint in particular for the data reported herein. Did diffraction data for any\ngiven timepoint dataset come from different beamtimes, and if so what fractions of the datasets for the different beamtimes\nwere merged?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Point-by-point response to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nIn Fig.3a, is the position where Phenylethylamine (2-PEA) enters during the reaction from TPQpsb to TPQamr\nbetween TPQamr and TPQsq?\nThank you for pointing out the incorrect position where 2-PEA enters into the reaction scheme shown\nin Fig. 3a. Based on our finding that 2-PEA triggers the transition from TPQ to TPQ (ref 27), 2-\namr sq\nPEA should enter the scheme after (but not before) the formation of PAA/TPQ and desorption of\namr\nPAA. Accordingly, we revised Fig. 3a.\nIn Fig.2 and Fig.3, the observed difference of 3mS from 22mS to 25mS seems to be larger than others. Are\nthere any reason of that in the measurement?\nThank you for the comment that we did not mention in the manuscript. This query appears to come\nfrom our inadequate description for how to set the delay time. Since the formations of TPQ and\nssb\nTPQ in crystals were assumed to be very rapid as in solution (887, 206 s−1, respectively,\npsb\ndetermined at 4 °C and pH 6.8; ref 23), a large structural change was probably observed for a short\nperiod (3 ms) in an early stage (Δt = 22–25 ms) of the reaction. We added the following sentence\nm\nat the bottom of the first paragraph of the Discussion section.\nThe formations of TPQ and TPQ in crystals were assumed to be very rapid as in solution (887,\nssb psb\n206 s−1, respectively, determined at 4 °C and pH 6.8)23, which could be the reason for a relatively\nlarge structural change observed for a short period (3 ms) in an early stage (Δt = 22–25 ms) of the\nm\nreaction (Figs. 2 and 3c).\nTo clarify the reason for setting the Δt at different time intervals, we also added the following\nm\nsentence in the first paragraph of the Results section.\nAssuming that the reductive half-reaction in crystals proceeds rapidly as in solution with the\ndecreasing order of rate constants (see Extended Data Fig. 1; k > k > k > k )23, the Δt was set\n1 2 3 4 m\nat increasing time intervals to capture rapid structural changes in an early stage of the reaction.\nIn Fig.3, how do you measure the average occupancy of TPQamr and TPQsq? Is the way different form that\nused in Fig.4?\nAs described in the Methods section, occupancies of alternate conformers found in several residues,\nincluding TPQ, were refined using occupancy refinement in Phenix, with the initial values set to\nequal occupancy (= 0.5) for each conformer. To clarify this, ‘including TPQ’ was replaced by\n‘including TPQ and Tyr296’ in the revised manuscript. The average occupancies of TPQ and\namr\nTPQ in the dimer, contained in the asymmetric unit, were used for indicating their relative amounts\nsq\nin Fig. 3c. The occupancies used in Fig. 4 (TPQ , green square) were identical with those of TPQ\nsq sq\nin Fig. 3c.\nLines 142-143\n\"The orders of the observed rates of these steps (TPQpsb → TPQamr [50–100 ms] and TPQamr → TPQsq\n[100–1000 ms]) were compatible with the rate constants (102 and 39 s−1, respectively) determined in solution\nat 4°C and pH 6.8 via stopped-flow measurements23 but slower by 10–50 fold.\"\nIs the expression of \"orders ---- were compatible with the rate constants\" correct?\nThank you for your comment. We corrected the sentence as follows.\nThe order of magnitude of apparent rate constants of these steps (TPQ → TPQ , 20 s−1 (1/[50\npsb amr\nms]); TPQ → TPQ , 1 s−1 (1/[1000 ms])) was compatible with that of the rate constants (102 and\namr sq\n39 s−1, respectively) determined in solution at 4 °C and pH 6.8 via stopped-flow measurements23,\nalthough the rates in crystals were slower by 5–40 fold even at a higher temperature (26 °C).\nLines 239-241\n\"Collectively, these results suggest that the gradual transition from TPQamr to TPQsq during 100–1000 ms\n(Fig. 3c) is due to the slow replacement of PAA by 2-PEA depending on the diffusion-controlled rates of\ndesorption of PAA from the substrate-binding pocket and re-binding of 2-PEA.\"\nAt \"Line 87\", \"under anaerobic conditions at pH 9.0 so that the pH-dependent equilibrium between TPQamr\nand TPQsq shifts toward TPQsq24\" is written, while the gradual transition from TPQamr to TPQsq during\n100–1000 ms is due to the slow replacement of PAA by 2-PEA.\nAt least in crystals, this equilibrium reaction does not occur depending on pH?\nAs reported previously (Fig. 3 in ref 24), the equilibrium between TPQ and TPQ in crystals (also\namr sq\nin solution) shifts almost completely to TPQ at pH 9 and above, when 2-PEA is used as a substrate\nsq\nin the reductive half-reaction. This pH-dependent equilibrium was studied at an equilibrium state\n(i.e., Δt = ∞), whereas the present MISC study was conducted to capture transient structures before\nm\nreaching the equilibrium state. We employed the condition of pH 9.0 even in the present study to\naccelerate the transition from TPQ to TPQ .\namr sq\nIn Extended Fig.2, only data of 0 ms was measured in solutions without 2-PEA.\nIs it not appropriate to compare with the data from 22 minutes onwards?\nIn Extended Fig.2, actually, the length of a-axis in data of 0 ms is different from each of those of (L) and (S).\nIt is better to show data of 0mS and that of 22mS-1000mS, respectively.\nThank you for your important comment on the diffraction data obtained at different delay times.\nIndeed, it may not be appropriate to compare the data obtained by the single-flow (Δt = 0 ms) and\nm\nmix-and-inject measurements (22–1000 ms). However, measurements at 0 time of mixing are\ntechnically impossible in MISC (even in mixing with the buffer without 2-PEA) and also in the\ncontinuous-flow and stopped-flow measurements in solution. Therefore, we employed the single-\nflow SFX method to obtain the data for the initial structure before starting the reaction and this data\nset was defined as Δt = 0 ms. We revised the sentence for the single-flow measurement in the first\nm\nparagraph of the Results section and newly defined the data set at Δt = 0 ms, as follows.\nm\nTo obtain the initial enzyme structure (containing TPQ ) before starting the reaction, single-flow\nox\nSFX29,30 of the crystal suspension in a buffer without 2-PEA was conducted and the resultant data\nset was defined as Δt = 0 ms.\nm\nAccording to Reviewer’s suggestion, we showed the data set at 0 ms separately from the other data\nsets of 22–1000 mS in Extended Data Fig. 2. In addition, we described that the 0-ms data set derives\nfrom the initial structure in the buffer without 2-PEA in the legends of Extended Data Fig. 2, as\nfollows.\nExtended Data Fig. 2 | Distribution of unit cell parameters. Cell dimension values (a, b, c, α, β,\nand γ) determined by processing the diffraction images of the initial enzyme containing TPQ\nox\nmeasured by the single-flow SFX (Δt = 0 ms, upper panel) and those measured by MISC at each\nm\ndelay time point (Δt = 22–1000 ms, lower panel) are shown in the histograms. Two red thin lines in\nm\nthe histograms at Δt\nm\n= 22–1000 ms indicate two peaks (193.61 and 192.92 Å) of the a-axis\ndistribution in the data set at Δt = 50 ms.\nm\nAs for the difference in a-axis length between data of 0 ms and each of those of (L) and (S), we\nwould like to comment that the lengths of crystallographic axes differ slightly in each beamtime at\nSACLA because of manual adjustment of the detector camera (see red lines in Extended Data Fig.\n2), as also described later in this message.\n\"Method section\" (Line 356-424\" is described between \"References section\": (Lines 263-354) and (Lines 426-\n460).\nIs it OK?\n\"References section\" was combined and placed after \"Method section\" in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nSI\n• Additional information on the pH/ pKa and rates of the reactions in solution in the scheme in Extended Data\nFig. 1 would be helpful.\n• Additionally, the stopped flow data that has been used to calculate these rates and is referred to several times\nin the text should really be in the SI, and the details should be in the methods. Or if this is already published\ndata then it needs a reference.\nThank you for a good suggestion. We revised Extended Data Fig. 1 and its figure legends to show\nthe rate constants of the corresponding steps determined previously by the spectrophotometric and\nstopped-flow experiments of the reductive half-reaction in solution with the corresponding reference\nas follows.\nExtended Data Fig. 1 | Catalytic mechanism of copper amine oxidase. Abbreviations for TPQ:\nTPQ , oxidized form; TPQ : substrate Schiff base; TPQ : product Schiff base; TPQ :\nox ssb psb amr\naminoresorcinol form; TPQ : semiquinone radical form; TPQ : iminoquinone form. Reductive and\nsq imq\noxidative half-reaction steps are indicated by blue and red arrows, respectively. Rate constants in\nthe reductive half-reaction determined previously in solution at 4 °C and pH 6.8 via stopped-flow\nmeasurements23 are k = 887 s−1, k = 206 s−1, k = 102 s−1, and k = 39 s−1. The pH-dependent\n1 2 3 4\nequilibrium between TPQ and TPQ was also reported to have pK values of 5.96 and 7.74 with\namr sq a\nthe ionizable group with pK\na\n= 5.96 contributing predominantly to the equilibrium shift23.\n• Could you add a figure giving the chemical structures of PAA and 2-PEA.\nThe chemical structures of phenylacetaldehyde (PAA) and 2-phenylethylamine (2-PEA) are already\nshown in Fig. 3a of the original manuscript. We also gave the reason for the indistinguishability of\nPAA and 2-PEA by X-ray crystallography in the last paragraph of the Results section, as follows.\nNotably, substrate-derived electron densities in all structures refined for the data sets at Δt =\nm\n100–1000 ms were disconnected from those of the reduced cofactors (Fig. 3b) and could be\nassigned as the reaction product, phenylacetaldehyde (PAA), or amine substrate, 2-PEA (Fig. 4a).\nThe two ligands are almost indistinguishable by X-ray crystallography because of very similar\nelectron densities derived from the aldehyde oxygen and amine nitrogen atoms, but our recent…\n• Extended figure 5 & 6: 2-PEA diffusion in solvent channel: there is a program that allows the user to visualize\nthe solvent channel: MAP_CHANNELS by Juers and Ruffin (J. Appl. Cryst 2014). This program or maybe a\nnewer program could help to measure the solvent channel, and it could help to support or explain why 2D\ndiffusion model is preferred.\nThank you for your excellent suggestion. Comments on the 2D diffusion model were also provided\nby the team review, as described later. Accepting the Reviewer’s suggestion, we visualized the\nsolvent channel using the program MAP_CHANNELS (Juers, D. H. & Ruffin, J. (2014) J. Appl.\nCrystallogr. 47, 2105−2108). The map obtained for the solvent channel was contoured with the\nAGAO dimer and its symmetric mates aligned in the crystal lattice. We employed this figure\nillustrating the solvent channel as a new figure in the main text (new Fig. 5). (Fig. 7 of the original\nmanuscript was transferred to Extended Data Fig. 7.) Interestingly, the map exhibited a planar\nsolvent channel that expands to the direction parallel to the b- and c-axes, clearly showing that the\namine substrate easily diffuses into the crystals in the two directions. The entrances to the substrate\nchannel (Extended Data Fig. 6) are connected to this solvent channel (shown by red arrows in Fig.\n5). By contrast, the map indicates that the substrate can hardly diffuse in the direction parallel to the\na-axis due to the discontinuous solvent channel interrupted by a layer of enzyme molecules.\nAlthough the relationship between the crystal lattice and overall crystal shape is unknown at present,\nthe visualized solvent channel well supports the two-dimensional diffusion model for the substrate.\nThese points are described in the first paragraph of Discussion in the revised manuscript with a new\nFig. 5, as follows.\nDiscussion\nTime-dependent decrease in the number of diffraction images of 22–50 ms (L) and concomitant\nincrease in that of 22–50 ms (S) (Fig. 3) reflect the progress of substrate diffusion into the\nmicrocrystals. As shown in Fig. 5, the map calculated by the program MAP_CHANNELSref exhibited\na planar solvent channel that expands to the direction parallel to the b- and c-axes, suggesting that\nthe amine substrate easily diffuses in the two directions. Moreover, the entrances to the substrate\nchannel (Extended Data Fig. 6) are connected to this solvent channel (shown by red arrows in Fig.\n5). By contrast, the map indicated that the substrate can hardly diffuse in the direction parallel to the\na-axis due to the discontinuous solvent channel interrupted by a layer of enzyme molecules.\nAssuming that substrate diffusion follows a two-dimensional model, in which diffusion across the\ncrystal thin section is negligible (Extended Data Fig. 5), amine substrate requires approximately 120\nms to diffuse into the entire volume of the microcrystals.\nAdditional reference: Juers, D. H. & Ruffin, J. MAP_CHANNELS: a computation tool to aid in the\nvisualization and characterization of solvent channels in macromolecular crystals. J. Appl.\nCrystallogr. 47, 2105−2108 (2014). 10.1107/S160057671402281X, PubMed: 25484846.\nFig. 5 | Solvent channel in the crystal lattice. Solvent channels in the crystal lattice were\nvisualized using a program MAP_CHANNELSref with the initial enzyme structure (containing TPQ )\nox\nbefore starting the reaction (Δt = 0 ms). In the upper left panel, the solvent channels (cyan mesh\nm\nmap) are contoured with the AGAO dimer and its symmetric mates (light brown and grey surface\nmodels) aligned in the lattice. In the upper right panel, only the solvent channels are shown. A single\ncrystal lattice is shown in thin yellow lines with labels indicating a-, b-, and c-axes. Small black arrows\nindicate the direction of the unit cell axes in the abc coordinate system. Red arrows in an enlarged\nview (lower panel) indicate the two entrances to the substrate channel of the AGAO dimmer.\nManuscript\n• Abstract\no Line 38-39: \"active-site conformational changes that are unobservable by studies in solution\" is quite a\nbroad statement and may not be true, maybe NMR in solution could help. Could you please comment.\nWe agree that the phrase “that are unobservable by studies in solution” may not be true. We revised\nthe phrase to \"that are hardly scrutinized by studies in solution\".\n• Results / MISC measurement and data processing section\no Line 86: A reference is missing for \"(2-PEA; the most preferred substrate)\"\nWe cited a reference showing the highest activity of AGAO toward 2-PEA after “the most preferred\nsubstrate” with K and k values.\nm cat\nThe crystal suspension and substrate solution containing 2-phenylethylamine (2-PEA; the most\npreferred substrateref with K = 2.5 μM, k = 76 s−1 determined at pH 6.8, 30 °C16) were prepared\nm cat\nunder anaerobic conditions at pH 9.0 …\nAdditional reference: Shimizu, E., et al. Purification and Properties of Phenylethylamine Oxidase\nof Arthrobacter globiformis, Biosci. Biotechnol. and Biochem. 61, 501–505 (1997).\n10.1271/bbb.61.501.\n• Discussion section:\no Since there are 2 different redox states of Cu between TPQamr and TPQsp, could X-ray Emission\nSpectroscopy help to corroborate some hypothesis about the slow replacement of PAA by 2-PEA? If you had\naccess to this technique, would you use it, and would it help?\nThank you for your excellent suggestion. Unfortunately, we have currently no access to X-ray\nEmission Spectroscopy applicable to the mix-and-inject experiments.\no The temperature differences between the stopped flow experiments (15 C) and MISC experiments (26 C)\ncould also be a reason for differences in the rates.\nAs suggested by the Reviewer, the temperature differences between the stopped-flow experiments\n(4 °C) and MISC experiments (26 °C) could also be a reason for differences in the reaction rates.\nHowever, it is general that the reaction rates (both enzymatic and non-enzymatic) become faster at\nhigher temperatures. Thus, if we conduct both experiments at the same temperature (ex. at 26 °C),\nthe rate constants in solution obtained by the stopped-flow method will increase, and thereby the\ndifference further increases. We estimate that the rate constants in crystals are still smaller than\nthose in solution even at the same temperature.\nMethods\n• We would like a bit more details on the sample consumption. On average how many ml / ul (or mg of protein)\nhave been used for each time point?\nThank you for pointing out an important point on planning the MISC experiments in practice. As an\naverage of the total measurements, the protein concentration of microcrystal suspension of AGAO\nwas about 30 mg/mL. The time needed to collect diffraction images for the single data set was about\n8 h including adjustment time with 0.02 mL/min of the suspension flow rate. From these values, it is\nlikely that about 300 mg of protein was required (30 mg/mL × 0.02 mL/min × 480 min). This\ninformation was described in the Method section of the revised manuscript in the end of “Data\ncollection and processing” of the Method section.\nAs an average of the MISC measurements, operation time of about 8 h including the adjustment\ntime and a total of 300 mg protein were required for collecting the diffraction images sufficient for a\nsingle data set.\n• The MISC sample delivery method has been used quite extensively in the US with Schmidt and collaborators.\nWe were not aware that SACLA is offering this sample delivery method. Could you please give more details\non MISC: is it provided by the facility? Or by collaboration? Is it the same nozzle design used by Schmidt et\nal?\nBy collaboration with SACLA, we used the MISC system including the nozzle design that was\ndeveloped in SACLA referring the Schmidt system. The mixing injectors were fabricated in the same\nmanner as described in Ref. 40 and provided at SACLA.\nAdditional comments from the team review\nLine 28 (within the abstract)\nPlease state somewhere that the time-revolved studies are for the reductive half reaction.\nLine 32:\nPlease state the average crystal size used for the tr-SFX work\n(see line 84 for values; 2.5 × 4 μm parallelogram plate × < 1 μm thick).\nLine 33: “amine substrate”\nambiguous, please state what substrate was used for this study (2-phenylethylamine).\nWe would like to accept these suggestions for Abstract from the team review. Since Abstract is\nlimited to approximately 150 words in Nature Communications, we added a minimum number of\nwords as follows.\nAbstract\nProtein dynamics play a crucial role in various physiological functions, including enzyme catalysis.\nTo explore conformational changes during enzyme catalysis, we conducted mix-and-inject serial\ncrystallography, an advanced technique to capture time-resolved protein structures in real time,\nusing the microcrystals of bacterial copper amine oxidase containing a protein-derived quinone\ncofactor. Within 50 ms of mixing the microcrystals (< 4 μm) with a preferred substrate (2-\nphenylethylamine) under anaerobic conditions (reductive half-reaction), we observed domain\nmovements associated with substrate binding and formation of a metastable reaction intermediate,\na product Schiff-base of the quinone cofactor. At 100–1000 ms after mixing, conformational\ntransition from aminoresorcinol to the semiquinone radical forms of the reduced cofactor progressed\ngradually, likely depending on the replacement of the product aldehyde by the next-cycle amine\nsubstrate that triggers the cofactor conformational change. Overall, this study provides structural\ninsight into enzyme catalysis accompanying the active-site conformational changes that are hardly\nscrutinized by studies in solution.\nLine 54\na microcrystal suspension of an enzyme is mixed with the substrate and ejected to the XFEL emission point,\n“emission” should probably be “interaction”\nThank you for your suggestion. We replaced “emission” with “interaction” in the revised\nmanuscript.\nLines 56 – 57\nNamely, MISC is a crystallographic version of a classical continuous-flow or stopped-flow technique used for\ndetection of time-resolved reaction intermediates formed during enzyme catalysis in solution15,16.\nReads better as:\nNamely, MISC is a crystallographic version of a classical transient kinetic, spectroscopic analysis of\ncontinuous-flow or stopped-flow technique used for detection of time-resolved reaction intermediates formed\nduring enzyme catalysis in solution15,16.\nWe revised the manuscript according to the Reviewer’s comment as follows.\n“Namely, MISC is a crystallographic version of a classical continuous-flow or stopped-flow technique\nused for spectrophotometric detection of time-resolved reaction intermediates formed during\nenzyme catalysis in solution15,16.”\nLine 86 “(2-PEA; the most preferred substrate)”\nPlease provide KM, and kcat values for relevant pH values here and provide appropriate references.\nAt pH 9.0 used in MISC, K and k were 0.56 μM and 24 s−1, respectively, for 2-PEA (30 °C,\nm cat\nunpublished data). Under our standard assay conditions (pH 6.8, 30 °C), K and k values were\nm cat\nreported to be 2.5 μM and 76 s−1, respectively (ref. 16). The catalytic efficiencies shown by k /K\ncat m\nwere rather higher at pH 9 than at pH 6.8 (ref. 16). These kinetic parameters indicate that AGAO\nhas enough activity at pH 9.0. We added the kinetic data at the corresponding position of the\nmanuscript.\nThe crystal suspension and substrate solution containing 2-phenylethylamine (2-PEA; the most\npreferred substrateref with K = 2.5 μM, k = 76 s−1 determined at pH 6.8, 30 °C16) were prepared\nm cat\nunder anaerobic conditions at pH 9.0…\nLine 96: “we noted unusual distributions of”\nAs you state a few lines later, this is fit to a simple bimodal distribution; therefore, please replace “unusual”\nwith “bimodal”.\nAccording to the Reviewer’s suggestion, we replaced “unusual” with “bimodal” in the revised\nmanuscript.\nLine 101 and following sentences: “indicating the presence of two microcrystal regions with unit lattices of\ndifferent a-axis lengths...”\nThis sentence implies two different microdomains within the microcrystals, which may be true and could be\nconsistent with the partially bleached crystals shown with macrocrystals in Extended Figure 3. However, the\nbimodal distribution only demonstrates that successful indexing of the tr-SFX images reveals that there are\ntwo, non-isomorphic populations in the whole slurry. The illustration in Extended Data Fig. 3 suggests that\nthe two species result from X-ray a narrow X-ray beam passing through the flat face of the crystal plate. The\nX-ray beam shape is Gaussian, not a top-hat profile, and so there is not such a big difference between the\ncrystal and X-ray beam sizes. Indeed, the thin plate dimension is smaller than X-ray beam. Moreover, the X-\nray beam passed through randomly oriented crystals; it is possible that indexing may fail if/when the X-\nray beam passes through the bleached and non-bleached regions of the microcrystals. Given that the unit cell\nchanges by such a small amount (~0.3%), it is likely that indexing probably still succeeds in these cases too.\nInitially, we also had similar concerns with the review team: double hits of XFEL beam to both\nunreacted and reacted area of microcrystals. To confirm successful image separation based on the\na-axis length, we re-examined whether a single diffraction image in our saved image data\nsimultaneously includes the spots derived from both long (L) and short (S) a-axes lattices. We again\nfiltered diffraction images using program Cheetah with “multi” option, in which double indexing is\npossible for a single image. However, we were unable to find a doubly indexed image. This result\nsuggested two possibilities. (i) The images including both (L) and (S) lattices were discarded through\nthe SACLA data-processing pipeline because of unsuccessful indexing. (ii) The images including\nboth (L) and (S) lattices were indexed as intermediary lattice between (L) and (S). Case (i) means\nthat two lattices can be successfully separated into (L) and (S) data sets. In case (ii), to avoid mixing\n(L) and (S) lattices, we did not use data sets having intermediary a-axis length as described in Fig.\n2. Finally, structure determination with the (L) and (S) data sets provided the unreacted oxidized\nform and TPQ intermediate, respectively, clearly exhibiting successful image separation from\npsb\nbimodally distributed data set and data collection for the (L) and (S) lattices. In any case, we could\nnot explain why two forms of microcrystals with different a-axis lengths exist simultaneously in an\nearly stage of the reductive half-reaction, until we arrived at the interpretation that two microcrystal\nregions with unit lattices of different a-axis lengths are present within the same microcrystals due to\nthe difference in the extent of substrate diffusion in the peripheral and core regions.\nAnalysis of the data from Extended Data Table 1 indicates that the a-axis unit cell edge for all of the timepoint\ndatasets is different compared to the t = 0, ground state. The % change for the “Long” cell datasets are all\nsmall and positive 0.295, 0.348, and 0.191 for the 22, 25, and 50 ms datasets, respectively. However, the %\nchange for the “Short” cell species and all of the longer timepoints across the series are also all small but\nnegative (-0.124, -0.202, -0.197, -0.191, -0.217, -0.238, -0.243, -0.321, -0.295). The distribution for first three\ntimepoints are clearly bimodal, but more homogeneous for the later six timepoint data.\nWe sincerely thank the team review for very precise analyses of the a-axis unit cell edge for all of\nthe timepoint datasets compared to the dataset of the initial enzyme containing no substrate (defined\nas 0 ms). The small and positive % changes for the “Long” cell datasets (22, 25, and 50 ms) against\n0 ms dataset remain to be explained but might be derived from the difference in measurements\n(single-flow SFX vs. MISC) or from slight differences in the camera length in each beamtime (see\nExtended Data Fig. 2, red lines). In contrast, the small but negative % changes for the “Short” cell\nspecies and all of the longer timepoints indicate the shortening of a-axis length in the crystal\nstructure containing TPQ intermediates (TPQ , TPQ , and TPQ ) with bound PAA or 2-PEA.\npsb amr sq\nThese findings led to the most important conclusion of the present study, and we extensively\ndescribed them in Discussion.\nFigure 2 and Extended Data Table 1:\nPlease annotate the image (or indicate in the legend) the unit cell dimension for the peak of each species.\nAre these the same values reported in Extended Data Table 1?\nAccording to the suggestion by the team review, we indicated the a-axis length for the peak of each\nspecies (22 ms: (L) 193.82, (S) 193.13; 25 ms: (L) 193.69, (S) 193.01; 50 ms: (L) 193.61 (S) 192.92)\nin the legends of Fig. 2. These values are essentially identical with those shown in Extended Data\nTable 1, but deviate slightly because each group of images were refitted after image separation and\nthe a-axis lengths were calculated by the peak analyses.\nFig. 2 | Distributions of a-axis lengths and grouping of the diffraction data sets. Number of\nindexed images obtained from the diffraction data at Δt = 22 (a), 25 (b), and 50 (c) ms is plotted\nm\nagainst the a-axis length. Histograms were fitted to two normal distribution curves (red) using the\nIgor Pro version 6 software (Wave Metrics, Inc.; middle), with residuals showing the difference\nbetween the observed number of images and fitted curve (top). Deconvoluted distribution curves\nare shown for the data sets at Δt = 22−50 ms (S) and (L) in purple and orange, respectively (bottom).\nm\nColored thick bars indicate the areas of indexed images used for structure refinement, which are\nenclosed by dotted lines representing the cut-off a-axis lengths to minimize the contamination of the\ncounterpart distribution peaks (< 10%). The a-axis lengths recalculated for the peak of each species\nare 193.82 Å for 22 ms (L), 193.13 Å for 22 ms (S), 193.69 Å for 25 ms (L), 193.01 Å for 25 ms (S),\n193.61 Å for 50 ms (L), and 192.92 Å for 50 ms (S).\nExtended Data Fig. 3\nPlease comment on why the image of the large macrocrystal indicates that the crystal had not fully reacted\neven after 30 minutes. The tr-SFX data assumes many thousands of random orientations to sample the whole\nEwald sphere and thereby build up the complete dataset. So, the tr-SFX results with microcrystals indicates\nthat only about 1 second is needed for a complete reductive half reaction?\nThank you for your reasonable question. We are sorry we did not describe the experimental details\nof substrate soaking of the macrocrystal shown in Extended Data Fig. 3d. An AGAO crystal with a\nhuge size (3–4 mm) was grown inside a dialysis button and was anaerobically soaked in the\ncrystallization buffer containing the amine substrate (4 mM 2-PEA) through the dialysis membrane.\nTherefore, the substrate concentration inside the button is initially ‘0’ and it must diffuse slowly and\ngradually into the chamber of the dialysis button through the semipermeable membrane. Thus, the\nsubstrate diffusion into the macrocrystal placed inside the button takes longer time than direct\nexposure of crystals to the substrate-containing buffer as in the MISC experiments. If we apply the\nsubstrate-diffusion rate (ca. 120 ms) of microcrystals with a micrometer size to a macrocrystal with\na millimeter size, the macrocrystal should fully react with substrate in 120 seconds (2 min) by direct\nexposure to the substrate at the same concentration. In Extended Data Fig. 3d, we intended to show\nthe pattern of substrate diffusion into crystals (peripheral to core regions) rather than the difference\nin the diffusion rate. We described the method of macrocrystal soaking in the legends of Extended\nData Fig. 3 of the revised manuscript, as follows.\nExtended Data Fig. 3 | Schematic of X-ray free-electron laser (XFEL) irradiation. Illustration of\n17 the XFEL beam irradiating the (a) central core and (b) peripheral regions of the microcrystals. (c)\nreddish-19 pink color of the oxidized form of the cofactor (TPQox), with λmax of approximately 480\nnm in a single-20 crystal absorption spectrum2; scale bar, 2 mm. (d) Photomicrograph of a crystal\nsoaked in 2-PEA for 21 approximately 30 min under anaerobic conditions; scale bar, 2 mm. Crystal\nsoaking was performed by transferring the dialysis button containing the macrocrystal to the\ncrystallization buffer containing 4 mM 2-PEA; the substrate diffuses slowly and gradually into the\nchamber of the dialysis button through the semipermeable membrane. Peripheral and core regions\nare colored differently depending on the extent of substrate diffusion; pale yellow color of the\nperipheral region derives from TPQ with λ values of approximately 430 and 468 nm (TPQ is\nsq max amr\ncolorless)3; scale bar, 2 mm. Substrate diffusion across the thin section of the crystal was negligible,\nindicating the strict directionality of substrate diffusion.\nPlease support the statement, “strict directionality of substrate diffusion” with an analysis and appropriate\nfigure(s) of the crystal packing and solvent channels with respect to the observable crystal parallelogram plate\nmorphology. If this analysis has been done in another paper, then it should be referenced. For instance,\nexamination of the lattice packing in PDB code 3WA2 (with different unit cell edge lengths in C121 space\ngroup) clearly reveals two ~ orthogonal dimensions with rather large solvent channels and a third orientation\nwith much smaller channels.\nWe received a similar comment from Reviewer 2. As described above, an additional analysis with\na MAP_CHANNELS program has been added in the revised manuscript (new Fig. 5).\nExtended Data Fig. 6\nThis figure is a good place to show the crystal lattice packing and how it impacts access to the active sites\nwithin the lattice.\nWe appreciate a valuable suggestion by the review team. As described above, we have shown the\ncrystal lattice packing together with the calculated map for solvent channel and the entrance to the\nsubstrate channel as an independent new figure (Fig. 5). This figure was viewed from a similar\ndirection with Extended Data Fig. 6a.\nLine 464 – 466: “(proposal 2017B8038, 2020A6507, 2020A8013, 2021A6606, 2021A8613, 2021B6606,\n2021B8029, 2022A6705, 2022A8020, 2022B6705, 2022B8013, 2023A6805, 2023A8009, 2023B6805,\n2023B8013, 2024A8021, and 2024B8016)”\nThis suggests that the team accessed SACLA during 17 different beamtimes spanning about 7 years to collect\nthe data, which is fine and it is appropriate to indicate which SACLA beamtimes contributed to the publication.\nHowever, it is very likely that some of the time was used for testing and development, while some of the time\nwas used for the actual tr-SFX data collection reported in this manuscript. Please indicate which of the\nproposals were used to collect the reaction timepoint datasets and how long it took to collect each timepoint\nin particular for the data reported herein. Did diffraction data for any given timepoint dataset come from\ndifferent beamtimes, and if so what fractions of the datasets for the different beamtimes were merged?\nAs guessed by the team review, these beamtimes were used for both of the data collection in SACLA\nand preliminary experiments to develop the MISC system and to search the experimental conditions\nin SACLA and SPring-8. Details of the beamtimes for the data collection were indicated in the\nMethod section of the revised manuscript (the subsection “Data collection and processing”). All\ndiffraction data for a given timepoint dataset came from a single beamtime. In addition, we corrected\na mistake of the proposal number (2021A8613 → 2021A8013) in the Acknowledgement section.\nData sets for a single-flow SFX of the substrate-free initial enzyme (0 ms) and MISC at Δt = 500\nm\nand 1000 ms were collected first (proposal number of the Japan Synchrotron Radiation Research\nInstitute: 2020A8013), then those at Δt = 22–50 ms (2021A8013), and lastly those at Δt = 100–\nm m\n400 ms (2021B8029).\nPoint-by-point response to the reviewers’ comments\nReviewer #2 (Remarks to the Author):\nThe authors have done an excellent job revising their manuscript; thank you.\nThe addition of the MAP_CHANNELS analysis is as anticipated and greatly appreciated. I have one additional\ncomment related to the soaking of 2-phenylethylamine substrate into the crystal to initiate the reductive half-\nreaction. This is an important part of the story. The authors have everything they need to resolve the\nrelationship between the monoclinic crystal lattice in the reciprocal space to the crystal packing and\nmorphology in real space. This analysis will/should correlate to the observed diffusion characteristics in the\ncrystals; therefore, they should do the analysis and include it in the manuscript. For instance, with access to\nlarge crystals that display very distinct crystal morphology and habit, one could simply align a big crystal to\nthe home X-ray source or a synchrotron X-ray beam (e.g. shooting perpendicular (through) the plate face\n&/or along a thin axis (e.g. ± 90 degrees to the plate face). Then collect a few diffraction patterns with ~ 1\ndegree rotation starting from an orientation aligned to the observable crystal habit. The diffraction pattern\nand unit cell dimensions (a = 194 Å, b= 65 Å, c = 158 Å; alpha = beta = 90 deg., beta = 117 deg.) will\nprobably reveal the relationship between crystal lattice, packing, and visible crystal plate morphology.\nAlthough the AGAO crystal is not orthorhombic, the real space packing of the monoclinic unit cells should be\napparent. Indexing the crystal lattice in reciprocal space will also confirm/provide the needed connection to\ncrystal packing in real space.\nWe sincerely appreciate your suggestion for additional experiments to reinforce the results of the\nMAP_CHANNELS analysis. To address your suggestion under circumstances that a home X-ray\nsource is unavailable at present, we performed additional synchrotron X-ray diffraction experiments\nof the large AGAO crystal to determine the relationship between the crystal lattice orientation and\nmorphology, using an extra allocated beamtime. Fortunately, the results showed that the a-axis of\nthe crystal lattice approximately aligns with the shortest dimension (thin section) of the crystal.\nConsequently, the b-c plane forms the broad faces of the crystal plate. This structural confirmation\ndirectly supports our conclusion, drawn from the observation of substrate diffusion into the\nmacrocrystal and also the MAP_CHANNELS analysis, that the substrate amine is poorly permeable\nthrough the broad faces (= across the thin section), being consistent with the proposed two-\ndimensional diffusion model. Therefore, we incorporated the new results as supporting evidence\ninto the Discussion section of the final revised manuscript (as described below), together with\nSupplementary Figure 6 (new figure) and a new paragraph in the Methods section.\nThe a-axis of crystal lattice roughly coincides with the direction of crystal thin section, as confirmed\nby an X-ray diffraction analysis of crystal morphology using an AGAO macrocrystal (Supplementary\nFig. 6). Thus, the slow crystal growth along the a-axis may also be explained by the poor contact of\nenzyme dimers separated by the solvent channel (Fig. 5)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have carefully reviewed the revised manuscript.\nThe authors have appropriately addressed all of my comments.\nI recommend the manuscript for acceptance.", "reviewer_2": "The authors have done an excellent job revising their manuscript; thank you.\nThe addition of the MAP_CHANNELS analysis is as anticipated and greatly appreciated. I have one additional comment\nrelated to the soaking of 2-phenylethylamine substrate into the crystal to initiate the reductive half-reaction. This is an\nimportant part of the story. The authors have everything they need to resolve the relationship between the monoclinic crystal\nlattice in the reciprocal space to the crystal packing and morphology in real space. This analysis will/should correlate to the\nobserved diffusion characteristics in the crystals; therefore, they should do the analysis and include it in the manuscript. For\ninstance, with access to large crystals that display very distinct crystal morphology and habit, one could simply align a big\ncrystal to the home X-ray source or a synchrotron X-ray beam (e.g. shooting perpendicular (through) the plate face &/or\nalong a thin axis (e.g. ± 90 degrees to the plate face). Then collect a few diffraction patterns with ~ 1 degree rotation starting\nfrom an orientation aligned to the observable crystal habit. The diffraction pattern and unit cell dimensions (a = 194 Å, b=\n65 Å, c = 158 Å; alpha = beta = 90 deg., beta = 117 deg.) will probably reveal the relationship between crystal lattice,\npacking, and visible crystal plate morphology. Although the AGAO crystal is not orthorhombic, the real space packing of the\nmonoclinic unit cells should be apparent. Indexing the crystal lattice in reciprocal space will also confirm/provide the needed\nconnection to crystal packing in real space.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter