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Electron spin echo studies of complex biological systems. An application to “signal II” of plant photosynthesis
CHEhlICAL PHYSICS LE’JTERS
Volume 58, number 2
15 September 1978
ELECTRON SPIN ECHO STUDES OF COMPLEX BIOLX)GICAL SYSTEM!% AN APPLIC.~TON TO “SIGNAL Ii” OF PLANT PHOTOSYNTHESIS N.N. NISHI, A.J. HOFF, J. SCHMIDT and J-H. VAN DER WAALS Centrefor the Study of the Excited State of Moiecuulesand BibphysicsDepartment. Huygens Lubomtotim State Universityof Lqyden. Leyden. Ihe Netherlands Received 9 June 1978
Electron spin-echo resonance can be used to unravel overktpping ESR signals from complex biological systems. The variation of experimental parame:crs, as repetition raze of the pulse sequence, delay time and pulsewidth, permirs the extracticn of signaIs with Werent Iongitudinal and transverse relaxation times- By way of illustration we report the investigation at 1.2 K of so-called signal II of plant photosynthesis. It is demonstrated that signal II is made up of at least three ‘dark” components that dizTerin their relaxation times. Upon illumination at 1.2 K two additional reversible light-induced components appear with different decay characteristics and relaxation behaviour.
i _ Introduction
2. Experimental
Pulsed ESI?, or electron spin-echo (ESE), spectrometry has been initiated by Kaplan et al_ [l] and Mims [2] _ Until now, ESE has mostly been applied to the study of well-defined species such as transition ions in crvstals and glasses (see e.g. refs. [2,3]), powders of some pure enzymes (see e.g. refs. [4,5] ) and organic crystals [6]. In this communication we demonstrate that ESE is a powerful technique to study overlapping ESR signaIs of complex biological systems by making use of its potential to discriminate between species differing in their longitudinal and/or transverse relaxation times. As an illustration we present some results on the
2.1. Design
so-called
signal II of plant
photosynthesis_
It is anti-
cipated, however, that the electron spin-echo technique in the form used here has a much wider applicability and might greatly aid in the unravelling of the ESR signals of other biological systems, for instance those arising from the electron tramport components in the respiratory chain.
164
of the experiment
The technique of ESE has been described in detail by Kaplan et al. [I] and by Mims [2]. Briefly, a 90” pulse of resonant microwaves tips the magnetization M, from the z-axis into the x--y plane, where it starts to precess with the iarmor frequency around the magnetic field HO. After a time 7, when the magnetization has disappeared owing to a spread in the focal magnetic fields, a 1 SO0 pulse is applied by which the magnetization is refocussed and a signal, the echo, is indated in the detector. The height of the echo, 1, depends on 7 and for a Iorentzian profile of the absorption line the dependence is exponential, I= I0 exp(-2r/TM). The phase-memory time Thi thus defined Is a measure for the transverse relaxation time T2 of the individual spin-packets [7]. In the experimental procedure we apply the twopulse sequence repetitively at a rate R and sample the echo height bjr a boxcar signal detector gated at time 27 (fig. 1). If there are seve_ralspecies present with different T1, then all species will contribute to the echo if R-1 exceeds the longest T1 present. However, ifR_I is much shorter than the TI of a given species, this particular species does not have the time to come
Volume 58, number 2
CHEMICAL
PHYSICS
15 September 1978
LEl-fERS
Boxcar gate l-l Mumwhve
Il.,
-ygJJ-$+4~i=+
L-
t
repetlhon
time _/
Fig_ 1. Schematic description of electron spin-echo detection. Pulse sequenaz and box-car detection of the echo.
to Boltzmann equilibrium during the time ,P-* separating the pulse pairs and its contribution to the echo intensity will be decreased by a factor 1 exp(-l/RT1)Therefore by increasing R, one can selectively depress components with the longer TI. The pulsewidths yielding optimal echo intensity differed somewhat for the various paramagnetic species encountered in the photosynthetic material studied. This provides another means of discrimination between different components in a complex spectrum. The ESE technique is also suited for the measurement of +-hespin--lattice reJaxation time Tr _ To this end a three-pulse sequence is employed. First, a 180” pulse is given. Ihis pulse inverts the magnetization Al, to --Afz. After a variable time interval tS, a 90”180” pulse sequence (separation r) is given and the ensuing echo monitored with the boxcar gate set at rs + 2~. During the interval tS the spin system returns to equilibrium at a rate Til, and the echo height plotted against fs thus yields a saturation recovery curve. The slope of a semi-logarithmic plot of this curve gives the spin-lattice relaxation rate, Ti’. 2_2_ Apparatus
X-band (9 GHz) cw ESR (100 kHz field modulation, super-heterodyne detection) and ESE measurements were carried out at 1.2 K in a liquid helium immersion type cryostat. By a simple change the spectrometer could be converted from cw ESR to ESE mode of operation, without affecting the sample or its temperature. This permitted direct comparison of the cw ESR spectrum -with the ESE spectrum. Illumination of the sample was carried out with a Philips SP 1000 W high pressure mercury arc, suitably filtered by a combination of a solution of 100 g/ml
CuSO, in water (10 cm), a Schott KV 520 filter and a BaLze. CAflex Cheat reflecting filter (power incident on the cavity 40 mW/cm2). Check experiments with pammagnetic dummy samples having similar absorption characteristics as the chloroplasts suspension showed the temperature rise due to illumination to be negggible as measured by the paramagnetic susceptibility at 1.2 K. 2.3. Materials Chloroplasts
were
prepared
from
market
spinach
as described [8], suspended in a medium containing 50 mM tricine @H 7.8), 0.4 M sucrose, 10 mM KC1 and 2 mM MgC12, and then stored on ice in the dark until use. In order to avoid crystallization 50% v/v glycerol was added before ftig the ESR sample tubes with the dark-adapted chloroplast suspension (about 5 mg Chl/ml). Ail samples are exposed during handling for about one minute to weak white li$t (<50 gW/crn2), then left in the dark for several minutes, and subsequently frozen in a stream of cooled. N2 gas (=lOO K). For some experiments 30 fl of the electron transport inhibitor 3(3,4dichlorophenyl)l,ldimethylurea (DCMU) was added.
3. ResuIts We measured the cw ESR and ESE spectra at 1.2 K under two different conditions: (i) after adaptation in the dark, (ii) directly after five minutes of illumination at 1.2 K. The ESE spectra taken over a wide field range showed various signals at g = 6,g = 4.3,,0=2-05, g = 2.00 andg = 19-l -8. in this report we concentrate on the signals near g = 2.00. The work on the other sig165
Volume 58, number 2
- CHENICAL PHYSICS JXERS
nals, attributed to various elecrron carriers in the photosynthetic chain, will be presented elsewhere [9]. 3. I. L&k spectnz In fig- 2 we show the cw ESR and E!!X spectra in the region around g = 2.OC, centered around 3200 G, taken in the dark before low temperature illumination‘Ihe cw JZSR spectrum represents the so-calIed ‘signal II? [lo]. The ESE spectrum (which is a representation of the absorption line x” versus Ho, in contrast to the derivative dx”/d.&, versus H, observed in cw ESR is seen to correspond to the integral of the cw ESR spectrum of s&nal II. In the echo spectrum signal II is superimposed on a very broad background (rang&g between 2500 and 4000 C, not shown in fig. 2) of i&r&y about 20% of signal II, which must be due QxhulbndEPR
@
_‘,_=
_,.-*
.-
/“”
to other electron carriers (plastocyanin and ferredoxins). ‘Ihe addition of the electron transport inhibitor DCMU, known to decrease the intensity of cw ESR signal II appreciabj [I 1,121, lowered the amplitude of the ESE spectrum of signal II by a factor of about 10 (data not shown). 3.2. Spin--lattice re2iucation In fig_ 3 plots of the saturation recovery are displayed. It is seen that the “dark” spectrum of signal II contains at least three components, with Tl of abol-lt 0.6,3-O and 20 ms. The measurements were carried out at a repetition rate of 10 Hz, which eliminates components with Tl > 200 ms. The 0.6 ms component, posdbly, contains even faster relaxing species, but these could not be resolved. In what follows these components are included in the “T1 = 0.6 ms” cornponent. The slowest component has a T1 = 20 ms, that is close to the Tl for the background signal (1822 ms) as measured at 3300 G and 3 100 G respectively. Ihe amplitude of the 20 ms component measured at 3200 G, however, is much larger than the intensity of the background signai, so that the 20 ms component must be part of signal II- By varying the repetition rate R we can seIectiveIy study either all components (R = 50 Hz), mainly the components with T1 of 3.0 and 0.6 ms(R=l kHz),oronlythatwithT1 of0_6ms(R> 5 kHz>.
3.3. Base-meaty
I
1 3Iw
I
I
3193
I
I
3200
t
1
3210 Uaqnzhc
,L 3239 heklOku-xsl
Fig. 2 _ comparison of electron spinecho spectra @I) with cw ESR spectra (a) of untreated chIcroplasts at 1.2 K. Drawn lines: dark spectra. Dotted iines: spectra taken after 5 mbt of iIluminationat 1.2 K. The corresponding LW ESR spectrum has been reduced by one half and the ESE spectrum has becu scald to the dark spectrum. Instrument settins: cw ESR, microwave Power, 0.1 JJW.moduIation, 2 G at It30 kHz, scan rate 5 G/min; ESE., microwave peak power, 1 ‘X, repetitio6 rate, 50 Hz, 90” p&se duration, 200 ns, delay time r, 1.2 fi.
166
15 Seprember 1978
times
In fg. 4 the decay envelope of the two pulse echoes of signal II is shown for three values of R. If the echo signal originated from one species, than the decay envelope would be independent of R and only the relative intensity would change because of spinlattice IAaxation. By contrast, in fig. 4 it is seen that at R = 2 kHz the decay envelope is much steeper (TM = 0.9 JLS)than at R = 50 Hz (TM * 2.2 I.CS).With the latter rate all components are present, whereas at the former rate only the component with T, = 0.6 ms is measured. The decay envelopes show modulation due to hyperfine interactions. _4t R = 2 kHz, a strong modulation is present with a period in the order ofabout 0.5 p, which is absent for R = 50 Hz_ The phasememory times of the background at g= 2.0 are bnger (about 3 m), they do not contribute significantly to the curves dis&yed in fig- 4.
Volume
number 2
CHEMICAL
PHYSICS
LElTERS
15 September 1978
Fig. 3. Saturation recovery curves of signal II at 1.2 K for dark-adapted (0) and illmninated (A) samples. The recovery curves are decomposed iota three components by linear regression. Instrument settings: repetition rate, 10 ,rIz, 90” pulse duration, 200 ns, delay time, 1.2 fls.
3.4. Effects of il!umimtion a? 1.2 K After taking the “dark” spectra of signal II, the sample was illuminated for 5 minutes at 1.2 K. The cw ESR spectrum taken in the dark after this period of illumination is indicated by the dotted curve in fig. 2. It is seen that a strong signal corresponding tog = 2.0025 has been generated. This signal, which is ir-
, ,
)
32OOGouss
,
,
,
,
1
L
)
,
)
,
,
,
,
-__
z_“a
,
,
I
I
,
,rn”_
-..-
10 =
4i
Fig. 4. Echo intensity of signal II as a function of the delay time 7 at 1.2 K. Frequencies indicate rcpetitiotl rate R
reversible at 1.2 K, is called signal I and it is due to the oxidized primary donor of photosystem I, P700+ (for recent reviews see. refs. [13-15])Ihe ESE spectrum of the illuminated sample, again taken in the dark, is also shown in fig_ 2. Strikingly, this spectrum does not show any trace of signal I. Apart from an increase of about 40% (_$ = 50 Hz), the spectral shape is almost the same as that taken before illumination except for a slightly enhanced relative intensity at 3207 G (g = 2.000) @ii_ 2b, dotted line). We ha- varied thz repetition rate of the ESE sampling from 10 kHz to 10 Hz, without detecting a change in intensity at g = 2.0025 compared to the first dark ESE spectrum. We thus conclude that it is not a very long Tl which is responsrhle for the absence of the P700* signal, but that P700+ must have a very short phase-memory time Thf. Our current instrumental time resolution, limited by “cavity ringing”, is 1.1 ps, so that ThI (P7GO*) 55 0.5 ws. This would be in line with the short phase-memory time, TM = 0.5 ys, which we have measured in zero fiefd for the triplet state of ihe primary donor of the photosynthetic bacterium Rps. sphaeroides 1161. The possibility to discriminate against signal I with the ESE technique allows one to draw conclusions about low temperature electron-transport processes involving the precursors to signal II [9]. This is illustrat167
Volume 5 8. number 2
15 September 1978
CHEMICAL PHYSICS L?Z?ZRS
i.t..I~...l...,I,.,.J
-20
-10
0
10 AH Gaussl
20
Fig. 6. Drawn line: amPEtude of the rapid decay component of fii. 5 as a function of the magnetic field. Repetition rate A = 5 IrHz. Dashed line: difference spectrum light on - light off at R = 1 kHz. 90” pulse duration, 200 ns.
Fig. 5. Light-induced reversibfe components of signal II zt
1.2 K. Intensity ofilIuminatron 40 mW/cmz. Cumtion of 90° pulse, 200 ns, dehy time I, 1.2 gs- IO denotes the backgroltnd echo intensity measured at 317.5 C. The slow decay has a chamcteristic time of 4.4 min. Arrows indicate opening (up ~zrd) and ciosin,o of shutter. ed by our study of light-induced changes in the ESE
spectrum of signal Ii. These changes turn out to be dependent on the repetition nteR. At 50 Hz the first period of illumination causes the intensity of the ESE spectrum to increase by 40% of the initial dark intensity- This increase is irreversible. However, at a repetition rate of 5 kHz (only the component of signal If with Tl = 0.6 MS then is observable), a reversible change is observed. This is illustrated in fg. 5, where the ~~ht-induced reversilile change is displayed for repetition rates of 500 Hz and 5 l&z. At 5 kHz 35% of the light-induced increase decays very rapidiy, whereas the remaining signal decays with the very slow characteristic time of 4-4 min. The intensity of this slow component was reduced by 20% after 3 h of continuous iUuminatbn_ It is seen that the intensity of the rapid decay component is dependent on the repetition rate: at R = SCM3 Hz this component has disappeared. In fig. 6 the amplitude of the rapid decay component is plotted as a function of the magnetic field (drawn line). The spectral shape is different from that of the light-induced difference spectrum measured at R = 1 kHz to which all TX components contribute (fig. 6, dasbecl line). 168
When comparing the intensities of the slow and the fast decay component for various values of R with the intensity of the dark signal measured at R = 500 Hz, a maximum is-found at 2 kHz for the faster decay component, whereas the slower component exhibits its ma&urn at 500 Hz. These values correspond to Tl values of 0.5-0.7 and 2-3 ms respect?dely.
d Discussion According to previous investigations by cw ESR at room temperature three different types of signal II can be ~t~gu~h~. These differ in their induction kinetics and decay times, but not in their spectra1 shape [12,15,17-201, Signal II,, (S II,) is always present in the dark, even after prolonged periods of dark adaptation. Signal I&low (S II,) is induced by iilumination at room temperature (rise time 1 s) on top of S fi, and to an equal extend as S II,, it decays in l-12 h depending on the preparation. Signal IIv,,v W [21,22], and a similar form, signal I& [19,20], have rapid rise and decay kinetics- Since the latter two are connected to the oxygen evolvmg complex of photo. system 2 which stops functioning below -6O*C i23, 241, these signals need not be considered in this work. it has been demonstrated that S I& and 8 IIs are due to oxidation of a premraor [ 17,25,26~, suggested
Volume 58, number 2
CHEhHCAL PHYSICS LETlXRS
to be a chromanoi derivative of plastoquinol [2728] _ Esser [29] and Ruuge et al. [3Q] found that the shape of signal II is dependent on microwave power, and attributed this to the presence of two different corn ponents. Babcock and Sauer [X8] found that the width of signal II varied from 16 to 19 G depending on the ionic strength of the medium- The present work shows that our dark signal II (composed of S II, and S lls) contains at least three different components with relaxation times T1 = 0.6,3 and 20 ms respectively. That several species contribute to S II is further demonstrated by the fact that our component with T1 = 0.6 ms also has a different phase-memory time, viz. 0.9 MSas compared to the 2.2 crs for the other two components. Moreover, its decay envelope is modulated by hyperfine interaction, corresponding to a hyperfine coupling constant of the order of 0.7 GLarger hyperfine couplings were difficult to measure because of the limitations on our instrumental time resolution. The other two components with the longer Tl’s do not show such a pronounced modulation. The diversity in signal Ii we have detected by ESE cannot be explained by assuming that S II is due to just one radiml species- The difference in Thl and the nuclear modulation shown in fig. 4 indicates that at least two radicals are present. Both these radicals exhibit various spin-lattice relaxation times, presumably due to differences in the environment. If we accept the evidence that signal II is due to an oxidized piastoquinol derivative [27,28], then we must assume that these molecules from a heterogeneous population with different locations with respect to photosystems 1 and 2. To account for the variability in spectral shape and in Thl and nuclear modulation, we propose that at room temperature plastoquinol is in dynamic equilrbrium with its chromanol anaiogue. Such ~u~~~~ can easily be shifted by photo-oxidation of the quinol, which facilitates reversibility of the ring-closure reaction [31]. Thus, under our conditions, sig,nal II is attributed to a mixture of a phenoxyl and a semiquinone type spectrum. The maximum in intensity at g =2.005 of the R = 5 kHz spectrum of fig. 6 would then be due to an enhanced contribution of the narrow semiquinone radical. The implications of this proposal for electron transport in plant photosynthesis are discussed elsewhere [9] ‘Ihe above hypothesis can be tested by performing studies with quinols of different side-chain structure,
15 September 1978
either in vim or by extraction/reconstitution experiments analogous to those of Kohl et al- [27]. The ESE technique would seem to be a valuable aid in such investigation.
Acknowledgement We are grateful to Dr. J. Lugtenbug for a helpful discussion on the conversion of quinols to chromanols. This work was supported by the Netherlands Foundation for Chemical Research (S.O.N.) with furancial assistance from the Netherlands Organization for the Advancement of pure Resear~ (Z.W_O_)_
References Ill D-E. Kaplan, WE
Browne and J.A. Cowen, Rev. Sci. In&. 32 (1961) 1182. 121 W-B. Mims, Rev_ Sci_ Instr. 36 (1965) X472_ 131 W-B. hfims and J-L. Davis, J. Cbem. Phys. 64 (1976) 4836. 141 W-3. Mims and J_ Peisxh, Biochemistry 15 f1976) 3863. I53 B. Mondovi, hi-T_ G raziani, W.B. Mints, R. Oltzik and J_ Pcisacb, Biochemistry 16 (1977) 4198. 161 BJ. Botter, DC. Doetschman, J. Schmidt and J.H. van der WI&, hfol- Phys 30 (1975) 609. I71 W.B. Ilfims, in: Eiecc_ronpammag~tic resonance, ed. S. Geschwind
X69
Volume 58, number 2
CX-IEMICAL PHYSICS
RE. BIanIcenship, G-T. Babcock, J.T. Warden and K- Sawr, FEBS Letters 51 (X975) 287. (22 J R.E. Bknkenship. A. McGuite and K. Sauer, Biochim. Biophys. Acta 459 (1977) 617. 1231 3-T. Warden, RE. Blankenship and K. Sauer, Biochitt~ Biophys Acta 423 (1976) 462_ [24 J k Joliot, Biochim. Biophys. Act3 357 (1974) 439. 1251 A-F. Esser, Photochem. Photobiol. 20 (1974) 173. (261 J-T. War&m and J-R_ BoXton, Photochem. PhotobioL20 (1974) 245. [21]
170
LETIERS
15 September 1978
1271 D-H. Kohland P-M- W_ood, Pkmt PhysioL 44 (1969) 1439, 1281 D-I-I. Kohl, J.R. Wright and M. We&man, Eio@im. Biophys. Acta 180 (1969) 536. 1291 A-F. Esser, Photochem. Photobiot 20 (1974) 167. 1301 E-K- Ruuge, A-V_ Z”iionov and L-k Blyumenfeld, Biofii 19 (1974) 1034. 13x1 R- Bent&y and I.&f. CampbeII, The chemistry of quinoid compounds, Part II Wey, New York, 1974) ch. 13, p. 683.
Volume 58, number 2
15 September 1978
ELECTRON SPIN ECHO STUDES OF COMPLEX BIOLX)GICAL SYSTEM!% AN APPLIC.~TON TO “SIGNAL Ii” OF PLANT PHOTOSYNTHESIS N.N. NISHI, A.J. HOFF, J. SCHMIDT and J-H. VAN DER WAALS Centrefor the Study of the Excited State of Moiecuulesand BibphysicsDepartment. Huygens Lubomtotim State Universityof Lqyden. Leyden. Ihe Netherlands Received 9 June 1978
Electron spin-echo resonance can be used to unravel overktpping ESR signals from complex biological systems. The variation of experimental parame:crs, as repetition raze of the pulse sequence, delay time and pulsewidth, permirs the extracticn of signaIs with Werent Iongitudinal and transverse relaxation times- By way of illustration we report the investigation at 1.2 K of so-called signal II of plant photosynthesis. It is demonstrated that signal II is made up of at least three ‘dark” components that dizTerin their relaxation times. Upon illumination at 1.2 K two additional reversible light-induced components appear with different decay characteristics and relaxation behaviour.
i _ Introduction
2. Experimental
Pulsed ESI?, or electron spin-echo (ESE), spectrometry has been initiated by Kaplan et al_ [l] and Mims [2] _ Until now, ESE has mostly been applied to the study of well-defined species such as transition ions in crvstals and glasses (see e.g. refs. [2,3]), powders of some pure enzymes (see e.g. refs. [4,5] ) and organic crystals [6]. In this communication we demonstrate that ESE is a powerful technique to study overlapping ESR signaIs of complex biological systems by making use of its potential to discriminate between species differing in their longitudinal and/or transverse relaxation times. As an illustration we present some results on the
2.1. Design
so-called
signal II of plant
photosynthesis_
It is anti-
cipated, however, that the electron spin-echo technique in the form used here has a much wider applicability and might greatly aid in the unravelling of the ESR signals of other biological systems, for instance those arising from the electron tramport components in the respiratory chain.
164
of the experiment
The technique of ESE has been described in detail by Kaplan et al. [I] and by Mims [2]. Briefly, a 90” pulse of resonant microwaves tips the magnetization M, from the z-axis into the x--y plane, where it starts to precess with the iarmor frequency around the magnetic field HO. After a time 7, when the magnetization has disappeared owing to a spread in the focal magnetic fields, a 1 SO0 pulse is applied by which the magnetization is refocussed and a signal, the echo, is indated in the detector. The height of the echo, 1, depends on 7 and for a Iorentzian profile of the absorption line the dependence is exponential, I= I0 exp(-2r/TM). The phase-memory time Thi thus defined Is a measure for the transverse relaxation time T2 of the individual spin-packets [7]. In the experimental procedure we apply the twopulse sequence repetitively at a rate R and sample the echo height bjr a boxcar signal detector gated at time 27 (fig. 1). If there are seve_ralspecies present with different T1, then all species will contribute to the echo if R-1 exceeds the longest T1 present. However, ifR_I is much shorter than the TI of a given species, this particular species does not have the time to come
Volume 58, number 2
CHEMICAL
PHYSICS
15 September 1978
LEl-fERS
Boxcar gate l-l Mumwhve
Il.,
-ygJJ-$+4~i=+
L-
t
repetlhon
time _/
Fig_ 1. Schematic description of electron spin-echo detection. Pulse sequenaz and box-car detection of the echo.
to Boltzmann equilibrium during the time ,P-* separating the pulse pairs and its contribution to the echo intensity will be decreased by a factor 1 exp(-l/RT1)Therefore by increasing R, one can selectively depress components with the longer TI. The pulsewidths yielding optimal echo intensity differed somewhat for the various paramagnetic species encountered in the photosynthetic material studied. This provides another means of discrimination between different components in a complex spectrum. The ESE technique is also suited for the measurement of +-hespin--lattice reJaxation time Tr _ To this end a three-pulse sequence is employed. First, a 180” pulse is given. Ihis pulse inverts the magnetization Al, to --Afz. After a variable time interval tS, a 90”180” pulse sequence (separation r) is given and the ensuing echo monitored with the boxcar gate set at rs + 2~. During the interval tS the spin system returns to equilibrium at a rate Til, and the echo height plotted against fs thus yields a saturation recovery curve. The slope of a semi-logarithmic plot of this curve gives the spin-lattice relaxation rate, Ti’. 2_2_ Apparatus
X-band (9 GHz) cw ESR (100 kHz field modulation, super-heterodyne detection) and ESE measurements were carried out at 1.2 K in a liquid helium immersion type cryostat. By a simple change the spectrometer could be converted from cw ESR to ESE mode of operation, without affecting the sample or its temperature. This permitted direct comparison of the cw ESR spectrum -with the ESE spectrum. Illumination of the sample was carried out with a Philips SP 1000 W high pressure mercury arc, suitably filtered by a combination of a solution of 100 g/ml
CuSO, in water (10 cm), a Schott KV 520 filter and a BaLze. CAflex Cheat reflecting filter (power incident on the cavity 40 mW/cm2). Check experiments with pammagnetic dummy samples having similar absorption characteristics as the chloroplasts suspension showed the temperature rise due to illumination to be negggible as measured by the paramagnetic susceptibility at 1.2 K. 2.3. Materials Chloroplasts
were
prepared
from
market
spinach
as described [8], suspended in a medium containing 50 mM tricine @H 7.8), 0.4 M sucrose, 10 mM KC1 and 2 mM MgC12, and then stored on ice in the dark until use. In order to avoid crystallization 50% v/v glycerol was added before ftig the ESR sample tubes with the dark-adapted chloroplast suspension (about 5 mg Chl/ml). Ail samples are exposed during handling for about one minute to weak white li$t (<50 gW/crn2), then left in the dark for several minutes, and subsequently frozen in a stream of cooled. N2 gas (=lOO K). For some experiments 30 fl of the electron transport inhibitor 3(3,4dichlorophenyl)l,ldimethylurea (DCMU) was added.
3. ResuIts We measured the cw ESR and ESE spectra at 1.2 K under two different conditions: (i) after adaptation in the dark, (ii) directly after five minutes of illumination at 1.2 K. The ESE spectra taken over a wide field range showed various signals at g = 6,g = 4.3,,0=2-05, g = 2.00 andg = 19-l -8. in this report we concentrate on the signals near g = 2.00. The work on the other sig165
Volume 58, number 2
- CHENICAL PHYSICS JXERS
nals, attributed to various elecrron carriers in the photosynthetic chain, will be presented elsewhere [9]. 3. I. L&k spectnz In fig- 2 we show the cw ESR and E!!X spectra in the region around g = 2.OC, centered around 3200 G, taken in the dark before low temperature illumination‘Ihe cw JZSR spectrum represents the so-calIed ‘signal II? [lo]. The ESE spectrum (which is a representation of the absorption line x” versus Ho, in contrast to the derivative dx”/d.&, versus H, observed in cw ESR is seen to correspond to the integral of the cw ESR spectrum of s&nal II. In the echo spectrum signal II is superimposed on a very broad background (rang&g between 2500 and 4000 C, not shown in fig. 2) of i&r&y about 20% of signal II, which must be due QxhulbndEPR
@
_‘,_=
_,.-*
.-
/“”
to other electron carriers (plastocyanin and ferredoxins). ‘Ihe addition of the electron transport inhibitor DCMU, known to decrease the intensity of cw ESR signal II appreciabj [I 1,121, lowered the amplitude of the ESE spectrum of signal II by a factor of about 10 (data not shown). 3.2. Spin--lattice re2iucation In fig_ 3 plots of the saturation recovery are displayed. It is seen that the “dark” spectrum of signal II contains at least three components, with Tl of abol-lt 0.6,3-O and 20 ms. The measurements were carried out at a repetition rate of 10 Hz, which eliminates components with Tl > 200 ms. The 0.6 ms component, posdbly, contains even faster relaxing species, but these could not be resolved. In what follows these components are included in the “T1 = 0.6 ms” cornponent. The slowest component has a T1 = 20 ms, that is close to the Tl for the background signal (1822 ms) as measured at 3300 G and 3 100 G respectively. Ihe amplitude of the 20 ms component measured at 3200 G, however, is much larger than the intensity of the background signai, so that the 20 ms component must be part of signal II- By varying the repetition rate R we can seIectiveIy study either all components (R = 50 Hz), mainly the components with T1 of 3.0 and 0.6 ms(R=l kHz),oronlythatwithT1 of0_6ms(R> 5 kHz>.
3.3. Base-meaty
I
1 3Iw
I
I
3193
I
I
3200
t
1
3210 Uaqnzhc
,L 3239 heklOku-xsl
Fig. 2 _ comparison of electron spinecho spectra @I) with cw ESR spectra (a) of untreated chIcroplasts at 1.2 K. Drawn lines: dark spectra. Dotted iines: spectra taken after 5 mbt of iIluminationat 1.2 K. The corresponding LW ESR spectrum has been reduced by one half and the ESE spectrum has becu scald to the dark spectrum. Instrument settins: cw ESR, microwave Power, 0.1 JJW.moduIation, 2 G at It30 kHz, scan rate 5 G/min; ESE., microwave peak power, 1 ‘X, repetitio6 rate, 50 Hz, 90” p&se duration, 200 ns, delay time r, 1.2 fi.
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15 Seprember 1978
times
In fg. 4 the decay envelope of the two pulse echoes of signal II is shown for three values of R. If the echo signal originated from one species, than the decay envelope would be independent of R and only the relative intensity would change because of spinlattice IAaxation. By contrast, in fig. 4 it is seen that at R = 2 kHz the decay envelope is much steeper (TM = 0.9 JLS)than at R = 50 Hz (TM * 2.2 I.CS).With the latter rate all components are present, whereas at the former rate only the component with T, = 0.6 ms is measured. The decay envelopes show modulation due to hyperfine interactions. _4t R = 2 kHz, a strong modulation is present with a period in the order ofabout 0.5 p, which is absent for R = 50 Hz_ The phasememory times of the background at g= 2.0 are bnger (about 3 m), they do not contribute significantly to the curves dis&yed in fig- 4.
Volume
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CHEMICAL
PHYSICS
LElTERS
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Fig. 3. Saturation recovery curves of signal II at 1.2 K for dark-adapted (0) and illmninated (A) samples. The recovery curves are decomposed iota three components by linear regression. Instrument settings: repetition rate, 10 ,rIz, 90” pulse duration, 200 ns, delay time, 1.2 fls.
3.4. Effects of il!umimtion a? 1.2 K After taking the “dark” spectra of signal II, the sample was illuminated for 5 minutes at 1.2 K. The cw ESR spectrum taken in the dark after this period of illumination is indicated by the dotted curve in fig. 2. It is seen that a strong signal corresponding tog = 2.0025 has been generated. This signal, which is ir-
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Fig. 4. Echo intensity of signal II as a function of the delay time 7 at 1.2 K. Frequencies indicate rcpetitiotl rate R
reversible at 1.2 K, is called signal I and it is due to the oxidized primary donor of photosystem I, P700+ (for recent reviews see. refs. [13-15])Ihe ESE spectrum of the illuminated sample, again taken in the dark, is also shown in fig_ 2. Strikingly, this spectrum does not show any trace of signal I. Apart from an increase of about 40% (_$ = 50 Hz), the spectral shape is almost the same as that taken before illumination except for a slightly enhanced relative intensity at 3207 G (g = 2.000) @ii_ 2b, dotted line). We ha- varied thz repetition rate of the ESE sampling from 10 kHz to 10 Hz, without detecting a change in intensity at g = 2.0025 compared to the first dark ESE spectrum. We thus conclude that it is not a very long Tl which is responsrhle for the absence of the P700* signal, but that P700+ must have a very short phase-memory time Thf. Our current instrumental time resolution, limited by “cavity ringing”, is 1.1 ps, so that ThI (P7GO*) 55 0.5 ws. This would be in line with the short phase-memory time, TM = 0.5 ys, which we have measured in zero fiefd for the triplet state of ihe primary donor of the photosynthetic bacterium Rps. sphaeroides 1161. The possibility to discriminate against signal I with the ESE technique allows one to draw conclusions about low temperature electron-transport processes involving the precursors to signal II [9]. This is illustrat167
Volume 5 8. number 2
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CHEMICAL PHYSICS L?Z?ZRS
i.t..I~...l...,I,.,.J
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Fig. 6. Drawn line: amPEtude of the rapid decay component of fii. 5 as a function of the magnetic field. Repetition rate A = 5 IrHz. Dashed line: difference spectrum light on - light off at R = 1 kHz. 90” pulse duration, 200 ns.
Fig. 5. Light-induced reversibfe components of signal II zt
1.2 K. Intensity ofilIuminatron 40 mW/cmz. Cumtion of 90° pulse, 200 ns, dehy time I, 1.2 gs- IO denotes the backgroltnd echo intensity measured at 317.5 C. The slow decay has a chamcteristic time of 4.4 min. Arrows indicate opening (up ~zrd) and ciosin,o of shutter. ed by our study of light-induced changes in the ESE
spectrum of signal Ii. These changes turn out to be dependent on the repetition nteR. At 50 Hz the first period of illumination causes the intensity of the ESE spectrum to increase by 40% of the initial dark intensity- This increase is irreversible. However, at a repetition rate of 5 kHz (only the component of signal If with Tl = 0.6 MS then is observable), a reversible change is observed. This is illustrated in fg. 5, where the ~~ht-induced reversilile change is displayed for repetition rates of 500 Hz and 5 l&z. At 5 kHz 35% of the light-induced increase decays very rapidiy, whereas the remaining signal decays with the very slow characteristic time of 4-4 min. The intensity of this slow component was reduced by 20% after 3 h of continuous iUuminatbn_ It is seen that the intensity of the rapid decay component is dependent on the repetition rate: at R = SCM3 Hz this component has disappeared. In fig. 6 the amplitude of the rapid decay component is plotted as a function of the magnetic field (drawn line). The spectral shape is different from that of the light-induced difference spectrum measured at R = 1 kHz to which all TX components contribute (fig. 6, dasbecl line). 168
When comparing the intensities of the slow and the fast decay component for various values of R with the intensity of the dark signal measured at R = 500 Hz, a maximum is-found at 2 kHz for the faster decay component, whereas the slower component exhibits its ma&urn at 500 Hz. These values correspond to Tl values of 0.5-0.7 and 2-3 ms respect?dely.
d Discussion According to previous investigations by cw ESR at room temperature three different types of signal II can be ~t~gu~h~. These differ in their induction kinetics and decay times, but not in their spectra1 shape [12,15,17-201, Signal II,, (S II,) is always present in the dark, even after prolonged periods of dark adaptation. Signal I&low (S II,) is induced by iilumination at room temperature (rise time 1 s) on top of S fi, and to an equal extend as S II,, it decays in l-12 h depending on the preparation. Signal IIv,,v W [21,22], and a similar form, signal I& [19,20], have rapid rise and decay kinetics- Since the latter two are connected to the oxygen evolvmg complex of photo. system 2 which stops functioning below -6O*C i23, 241, these signals need not be considered in this work. it has been demonstrated that S I& and 8 IIs are due to oxidation of a premraor [ 17,25,26~, suggested
Volume 58, number 2
CHEhHCAL PHYSICS LETlXRS
to be a chromanoi derivative of plastoquinol [2728] _ Esser [29] and Ruuge et al. [3Q] found that the shape of signal II is dependent on microwave power, and attributed this to the presence of two different corn ponents. Babcock and Sauer [X8] found that the width of signal II varied from 16 to 19 G depending on the ionic strength of the medium- The present work shows that our dark signal II (composed of S II, and S lls) contains at least three different components with relaxation times T1 = 0.6,3 and 20 ms respectively. That several species contribute to S II is further demonstrated by the fact that our component with T1 = 0.6 ms also has a different phase-memory time, viz. 0.9 MSas compared to the 2.2 crs for the other two components. Moreover, its decay envelope is modulated by hyperfine interaction, corresponding to a hyperfine coupling constant of the order of 0.7 GLarger hyperfine couplings were difficult to measure because of the limitations on our instrumental time resolution. The other two components with the longer Tl’s do not show such a pronounced modulation. The diversity in signal Ii we have detected by ESE cannot be explained by assuming that S II is due to just one radiml species- The difference in Thl and the nuclear modulation shown in fig. 4 indicates that at least two radicals are present. Both these radicals exhibit various spin-lattice relaxation times, presumably due to differences in the environment. If we accept the evidence that signal II is due to an oxidized piastoquinol derivative [27,28], then we must assume that these molecules from a heterogeneous population with different locations with respect to photosystems 1 and 2. To account for the variability in spectral shape and in Thl and nuclear modulation, we propose that at room temperature plastoquinol is in dynamic equilrbrium with its chromanol anaiogue. Such ~u~~~~ can easily be shifted by photo-oxidation of the quinol, which facilitates reversibility of the ring-closure reaction [31]. Thus, under our conditions, sig,nal II is attributed to a mixture of a phenoxyl and a semiquinone type spectrum. The maximum in intensity at g =2.005 of the R = 5 kHz spectrum of fig. 6 would then be due to an enhanced contribution of the narrow semiquinone radical. The implications of this proposal for electron transport in plant photosynthesis are discussed elsewhere [9] ‘Ihe above hypothesis can be tested by performing studies with quinols of different side-chain structure,
15 September 1978
either in vim or by extraction/reconstitution experiments analogous to those of Kohl et al- [27]. The ESE technique would seem to be a valuable aid in such investigation.
Acknowledgement We are grateful to Dr. J. Lugtenbug for a helpful discussion on the conversion of quinols to chromanols. This work was supported by the Netherlands Foundation for Chemical Research (S.O.N.) with furancial assistance from the Netherlands Organization for the Advancement of pure Resear~ (Z.W_O_)_
References Ill D-E. Kaplan, WE
Browne and J.A. Cowen, Rev. Sci. In&. 32 (1961) 1182. 121 W-B. Mims, Rev_ Sci_ Instr. 36 (1965) X472_ 131 W-B. hfims and J-L. Davis, J. Cbem. Phys. 64 (1976) 4836. 141 W-3. Mims and J_ Peisxh, Biochemistry 15 f1976) 3863. I53 B. Mondovi, hi-T_ G raziani, W.B. Mints, R. Oltzik and J_ Pcisacb, Biochemistry 16 (1977) 4198. 161 BJ. Botter, DC. Doetschman, J. Schmidt and J.H. van der WI&, hfol- Phys 30 (1975) 609. I71 W.B. Ilfims, in: Eiecc_ronpammag~tic resonance, ed. S. Geschwind
X69
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RE. BIanIcenship, G-T. Babcock, J.T. Warden and K- Sawr, FEBS Letters 51 (X975) 287. (22 J R.E. Bknkenship. A. McGuite and K. Sauer, Biochim. Biophys. Acta 459 (1977) 617. 1231 3-T. Warden, RE. Blankenship and K. Sauer, Biochitt~ Biophys Acta 423 (1976) 462_ [24 J k Joliot, Biochim. Biophys. Act3 357 (1974) 439. 1251 A-F. Esser, Photochem. Photobiol. 20 (1974) 173. (261 J-T. War&m and J-R_ BoXton, Photochem. PhotobioL20 (1974) 245. [21]
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1271 D-H. Kohland P-M- W_ood, Pkmt PhysioL 44 (1969) 1439, 1281 D-I-I. Kohl, J.R. Wright and M. We&man, Eio@im. Biophys. Acta 180 (1969) 536. 1291 A-F. Esser, Photochem. Photobiot 20 (1974) 167. 1301 E-K- Ruuge, A-V_ Z”iionov and L-k Blyumenfeld, Biofii 19 (1974) 1034. 13x1 R- Bent&y and I.&f. CampbeII, The chemistry of quinoid compounds, Part II Wey, New York, 1974) ch. 13, p. 683.