Relaxation behaviour of the tyrosine YD radical in photosystem II:

evidence for strong dipolar interaction with paramagnetic centers

in the S1 and S2 states

Fikret Mamedov,a Paul J. Smith,b Stenbjorn Styringac and Ron J. Pace*b

aDepartment of Molecular Biomimetics, Uppsala University, Villavagen 6, 752 36Uppsala, Sweden

bDepartment of Chemistry, Faculty of Science, Australian National University, Canberra 0200,ACT, Australia. E-mail: Ron.Pace@anu.edu.au; Fax: þ61 2 61258997; Tel: 61 2 61254546

cDepartment of Biochemistry, Centre for Chemistry and Chemical Engineering, LundUniversity, Lund 221 00, P.O. Box 124, Sweden

Received 17th May 2004, Accepted 8th July 2004First published as an Advance Article on the web 27th July 2004

Inversion recovery (T1) and microwave power saturation studies have been performed, between 4 and 25 K, onthe EPR signal from the stable tyrosyl radical, YD

d, in photosystem II core complex preparations from higherplants. Measurements are reported from the dark stable S1 and first turnover S2 states of the photosystemcatalytic Mn cluster and in two cryoprotectant regimes; sucrose and ethylene glycol/glycerol. The inversionrecovery kinetics show a dominant, non exponential decay component which is well described by a through spacedipolar relaxation model, with a weak exponential decay background (Ban order of magnitude less than thedipolar rate). The dipolar relaxation rate is only modestly temperature dependent and shows no consistentdependence on S state or cryoprotectant. In contrast, the background rate shows a S state dependence, consistentwith an interaction between YD

d and the Mn cluster in the multiline S2 state, over a distance of B30 A. Thefraction of centers exhibiting the dipolar relaxation component appears to be temperature dependent, but S stateindependent and consistent with the presence of a fast relaxing species interacting with YD

d. The present resultsand the possible nature of this interacting species are discussed in comparison with earlier YD

d relaxationstudies on photosystem II membrane samples.

Introduction

The photo-oxidisable tyrosine on the D2 peptide of the reac-tion center complex in photosystem II, called YD, has longbeen known to form a stable neutral radical species (YD

d),originally called Signal II (slow).1 Its nature and behavior havebeen extensively studied by a number of groups, with regard toits EPR spectroscopic properties, redox reactions withinphotosystem II (PS II) and interactions with the proteinenvironment (e.g., See ref. 2 for recent review). Because it isa relatively stable, magnetically isolated spin 1/2 center, withintrinsically slow relaxation rates at cryogenic temperatures, ithas provided an experimentally convenient system to explorelong range (tens of angstrom) dipolar interactions betweenitself and other paramagnetic centers within PSII.

The first such studies were performed by Rutherford, Styringand colleagues3,4 using conventional CW power saturation andspin echo (inversion recovery T1) measurements. The relaxa-tion behavior of YD

d was followed as a function of the turn-over state of the catalytic Mn cluster, which is the site of wateroxidation in PSII.5 The Mn site cycles through four quasi-stable oxidation state intermediates, called S states (labeledS0–3), in the overall water oxidase reaction. Both S0 and S2 arenet spin 1/2 paramagnetic states of the coupled Mn cluster,while the S1 and S3 states are even spin. S1 is the dark stablestate, which is anti-ferromagnetically coupled and diamagneticat sufficiently low temperatures.6 These studies were subse-quently extended by Evans et al.,7 Brudvig et al.6,8,9 andKawamori et al.10,11 using saturation recovery (also a T1

technique) and most recently pulsed electron-electron doubleresonance (PELDOR) to extract the long range dipolar

interaction components of the spin lattice relaxation or rotat-ing frame nutation behavior of the YD

d magnetization signal.An important question, which all of the above studies have

sought to address, is the apparent dipolar distance between theYD

d radical and Mn cluster in the S2 state. The latter exhibits awell known, hyperfine structured net spin 1/2 signal, called the‘multiline’ signal, which isB180 mT wide and centered close tog ¼ 2. From the recently available medium resolution crystalstructure information on cyanobacterial PSII complexes,12–14 itis now known that the distance from the phenoxide ring on YD

to the middle of the Mn cluster density region is B29 A. Theelectron–electron dipolar interaction has a simple, character-istic angular dependence with respect to the external magneticfield (e.g., see ref. 8). This ‘signature’ may be used to isolate thedipolar interaction in a spin lattice relaxation enhancement orPELDOR type experiment, in either oriented or powdersamples. The distance estimate from PELDOR generally in-volves fewer assumptions than that from relaxation measure-ments. If the magnetic moments of the interacting species(usually spin 1/2) are known, then the distance is given directlyfrom the interaction modulation frequency. Both the magni-tude (B27 A) and orientation of the Mn cluster–YD inter-action vector found from PELDOR are consistent with thecrystal structure.11 The situation with regard to the relaxationenhancement measurements is more complex. In one case,7 thetotal relaxation rate of YD seen in either the S1 or S2 states wasrelatively slow (B50 s�1 or less), for temperaturesB4 K. In thestudies of Brudvig et al., the rates observed under theseconditions were much higher (B103 s�1 or greater), but againquite similar for the S1 and S2 states.

6,8,9 The results of Eveloet al.4 show a combination of these behaviors. These authors

R E S E A R C H P A P E R

PC

CP

ww

w.rsc.o

rg/p

ccp

DOI:10.1039/b407329k

4890 P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 4 8 9 0 – 4 8 9 6 T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

found that the YDd relaxation was composed of two distinct

components, from roughly equal populations in their PSIIcenters. One rate component (fast) had a magnitude andtemperature dependence generally similar to that seen in refs.6,8,9. The other component (slow) had rates that wereabout an order of magnitude slower and similar to that seenin ref. 7 at low temperatures. The slow components, fromeither ref. 7 or ref. 4, consistently resolved a difference(increase, tens of s�1) between the S1 and first turnover S2states. Using plausible assumptions for the relevant multilinestate magnetic parameters,9 these lower YD relaxation ratescorrespond to a Mn cluster–YD distance approaching 30 A,generally consistent with the PELDOR and crystal structureresults. The higher rates correspond to a distance of o20 A.This was recognized as improbable and alternative interpreta-tions, involving factors possibly related to the spin dynamics ofthe multiline center were discussed.9

Another explanation of the high relaxation rates is that someother paramagnetic center, magnetically separate from themultiline Mn cluster, may interact with YD in the S1 and S2states. This center would be variably ‘switched on’ by factorsyet to be identified. All of the above studies were carried out inhigher plant (usually spinach) sourced PSII. While there aresome spectroscopic differences between PSII from plants andcyanobacteria (the latter do not exhibit a conventional g4.1signal),15,16 the S2 multiline signals from these species areessentially identical. From our current knowledge of thecyanobacterial crystal structure, there is no obvious candidatefor a paramagnetic relaxation center, other than the Mncluster, that would be common to plants and cyanobacteriaand be o30 A from YD

d Nonetheless, there are a number ofobservations, going back almost two decades, which suggestthat both the multiline center and YD might experience avariable relaxation enhancement interaction with some otherparamagnetic species, which is much closer to the multilinecenter than to YD

d These include the original observation of‘active’ and ‘resting’ state forms of the multiline Mn cluster, inboth the S1 and S2 states6,17 and more recently the demonstra-tions by Peterson et al.18 of a turnover dependence of themultiline and YD signal relaxation properties and the results ofAhrling and Peterson19 that such an effect may be related to thereduction state of the quinone pool in PSII membrane particles.

One difference between the experimental protocols givingrise to fast6,8,9 and mixed or slow4,7 YD

d relaxation behavior isthe cryoprotectant employed with the PSII membrane parti-cles. In the studies of Brudvig and co-workers, poly-alcohols(EG at B30% v/v) seems to have been consistently used, whilethe other studies, reporting mixed or slow rates, used sucrose(B400 mM)7 or mixtures of sucrose and ethylene glycol3,4 asthe cryoprotectant. It has long been known that these cryo-protectant regimes have some influence on Mn derived signalproperties in the S2 state (e.g., see ref. 20). Thus a g4.1 signalmay be generated by visible light illumination from S1, attemperatures below B150 K, only in polyalcohol type buffers.The basis of this has never been satisfactorily explained.

In this paper, we report on studies of the relaxation behaviorof the YD

d radical in PSII core complexes from spinach, whichseek to resolve some of the matters raised above. The experi-ments involve principally inversion recovery pulsed EPR. Thecore complex preparation is that which we have recentlydeveloped for optical spectroscopy and other purposes.21 Thismaterial is very catalytically active and generates EPR signalsin high yield (B1.0 multiline per reaction center).

Materials and methods

PSII samples

All preparative procedures were carried out at between 2 1Cand 6 1C under dim green light. Fresh market English spinach

was used for all sample preparations in this study. PSIIcontaining thylakoid membrane fragments (PSII membranes)were isolated according to the Triton X-100 solubilisationmethod of Smith et al.22 and stored at 414 mg ml�1 chlor-ophyll at 77 K in a buffer consisting of 0.4 M sucrose, 0.02 MMES, 0.015 M NaCl, 0.01 M MgCl2 pH 6.0 (NaOH) (storagebuffer). PSII membrane samples were maintained frozen andused within six months of storage. They were thawed fresh forthe PSII oxygenic core complex isolation procedures.Isolation of PSII oxygenic core complexes was undertaken

as in ref. 21, after dilution and homogenisation of the PSIImembrane samples into a buffer containing 0.4 M sucrose,25 mM Bis-Tris, 20 mM MgCl2, 5 mM CaCl2, 10 mM MgSO4

and 0.3 g.l�1 b-D-dodecyl maltoside (bDDM), pH 6.5 NaOH(BTS400 system). Core complexes were eluted from the FPLCcolumn using 85 mM MgSO4 in the BTS400 buffer. The corecomplex containing fractions finally collected from the FPLCcolumn were pooled and concentrated using Amicon Centri-prep (10 kDa cutoff) centrifugal concentrators. The PSII coresamples maintained viability and stability over a 12 monthperiod without loss of initial activity (43500 mmol O2 (mgchl)�1 h�1). The Ca and Cl� ion levels compensate for theabsence of the low molecular weight extrinsic proteins (16 and23 kDa) mobilised during the preparation.Chlorophyll content was determined using the method of

Porra et al.23

EPR samples

For EPR studies, the core complex samples were thawed indarkness and then diluted to give a final chl content of about0.7 to 1 mg ml�1 chl. For this study, the samples were dilutedinto BTS System buffer or this mixed with ethane-diol (EG)and glycerol (Glyc) (50:50 v:v) to a final concentration of 40%v:v poly-alcohol. The poly-alcohol mixture is that used forglass formation in optical experiments.21,24 Samples were pre-illuminated for 10 s at room temperature to pre-oxidise Tyr-osine D (YD

d). The samples were then left for 10 min in thedark at B20 1C to relax back to the S1 state before furthersample illumination procedures. In our experience, this pro-duces B100% of centers in S1 (based on S2 signal quantitationfollowing single turnover).

EPR spectroscopy

CW EPR spectra were recorded at X band (9.42 GHz) on twoinstruments: (i) Bruker ESP 300E spectrometer using an Ox-ford instruments ESR 9 flow cryostat with ITC4 temperaturecontroller and (ii) a Bruker E500 ELEXSYS spectrometerequipped with a SuperX EPR049 microwave bridge, anST4102 cavity and Oxford Instruments ESR 900 cryostat andITC 503 temperature controller.The pulsed EPR experiments were performed with a Bruker

E580 ELEXSYS spectrometer equipped with ER4118XMD-5-W1 dielectric resonator. The system was fitted with an CF 935cryostat and ITC 503 temperature controller (Oxford Instru-ments Ltd., UK). The electron spin–lattice relaxation rateswere measured with an inversion recovery pulse sequence p–T–p/2–t–p, where the time t between the p/2 (16 ns) and p (32 ns)pulses was held constant at 200 ns. The measurements wereperformed at microwave frequency of 9.69 GHz at the fieldposition of 3346 G where the maximum absorption for YD

d

was observed. The repetition rate was never less than 26 msand data were acquired with 100 or 20 shots per point.

Sample illuminations

Samples were illuminated using a white light source in anitrogen gas flow cryostat. Green light filtering for 250 K (S1to S2 multiline) turnover was by a combination of blue and

P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 4 8 9 0 – 4 8 9 6 4891T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

orange filters and 15 cm of water. (IR totally excluded). Thisproduces B100% turnover to the S2 state, but B10–15% ofcenters may exhibit the g4.1 form of this state (e.g., see ref. 21).

Data fitting

The inversion recovery amplitude response, AY(t), for the YDd

spin lattice relaxation was fitted to a modified form of thedipolar interaction model, as discussed in ref. 8. This is of theform;

AY(t)/AY(0) ¼ F[dipolar model] þ (1 � F)[exp(�kbackgroundt)] (1)

AY(0) is the initial amplitude of the time dependent spin echosignal, determined as below. F is a fraction (0–1.00), whichrepresents the fraction of centers experiencing evident dipolarrelaxation interaction with another, specific paramagnetic cen-ter within the PSII complex. It was found necessary to invokethis term (not used in ref. 8), as the data clearly indicate thatnot all centers experience such interaction in our measurements(see Results). kbackground is a background, first order rate,which is presumed to operate in centers not experiencing thespecific dipolar interaction and includes the effects of all other(including weak dipolar) relaxation mechanisms. Within thelimits of our analysis, these can be adequately represented by asingle first order term.

The dipolar model, I(t), is of the form;

IðtÞ ¼Zp =2

0

sin y exp½�ðk1;scaler þ k1yÞt�dy ð2Þ

and represents a powder average of the angular dependentdipolar relaxation interaction, given by k1y, plus a first orderscalar background interaction, k1,scaler. In the fitting, thek1,scaler and kbackground terms were assumed to be equivalent,in a minimal model. This effectively assumes that all centersexperience the same background relaxation environment butthat in some (fraction F), an additional relaxation interactionis present.

The angular dependence of the k1y term was taken as;

k1y ¼ k1d(1 � 3 cos2y)2 (3)

This assumes that the spin lattice relaxation enhancement ofYD

d by the specific fast relaxing center is dominated by theleading B term in the dipolar interaction expression (see ref. 8).k1d is determined by the nature of the interacting spins andtheir separation (see Discussion). The inversion recovery data,as recorded, has an instrumental offset, which must be sub-tracted to obtain the true infinite time asymptotic response.This then allows determination of AY(0). The offset value wasdetermined to give the best linearity of the long time logresponse of the data, after subtraction of the offset. Thefractional uncertainty in the inferred parameter values forany individual fit isB5–7%, for the three dipolar model terms,and B1% for AY(0) (see Results).

Results

Studies were performed using PSII core complexes in bothsucrose and poly-alcohol cryoprotected buffers. No artificialacceptors were employed and under these conditions turnoverby brief (B15 s), 250 K green illumination produces the S2(multiline) QA

� state in high yield.21 Dark relaxation of suchsamples for B10 min. on ice or room temperature regeneratesthe S1 QA state quantitatively (see Fig. 1). These samples haveno EDTA or similar chelator added, so all background, nonspecific Mn present is presumably visible as ‘6 line’ Mn21. Thisspecies is variably present in the g2 region of the S1 spectra ofour core complex preparations and corresponds typically to

less than 1% of the total EPR active reaction centers. Althoughboth samples have the same nominal chlorophyll concentra-tions, the total number of EPR visible centers appears some-what greater in the EG/Gly samples (see also Fig. 4 below).The basis of this is currently not clear.The PSII core complexes totally lack the 16 and 23 kDa

luminal peptides. This appears to have no effect on catalyticactivity or the conventionally observed Mn derived EPRproperties (see ref. 21), but might conceivably have someinfluence on the protein matrix (solvent accessibility etc.)around YD. This appears not to be so, the spectral shape andpower saturation properties of YD

d in the core complexes aresimilar to that seen in PSII particles from the same source,when measured on the same spectrometer.19 The YD

d P1/2

Fig. 1 Wide scan CW spectra for PSII core complex samples in the S1(top) and S2 (multiline, bottom) states, for A: sucrose and B: EG/Glycryoprotected samples. These were generated as described in Methodsand below. Insets show the field swept, pulsed absorption spectra of theYD

d region, with the field position used for the inversion recoverymeasurements indicated (arrows). This lies within the regions aroundg ¼ 2 excised in the wide scans. Some free Mn21 is visible in thesesamples (o0.01 Mn21 per EPR visible reaction center). Both sampleshave the same nominal (1.0 mg ml�1) total chlorophyll concentration,but the total number of EPR visible centers (multiline, Cyt.b559, YD

d)appears to be greater in the EG/Gly sample (see also Fig. 4). Measure-ment details: S2 state generated by green illumination at 250 K for1 min and S1 obtained after annealing of illuminated spectra for10 min at room temperature (see Methods). Spectrometer conditions:CW spectra: microwave frequency 9.44 GHz, microwave power 5 mW,modulation amplitude 2 mT, modulation frequency 100kHz, tempera-ture 8 K. Field swept spectra: microwave frequency 9.69 GHz andtemperature 4 K. The pulse sequence p/2 (16 ns)–t (200 ns)–p–24 ns)was used.

Fig. 2 Typical behavior range of CW half saturation power (P1/2) forYD

d, as a function of temperature, in the PSII core complexes usedhere. Both data sets are for the S2 state. P1/2 values determined as inref. 19. Spectrometer conditions: microwave frequency 9.42 GHz,modulation amplitude 0.2 mT, modulation frequency 100 kHz.

4892 P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 4 8 9 0 – 4 8 9 6 T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

values typically lie within the range shown in Fig. 2 and arebroadly similar to those reported earlier on PSII particles,3 butlower overall. These data, which involve more assumptions intheir interpretation than spin lattice (T1) relaxation measure-ments, are not used for detailed quantitative analysis here.

Fig. 3 shows representative examples of inversion recoverykinetics of the YD

d signal in sucrose and EG/Gly buffers, overthe temperature range (4–25 K) studied. Shown also are therelevant fits of eqns. (1)–(3) to the data. Fig. 4 shows Curieplots of the initial amplitude (AY(0)) values for the samples inboth S states. These latter indicate that the total YD

d signalbehaves as expected and that no significant loss of YD

d

population occurs on the S2 - S1 annealing. Several observa-tions are immediately apparent from Fig. 3, which in fact applyto all the results obtained here:

(a) The relaxation kinetics are highly non-exponential atshort times, but are mono-exponential at long times to goodapproximation.

(b) The modified dipolar relaxation model fits the datareasonably well over the full time range, but only if the valueof F is allowed to vary substantially from unity (see Fig 3A).

This fit has only three free parameters and is constrained to gothrough AY(0), which we feel is reliably determined indepen-dently. Slight systematic discrepancies between the data andfits are sometimes evident, which we suspect may be due tomosaic spread of the F value within the sample.Figs. 5–7 summarise the parameter values from fitting the

model in eqns. (1)–(3) to all the YDd relaxation kinetics for core

complexes. Fig. 5 shows the temperature dependence of the k1dvalues for core complexes in both cryoprotectants. Thesedipolar rates in cores are at least an order of magnitude fasterthan the slow rates seen in PSII particles (for S1, single turn-over S2

4,7), or the background rates seen here (Fig. 6). The k1dvalues are of a similar order (41000 s�1) to that seen byBrudvig et al. in PSII membranes6,8,9 and the fast rate compo-nents in a fraction of centers seen by Evelo et al.4 In the presentcase there appears to be no systematic difference between thek1d values for S1 and S2 over the temperature range studied.The only consistent difference we detect (within the reliabilityof the analysis), is in the background rates for the EG/Glysamples (see Fig. 6A). This pattern appears also in refs. 4 and 7,where the slow rate components most consistently exhibit anincrease on first turnover.Fig. 7 shows the results for the inferred values of the dipolar

relaxation fraction, F, for all samples. Remarkably, this ap-pears to display a distinct temperature dependence over theobservation range, although there is scatter in the trend for alldata, as in Fig. 5. Again there is no consistent differencebetween S1 and S2 for either cryoprotectant. Since a given

Fig. 3 Representative examples of the YDd inversion recovery decay

kinetics for PSII core complex samples over the range 4 K (panels A,B;sucrose buffer) to 22 K (panels C,D; EG/Gly buffer). These (points) areshown together with fits (solid lines) of the dipolar relaxation model, asdiscussed in the text. Panels A and C show the time dependence of lnA(t) and panels B, D are linear in A(t). In panel A, the S2 data show thedipolar relaxation model values for both F ¼ 0.55 (as fitted), and F ¼1.00, keeping all other parameter values the same. The requirement forF values substantially less than one to obtain adequate fits to the data isclear. Spectrometer conditions as in Methods, using 100 shots perpoint. The S1 data in A and C are vertically offset by B1 ln scale unit.

Fig. 5 Temperature dependence of the YDd dipolar relaxation rate

parameter, k1d, for PSII core complexes. Data are for S1 and S2, asindicated. The line through the k1d data points is the average responsefor both S states and cryoprotectants in the core complexes.

Fig. 4 Curie plots of the initial YDd signal amplitude parameters,

AY(0), against inverse temperature, for sucrose and EG/Gly cryopro-tected samples in S1 and S2 states. (see Methods). Indicated also are thebest linear fits. As in Fig. 1, the total number of EPR visible centers isless in the sucrose samples than the EG/Gly samples, by about 25%.

Fig. 6 Temperature dependences of the YDd background rate para-

meter (k1,scaler or kbackground, assumed equal, see text) for A; EG/Glyand B; sucrose cryoprotected core complex samples (same data set as inFig. 5). The S2 � S1 difference in this rate parameter for the EG/Glysamples is indicated in A.

P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 4 8 9 0 – 4 8 9 6 4893T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

sample was measured (several times) at each temperaturepoint, this represents a real variation of F with temperaturewithin the sample. The temperature dependence may be stron-ger for sucrose than for EG/Gly cryoprotectant. There is anapparent ‘plateau’ for F of B0.75 for temperatures betweenB7 and 20 K. Similar to this, it was also earlier observed thatthe relative amplitude of the fast phase (approximately corre-sponding to k1d) and the slow phase (corresponding toour background exponential relaxation) in ref. 4 varied withtemperature.

Discussion

A conclusion to be drawn from these and the earlier studies onYD

d spin lattice relaxation is that while the relaxation proper-ties may vary widely in the S1 state, there is no substantialchange in the fast dipolar rate component on the first advance-ment to S2, at least for temperatures belowB10 K. In contrast,the slow background rate varies with S state in a mannersimilar to that found earlier.4 This implies either that the Mncluster in the multiline S2 state interacts weakly with YD

d andthat some other variable interaction dominates YD

d relaxation,or that the Mn cluster interaction is always the same in S2 andS1, even though the S1 rate varies substantially. The secondalternative seems implausible for a single, closely coupledcluster, but might be possible if the cluster is ‘uncoupled’ intomagnetically ‘isolated’ units (e.g., a pair of weakly interactingdimers). In either case, the component of the cluster generatingthe spin 1/2 multiline state would have only a weak dipolarinteraction with YD

d.The functional form of k1d is (Gaussian units, ref. 25),

k1d ¼ g2sm2fT2f

6r6½1þ ðos � ofÞ2T22f �

ð4Þ

Here gs is the gyromagnetic ratio of the slow relaxing species(i.e. YD

d), taken to be the free electron value. mf is the magneticmoment of the fast relaxing interaction center (multiline etc.),given by mf

2 ¼ gf2b2Sf(1 þ Sf), where gf and Sf are the g value

and total spin respectively of the fast center and b the Bohrmagneton. r is the separation between the spin centers, of andos their Larmor frequencies (rad s�1) and T2f the spin–spinrelaxation rate of the fast center.

The magnitude of k1d is sensitively dependent on the ‘spec-tral separation’ term, (of � os), whenever the latter is greaterthan T2f

�1. For the multiline signal at temperatures B10 K,T2f

�1 is of order 106 s�1.9 This MHz separation corresponds to

a field separation in the resonance positions of o10�2 mT,much less than the partially resolved hyperfine spectral widthof the YD

d signal at X band (B2.5 mT). The hyperfinedominated spectral width of the multiline signal is B180 mTand the absorption envelope is roughly gaussian, with a widthat half height of B80 mT. Both signals are centered very closeto g ¼ 2.00. Under these circumstances, the dipolar relaxationinteraction of the multiline center and a radical species likeYD

d may be complex, but two limiting cases are simple toanalyse. In the first (slow spin diffusion) limit, as discussed inref. 9, it is assumed that the Mn nuclear relaxation is slow onthe time scale of the YD

d signal relaxation (i.e. is o10�2 s).Then the observed YD

d decay is a powder sum of individualdecays, as in eqns. (2)–(4), but with the value of (of � os)statistically varying for each decay according to the spectraldistributions, gf(of), gs(os) of these two centers (which may beapproximated as Gaussians, say). Eqn. (2) then becomes;

IðtÞ ¼Z 1

�1gfðofÞ

Z 1

�1gsðosÞ

Zp =2

0

sin y exp �ðk1;scaler þ k1yÞt� �

dydosdof

ð5Þ

In the second limit (fast spin diffusion), each YDd center

experiences the same averaged interaction with the wholemultiline envelope, as the Mn hyperfine level interchanges arefast compared to the YD

d decay. Then eqn. (2) remains un-changed, but eqn. (4) becomes,

k1d ¼Z 1

�1gfðofÞ

Z 1

�1gsðosÞ

g2sm2fT2f

6r6½1þ ðos � ofÞ2T22f �

dosdof

ð6Þ

In the present case, gf(of) is assumed to be a broad Gaussian,of width parameter w/rad s�1 and same central g value as theslow spin, while gs(os) is relatively narrow so that its width maybe ignored. Then gf(of) B exp[�Do2/w2] , where Do ¼ (of �os) and eqn. (6) may be explicitly evaluated. The importantbehavior, as a function of the dimensionless parameter (wT2f)is indicated in Fig. 8). This shows that k1d is proportional to T2f

when (wT2f) is small, but independent of T2f and proportionalto Op/w when the dimensionless parameter is large (note thatOp/w is effectively the total ‘inhomogeneous line width T0

2 ofthe hyperfine envelope).

Fig. 7 Temperature dependence of the dipolar fraction parameter, F,from the YD

d relaxation rate analysis in PSII core complexes (see text).Data are for S1 and S2, as indicated (same data set as in Figs. 5 and 6).The line through the data points is the average response for bothS states and cryoprotectants.

Fig. 8 Normalised behavior of eqn. (6) as a function of the dimen-sionless parameter, (wT2f). Vertical axis is log scale for k01d ¼k1d[6wr

6gs�2mf

�2]. The values of k01d in the two asymptotic regions areindicated.

4894 P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 4 8 9 0 – 4 8 9 6 T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

Koulougliotis et al.9 have evaluated eqn. (5), using appro-priate parameter values for the multiline center at 7 K andfound that r B 13 A corresponds to a k1d value of B103 s�1.Scaling to the crystal structure value of r B 29 A, this gives anexpected relaxation enhancement for YD

d of B10 s�1, due themultiline center, at temperatures in the 4–10 K region. This iscertainly of the order seen for the background rate enhance-ment on the S1 - S2 transition in the EG/Gly buffered corecomplexes (Fig. 6A).

Applying the fast spin diffusion analysis (eqn. (6)), Fig. 8),the multiline system is completely in the (wT2f) c 1 region atcryogenic temperatures (since w B 1010 s�1 and T2f B 10�6 s).Then the predicted value for k1d is temperature independentand B104 s�1. This is of the order seen for the highertemperature k1d estimates in Fig. 5, but these show nosystematic differences between the S1 and S2 states. To inter-pret all of the YD

d relaxation data in these terms (including thePSII membrane results), it would be necessary to assume thatthe g values and spectral widths of the paramagnetic states ofthe (same) Mn cluster in S1 and S2 were indistinguishable undersome conditions, but that cryoprotectant or other effects (seeref. 7) could sufficiently influence Mn nuclear relaxation ratesto move both S states simultaneously into or out of the fast spindiffusion limit (particularly for PSII membranes). Given thatwe can discern no systematic differences in even the mostresolved multiline hyperfine features between, say, sucrosemultiline spectra in PSII complexes and particles, the aboveinterpretation appears to us unlikely.

A second interpretation is that the multiline center is alwaysin the slow spin diffusion limit under typical observationconditions and that the weak enhancement effects (see refs. 4and 7, Fig. 6A) seen on the YD

d slow decay rates reflect thisand the Mn cluster’s B30 A separation from YD. Under theseconditions, some other center is responsible for the variable,strong dipolar interaction with YD

d. This cannot be QA� as

this recombines in the S1 state in our samples. Further, the factthat the highly non-exponential YD

d decay at early time is wellfit by the simple dipolar model (eqns. (2) and (3), see Fig. 3)suggests that interaction with a single species at a fixedseparation from the YD

d center is responsible. This makesnon specific components, like dissolved O2, improbable candi-dates. The latter is also unlikely as these samples are notmultiply turned over, even during the pre-illumination, as noexogenous acceptors are present.

The Mn cluster in the S1 state is known to be even spin anddiamagnetic at sufficiently low temperatures. However Brudviget al. have established that the antiferromagnetic coupling thatdetermines if paramagnetic, excited states are populated athelium temperatures, is variable. In the so called ‘active’ state,this coupling is sufficiently weak (few cm�1) that excited stateinteraction is observable as a strong dipolar relaxation en-hancement of YD

d, even at temperatures below 10 K. The ratesare comparable (B103 s�1) to those for k1d in Fig. 5. In the‘resting’ state, reported to be achieved only after long (hours)dark incubation at 0 1C,6 the YD

d relaxation rate has fallen tovalues similar to the background rates seen here (Fig. 6) andthe slow rates seen by Evelo, Evans and coworkers.4,7 As notedearlier, the experiments of Brudvig et al. employed 30% EG asthe cryoprotectant. It is unclear however, how variable thekinetics for the active to resting transition may be in the EG orsucrose buffers, as the ‘extensively’ dark-adapted material inref. 8 displayed YD

d relaxation rates at 20 K that were verysimilar in S1 and S2 and to the k1d data in Fig. 5. The S2 resultsfrom ref. 9 also for 4 h dark-adapted PSII particles, were againsimilar in magnitude and temperature dependence to the k1ddata in Fig. 5. A reasonable interpretation of the results ofEvelo et al. is that in sucrose containing buffer (even with EGpresent), 10 min dark relaxation at B20 1C is sufficient togenerate a substantial population of PSII centers in the restingstate. Therefore the cryoprotectant may influence the stability

of the active and resting forms in PSII particles. Brief (minutes)dark adaptation is sufficient to produce the resting state insucrose buffer, but in EG (and probably glycerol) buffers, theactive form may persist for extended times. In PSII corecomplexes, lacking the 16 and 23 kDa extrinsic proteins, theactive form appears stabilized in either cryoprotectant regime.The apparent temperature dependence of the dipolar relaxa-

tion center population (F) in Fig. 7 is then revealing. It impliesthat the ‘active’ form in S1 or S2 contains the same temperaturedependent population of fast relaxation centers interactingwith YD

d, in the same way. From the results of Ahrling,Peterson et al.18,19 it is likely that this same center interactsmuch more strongly with the Mn cluster producing the multi-line signal (i.e. is much closer to it, see ref. 18), but theseconclusions are presently based only on signal saturation P1/2

estimates. Since this putative relaxation center is present in theactive S1 state, which is diamagnetic at sufficiently low tem-peratures but has a thermally accessible (above B4 K) firstexcited (spin 1) state, it is tempting to interpret the data inFig. 7 as simply the progressive population of this and higherexcited states, influencing the YD

d decay rates. The fact thatthere is a temperature ‘plateau’ around F B 3/4 in the averageresponse in Fig. 7 is quantitatively consistent with this, if theanti-ferromagnetic exchange interaction is only B2 cm�1.(This follows since the triplet has a degeneracy of threeand the total degeneracy of this and the ground state is four-with all levels roughly equally populated). However such aninterpretation means that magnetic interchange between thedifferent even spin states of the relaxer’s own spin manifold isitself quite slow, on even the slowest YD

d decay rate time scale(B10�2 s). The relaxer center does not, on average, changeits spin identity as YD

d decays. In fact little appears to beknown about such total spin interchange rates in even spincoupled manifolds at low temperatures, the reactions being ofcourse formally spin forbidden. The well documentedexamples of the somewhat analogous Orbach relaxation me-chanism occur within the fine structure sub-states of paramag-netic ions of fixed total spin (e.g., Fe31 centers in proteins, seediscussion in ref. 26), although the Orbach mechanism hasbeen invoked for the T1 relaxation of the spin 1/2 multilinestate (e.g., see ref. 27), based on temperature dependencearguments.If the relatively weak temperature dependence of the fast k1d

rates seen in Fig. 5 and refs. 6 and 9 is taken to mean that thecenter that enhances YD

d relaxation is in the fast spin diffusion,(wT2f) c 1 region (see above), then some tentative conclusionsmay be drawn. Since this center is presumably near the Mncluster, rB 25–30 A and Sf ¼ 1 for temperatures in the 4–20 Krange. Then eqn. (6) and Fig. 5 give a signal width, w, estimateof B1011 s�1. This corresponds to a CW EPR line-width ofB500 mT, which would make the signal essentially undetect-able by conventional EPR techniques. Such a width is not at allimplausible however if the center were composed of a coupledpair of even spin paramagnetic ions (Mn(III), say). Cryopro-tectants might modulate the behavior of such a system inseveral ways, consistent with the phenomenology observedhere. The magnitude of the anti-ferromagnetic coupling couldbe influenced, it would need to become only a few tens of cm�1

for the center to become magnetically ‘silent’ below B50 K.Another intriguing possibility arises from the observations ofAhrling et al.19 on the multiline and YD

d relaxation ratedependences on the quinone pool reduction state in PSIIparticles and the observation of an apparent variability in themean Mn XANES edge energies of the S1 state (measured aspoints of inflection).28,29 This suggests that the actual redoxstate of the center may be variable, e.g., between Mn III–III andMn IV–IV, say. In the latter case (resting state), the center wouldalmost certainly be coupled silent at the temperatures exam-ined here. The manner in which cryoprotectants then alteredthe stability balance between these forms might be related to

P h y s . C h e m . C h e m . P h y s . , 2 0 0 4 , 6 , 4 8 9 0 – 4 8 9 6 4895T h i s j o u r n a l i s & T h e O w n e r S o c i e t i e s 2 0 0 4

mean, local dielectric environments within the protein matrix,or local protonation states.

Acknowledgements

SS and FM gratefully acknowledge financial support from theKnut and Alice Wallenberg Foundation, Swedish ResearchCouncil, DESS and the Swedish Energy Agency. RP and PSgratefully acknowledge financial support from the AustralianResearch Council.

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