Photosynthesis Research 20: 279-289, 1989 © 1989 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination HARRY A. F R A N K , 1 0 R J A N H A N S S O N 2 & P A U L MATHIS Service de Biophysique, D@artement de Biologic, Centre d'Etudes NuclOaires de Saelay, 91191 Gif-sur- Yvette, Cedex, France; 1Department of Chemistry, University of Connecticut U-60, 215 Glenbrook Road, Storrs, Connecticut 06268 USA; 2Department of Biochemistry and Biophysics, Chalmers Institute of Technology and University of G6teborg, S-41296, Ggteborg, Sweden Received 26 July 1988; accepted 7 September 1988

Key words: electron paramagnetic resonance, electron transfer, pheophytin, photosystem II, radical, triplet state Abstract. Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-l/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin. Abbreviations: EPR--electron paramagnetic resonance.

Introduction

The elucidation of the details of charge separation in Photosystem II in higher plants has been difficult owing in large part to the lack of a fundamental photochemically active particle in which to probe the primary photochemical events. This barrier has been largely overcome by Nanba and Satoh (1987) who have recently isolated a Photosystem II rection center complex consisting of two polypeptides (denoted D-1 and D-2 and having apparent molecular weights of 32-34 kDa), one cytochrome b559, five molecules of chlorophyll a, two pheophytin a and one/%carotene. The authors demonstrated photochemical activity of the preparation by the photoreduc-

280 tion of pheophytin (the primary electron acceptor) and by observing the electron paramagnetic resonance (EPR) spectrum associated with a lightinduced radical pair polarized triplet state (Nanba and Satoh 1987, Okamura et al. 1987). These observations proved that the site of the primary photochemical charge separation in Photosystem II is on the D-1 and D-2 subunits in analogy with the L and M subunits from purple photosynthetic bacteria (Okamura et al. 1982). The present work offers EPR and optical absorption spectroscopic measurements carried out on the D-1/D-2/cytochrome b559 complex in an attempt to understand its photochemical behavior at low temperatures. The EPR and optical results show that it is possible to produce a fraction of the reaction centers with the primary pheophytin acceptor reduced by low temperature illumination. The stabilization of the reaction center after electron transfer from the primary donor, P680, to the pheophytin is achieved by rereduction of P680 by a secondary donor molecule. Out of several possible candidates, cytochrome b559 and fl-carotene could be ruled out by the optical results. The EPR data indicate either a species having g = 2.0055 can act as the donor, or P680 oxidizes a chlorophyll molecule which is weakly magnetically coupled to the reduced pheophytin.

Materials and methods

Photosystem II reaction centers (D-1/D-2/cytochrome b559 complex) were prepared from spinach according to Nanba and Satoh (1987) in a final buffer containing 50mM Tris-HC1 (pH7.2), 50mM NaC1, 0.05% Triton X100 and 10-65% glycerol. Where indicated, 7 m M NazS204 (final concentration) was added before freezing the samples in the dark. Illumination of the EPR samples was performed at 77 K (liquid nitrogen), 200 K (ethanol-dry ice) or 273 K (ice water) using an 800 W tungsten lamp filtered by 3 Calflex B1K ~ filters and 2.5cm of water in a Pyrex container. This light source together with a lucite light guide was used for the generation of triplet state EPR signals. Optical samples were illuminated in the cryostat. EPR measurements were performed at X-band with a Bruker ER 200DSRC spectrometer equipped with an Oxford Instruments ESR9 helium flow cryostat. The microwave frequency was measured with an EIP model 545A frequency counter. The magnetic field was calibrated with the known g value, 2.0026, of the photooxidized primary donor of Rhodobacter sphaeroides R26 (McElroy et al. 1969). Spin quantification of EPR signals were made using standard techniques (Aasa and V/inng~rd 1975) with horseheart cytochrome c as a concentration standard. The concentration of cyto-

281 chrome c was determined with the differential extinction coefficient (reduced minus oxidized) of 21,100M -1 cm l at 550nm (Gelder and Slater 1962). Optical absorbance measurements were carried out on a Cary 17D spectrometer. In both the optical and EPR experiments a Tracor Northern TN 1710A averager was used for signal capture and processing.

Results

Figure 1A shows the steady state triplet spectrum observed if the reaction center complex is subjected to continuous illumination at 5 K in the E P R cavity. The strong triplet state signal is spin polarized according to the radical pair mechanism as evidenced by the a e e a a e (a = absorption; e = emission) spin polarization pattern (Hoff 1979). The polarization pattern was invariant with temperature between 5 and 30 K. The values of the zero field splitting parameters which characterize the triplet are ID] = 0.0288 _+ 0.0001 cm ' and [El = 0.0043 + 0.0001 cm -1 and are in agreement with previous determinations which ascribe this triplet to P680 (Okamura et al. 1987, Rutherford et al. 1981). Illumination of the sample for five minutes at 77,200 or 273 K prior to the triplet EPR experiment carried out at 5 K resulted in an attenuation of the triplet state signals. (See, e.g., Fig. lB.) The triplet state signal intensity observed after illumination of the sample in the presence of sodium dithionite was 63 + 5% of the original amplitude if the illumination was carried out at 77 K, 48 _+ 5 % for illumination carried out at 200 K, and 0% for illumination at 273 K; i.e. in the last case the signal was completely lost. In the absence of sodium dithionite, the observed triplet amplitudes were essentially unaffected by illumination carried out at 77 K. For illumination carried out at the other temperatures the remaining triplet signals were small, being ~ 10% of the original intensity for illumination at 200 K, and ~ 0% at 273 K. Raising the sample temperature to 273 K for 2 min and then lowering it to 5 K in the dark brought about a 19 + 5% recovery (relative to the original amplitudes) of the light-induced triplet signal intensity for the samples containing dithionite and illuminated at 77 or 200 K (Fig. 1C), but no recovery was accomplished in the sample containing dithionite and illuminated at 273 K; i.e. illumination at 273 K destroyed the ability of the reaction center to produce triplets. In the samples lacking sodium dithionite, after warming to 273 K and subsequent observation of the EPR signal at 5 K there was: Essentially no change in the triplet signal intensity for the sample illuminated at 77K; i.e. this sample was unaffected by illumination; a 10 + 5% recovery of triplet intensity for the sample illuminated at 200 K

282

Before 200 K illumination

D

A

After K 200 illumination

W% After 200 K illumination and thawing

I

I

I

I

2850

~

38 0 Field (gauss)

I

r

I

--

I

3300

I

3/,00 Field ( gauss )

Fig. 1. Triplet state and radical EPR signals observed in sodium dithionite-treated Photosystern II reaction centers. The spectra were taken under the following conditions: A. Before actinic illumination. EPR spectral conditions: tungsten lamp on; Microwave frequency, 9.45 GHz; Microwave power, 50/~W; Receiver gain, 3.2 x 105;Field modulation frequency, 100 kHz; Field modulation amplitude, 22 G; Time constant, 320 ms; Sweep time, 200 s; Temperature, 5 K. B. After 5 min of actinic illumination at 200 K. EPR conditions same as in A. C. After raising the sample used in B to a temperature of 273 K for 2min and refreezing in the dark. EPR conditions same as in A. D. Before actinic illumination. EPR experimental conditions; tungsten lamp off; Microwave frequency, 9.44GHz; Microwave power, 50/*W; Receiver gain, 5.0 x 10 6, Field modulation frequency, 100 kHz; Field modulation amplitude, 2.8 G; Time constant, 1.25 s; Sweep time, 100 s; Temperature, 20 K. E. After 5 rain of actinic illumination at 200 K. EPR conditions same as in D. C. After raising the sample used in B to a temperature of 273 K for 2 rain and refreezing in the dark. EPR conditions same as in D. a b o u t o n e h a l f o f t h a t o b s e r v e d for the d i t h i o n i t e - c o n t a i n i n g s a m p l e ; a n d , n o r e c o v e r y o f triplet i n t e n s i t y for the s a m p l e i l l u m i n a t e d at 273 K ; i.e. as i n the d i t h i o n i t e - c o n t a i n i n g s a m p l e , i l l u m i n a t i o n at 273 K d e s t r o y e d the a b i l i t y o f the r e a c t i o n c e n t e r to p r o d u c e triplets.

283 All the triplet spectra revealed small additional light-induced peaks appearing on the extreme low and high field sides of the P680 triplet signals (Fig. 1). These peaks may be assigned to a second triplet signal because they are light-induced, symmetrically distributed about the g = 2 region and have a splitting comparable to that expected for porphyrin (chlorin)-like triplets (Hoff 1979). The relative intensity of this second triplet signal versus that of the P680 triplet was not affected by temperature between 5 and 30 K. Also, the magnitude of the second triplet signal did not depend on the presence of sodium dithionite and was not affected by actinic illumination of the sample except when it was carried out at 273 K in which case all the triplet signals disappeared. (See above.) Assuming these small peaks maximize the fine structure splitting of another triplet species, a ]D] value of 0.0340 _+ 0.0002cm -1 can be assigned to this triplet. This is the same IDI value observed for pheophytin a in solution (Thurnauer et al., 1975), indicating that some pheophytin triplet is being light-induced. Owing to its lack of sensitivity to actinic illumination, in contrast to the P680 triplet, it is concluded that the pheophytin triplet is not being formed as a result of recombination of the primary radial pair. Concomitant with the reduction in signal amplitude of the P680 triplet brought about by illumination prior to the EPR experiment is the build up of a radical signal in the g = 2 region and observed at 20K. (See Fig. 1E.) The signal was optimally reproduced when the illumination was carried out at 200 K. A measurement of the double-integrated signal area against that obtained from a cytochrome c standard yielded approximately 1 radical spin per 21 chlorophyll molecules; i.e. a concentration of radicals equalling ~ 25% of the reaction enter concentration, assuming five chlorophyll molecules per reaction center (Nanba and Satoh 1987). The radical signal observed at 20 K disappeared upon raising the sample temperature to 273 K for 2min and then lowering it to 20K in the dark. (See Figs. 1E and 1F.) Extensive averaging revealed the detailed structure of this signal. (See Fig. 2A). It has an effective g value of 2.0046, a peak-to-peak width of 11.9 G and structure on the high field side which is suggestive of either spin coupling or more than one radical being present. The signal saturates uniformly over its lineshape and levels off at a microwave power of ~ 50/~W at 20 K. Figure 3A shows part of the EPR signal of the cytochrome b559 associated with the reaction center complex. Prior to the addition of sodium dithionite the EPR signal exhibits g value components at 2.92, 2.26 and 1.55. Reduction of the cytochrome b559 with sodium dithionite abolishes the signal. (See Fig. 3B.) Spin quantification of the cytochrome b559 signal v e r s u s a prepared standard of cytochrome c yielded one cytochrome b559 per 3.5 __ 0.4 chlorophyll molecules in the protein.

284

A•.

g = 2.0030

,: Z / \'". S \/

A = II.9G

g A= ll.6G I

33t,0

I

~j I

3360

I

I

3380

I

I

3?,00

Field ( gauss ) Fig. 2. Radical EPR signals from sodium dithionite-treated Photosystem II reaction centers. A. Experimental EPR spectrum (average of 10 sweeps) taken in the dark after 5 min of actinic ilumination at 200K. EPR experimental conditions: Microwave frequency, 9.4419GHz; Microwave power, 50 #W; Receiver gain, 5.0 x 106; Field modulation frequency, 100 kHz; Field modulation amplitude, 2.8 G; Time constant, 320 ms; Sweep time, 500 s; Temperature, 20 K. B. Computer-generated gaussian lineshape corresponding to a signal having a doubleintegrated area of approximately 66% of the experimental spectrum shown in A. A-B is the computer-generated difference spectrum between A and B.

Figures 3C and 3D show the effect of sodium dithionite on the 55 K optical absorption spectrum in the region between 535 and 610nm. The addition of the reductant causes a buildup (AA = 0.063) in intensity at 555 nm consistent with the cytochrome complex changing its oxidation state by chemical reduction. Using 15,000 M - 1cm- 1 as the differential extinction coefficient for cytochrome b559 (Nanba and Satoh 1987), and 58,900 M cm-1 as the extinction coefficient for chlorophyll "a at the red maximum in the D-1/D-2/cytochrome b559 complex (J. Duranton, personal communication), and 32,000 M ~cm -1 as the extinction coefficient for pheophytin a at its red maximum (Fujita et al. 1978), a ratio of one cytochrome b559 to 4.1 -t- 0.2 chlorophylls was obtained. This is in good agreement with the EPR-determined value of 3 . 5 - t - 0 . 4 given above and the ratio of

285

Untreated g value 2.92 i

i

i

2.26 [

I

A

I

I

J_

I

10-10

I

I

I

D it hionite - t r e a t e d 555

[

1700

I

I

Field (gauss)

I

i

2300

610

I

I

[

I

Wavetength (nm)

--[

535

Fig, 3. EPR and optical absorption spectra taken from Photosystem II reaction centers and

associated with cytochrome b559. The spectra were taken under the following conditions: A. Untreated reaction centers concentrated by centrifugation to an optical density of ~ 15 at 672 nm. EPR experimental conditions: Microwave frequency, 9.44 GHz; Microwave power, 0.5 roW; Receiver gain, 8.0 x 104; Field modulation frequency, 100 kHz; Field modulation amplitude, 22G; Time constant, 2.5s; Temperature, 15K. B. After addition of sodium dithionite. EPR conditions same as in A. C. Untreated reaction centers having an optical density of 1.24 at 672 nm. Experimental settings for the optical absorbance measurement: pen period, 1; Sweep rate, 2nm/s; Temperature, 55K. D. After addition of sodium dithionite. Experimental settings same as in C. 1:3.9 _+ 1.8 reported by N a n b a and Satoh (1987). N o p h o t o o x i d a t i o n o f c y t o c h r o m e b559 could be observed, either optically or by E P R , at any temperature, under a n y redox condition in this preparation. Figure 4 shows the effect o f 1 8 0 K illumination on the 3 0 K a b s o r p t i o n spectrum o f the reaction center complex. The spectrum presented in Fig. 4 is a difference between spectra t a k e n before a n d after actinic ilumination. The difference spectrum reveals that illumination causes a sharp bleaching at 681 nm, a less intense bleaching at 666 nm, a small but reproducible bleaching at 544 nm, a p r o n o u n c e d buildup at 446 nm, a zero crossing at 4 3 2 n m a n d a b r o a d bleaching at ~ 4 2 2 n m . The bleaching at 6 8 1 n m a p p e a r e d optimally when sodium dithionite was present and c o r r e s p o n d e d to an a b s o r b a n c e change (AA) o f 0.0092. W a r m i n g the sample to r o o m t e m p e r a t u r e and then lowering it to 30 K resulted in the d i s a p p e a r a n c e o f the sharp bleaching at 681 nm, the small bleaching at 544 n m and the b r o a d buildup at 446 rim. The 666 n m and 422 n m bleachings remained, however.

286 l

I

I

I

'

I

.,-0.010 !

'

l

~1,6 I

AA -0.010

681 l 700

,

[ 600

t

l 500

14}2

t, O0

Wavelength (rim) Fig. 4. Optical absorbance difference spectrum taken from Photosystem II reaction centers (optical density 0.4 at 672nm) before and after 5rain of actinic illumination at 180K. Experimental settings: Pen period, 1; Sweep rate, 2nm/s; Temperature, 30K.

(Data not shown.) In the absence of sodium dithionite an additional irreversible bleaching at 669nm occurred. No changes in the region 7501000 nm which could be attributed to the formation of a carotenoid radical cation were observed after actinic illumination of the samples.

Discussion Except for the irreversible bleaching at 666 nm, the shape of the optical difference spectrum shown in Fig. 4 bears a striking similarity to the lightinduced pheophytin a anion spectrum generated previously by Nanba and Satoh (1987) in the Photosystem II reaction center complex. Thus, illumination of the preparation at 200 K traps some of the pheophytin primary electron acceptor in its reduced state. Assuming the reaction center stoichiometry of Nanba and Satoh (1987); i.e. five chlorophyll a molecules, and using difference extinction coefficients for pheophytin a/pheophytin a- of 32,000M -1 cm -~ at 681 nm (Fujita et al. 1978) and after accounting for the absorption of chlorophyll a and pheophytin a in this region, the 0.0092 absorbance change shown in Fig. 4 corresponds to 13 _+ 5% of the reaction centers having the pheophytin acceptor in its reduced state. The reversible attenuation of the spin polarized triplet upon illumination of the sample provides additional evidence that the pheophytin acceptor is

287 trapped in its reduced state. This is because blocking the reaction center photochemistry by reducing the primary electron acceptor would lead to a decrease in the yield of triplet states born on P680 via the radical pair mechanism (Hoff 1979). One would also expect to observe a radical EPR signal if the primary acceptor were being phototrapped. Such a signal arises concomitant with the triplet attenuation. (See Fig. 1.) As mentioned in the Results section, a calculation of the number of spins corresponding to the g = 2.0046 radical signal resulted in a 25% yield of radicals in the reaction center complex obtained upon illumination. It should be emphasized that there is some amount of photodestruction of the reaction center complex induced by the actinic illumination. This is revealed by the lack of a total recovery of the spin polarized triplet after warming the sample to 273 K and refreezing it to 5 K in the dark. The photodestruction is most pronounced when the illumination is carried out at 273 K and may be associated with the irreversible bleachings at 669 nm, 666 nm and 422 nm in the optical absorbance spectra. However, no EPR signals appeared in conjunction with these bleachings. The fact that the photodestruction is less in the dithionite containing samples illuminated at 200 or 77 K is probably related to the ability of the reductant to react with molecular oxygen. The optical absorbance changes, radical buildup and triplet attenuation/ recovery experiments discussed above suggest that some part of the observed g = 2.0046 radical signal EPR spectrum can be attributed to pheophytin a - . Exactly how the pheophytin a- signal manifests itself in this EPR radical spectrum is still uncertain. Two interpretations are as follows: (i) It is known that the EPR signal of reduced pheophytin a has an unstructured gaussian lineshape, a g value of 2.0030 and a linewidth (A) of 13 G (Fujita et al. 1978, Fajer et al. 1980). Using values o f g = 2.0030 and A = 13.4G for the reduced pheophytin (values that fit the high field structure in the g = 2.0046 signal) and subtracting a simulated lineshape using these parameters (Fig. 2B) from the experimental spectrum (Fig. 2A) one obtains the spectrum of the remaining population of spins (Fig. 2 bottom). The remaining signal has a g value of 2.0055, a width of 11.6 G, displays unresolved structure, and could be a species that donates electrons to P680 + . The absence of photoinduced changes in the carotenoid cation absorption region (750-1000 nm) and in the cytochrome b559 absorption or EPR spectra rule out these species contributing to this lineshape as electron donors in the rereduction of P680 +. Another possibility is that the g = 2.0055 signal is a modified, phototrapped component of Signal II; i.e. D + or Z + , the stable or transient components, respectively, of Signal II (Babcock and Sauer 1975). The fact that the g = 2.0055 signal does not

288 occur in a 1:1 stoichiometry with the reduced pheophytin signal (the ratio of the double integrated area of t h e g = 2.0055 signal to that of the g = 2.0030 pheophytin a- signal is approximately 0.5:1) would then have to be explained by some amount of the g = 2.0055 signal being lost by reduction from another donor, possibly dithionite. However, the g value, 2.0055, is high and very likely out of the range of that for an assignment to D + . Also, the width of the g = 2.0055 signal (A = 11.6 G) is much lower than the width ( ~ 20 G) normally associated with D + or Z + . The D-l/D-2/ cytochrome b559 preparation may be highly modified structurally, however, and this may lead to different EPR signals for these species, as has been suggested to be the case for Photosystem II particles treated with potassium iridium chloride (Boussac and Etienne 1984). In making the assignment of this signal to D + one could argue that the signal should be stable in the dark in the absence of chemical reductant. It has been observed, however, that structural modifications of the Photosystem II donor side can lead to the disappearance of the D + signal, it being regenerated only after illumination (Boussac and Etienne 1982). Recent deuterium substitution experiments have shown that D + , and most likely Z + , arise from tyrosine radicals (Barry and Babcock 1987). The g value, 2.0055 of the remaining radical is too high to be consistent with this assignment. Also the rather narrow linewidth, 11.6 G, as compared to the native Signal II ( ~ 20 G) would have to result from either a peculiar redistribution of electron density in the tyrosine ring system or an ill-defined torsion angle of the bond between the -C(/~)H zgroup and the ring, neither of which is very likely. An oxidized amino acid residue other than tyrosine giving rise to the signal cannot be ruled out by these experiments, however. (ii) Alternatively, the g = 2.0046 signal could be due to reduced pheophytin magnetically interacting with another paramagnetic species. The most likely candidates as donors to P680 + are chlorophyll (often observed to be oxidized in illuminated Photosystem II preparations) or an oxidized amino acid. In this interpretation the pheophytin anion would be weakly (,-~ 2.5 G) exchange or dipolar coupled to one of these other species. In such a weakly coupled system, depending on the participating species and the nature of the coupling, one could observe an EPR spectrum that differed substantially in the observed g values from that expected for a noninteracting system; e.g. see Schepler et al. (1975). This is an attractive possibilty because one need not invoke magnetic properties of the paramagnetic species unlikely to be present under these experimental conditions. Further work is in progross to identify the molecular species responsible for the radical signals reported here.

289

Acknowledgements The authors wish to gratefully acknowledge the expert assistance of Jacques Duranton in carrying out the reaction center preparations, and Drs G. Babcock, G. Brudvig, A.W. Rutherford and T. Vfinnggtrd for helpful discussions. The stay of 13.H. in Saclay was supported in part by a postdoctoral fellowship from the Swedish Natural Science Research Council. This work was supported in part by grants to H.A.F. from the National Institutes of Health (GM-30353) and the Competitive Research Grants Office of the U.S. Department of Agriculture (86-CRCR-1-2016).

References Aasa R. and V/inngfird T. (1975) EPR signal intensity and powder shapes: A reexamination. J Mag Res 19:308-315 Babcock G. and Sauer K. (1975) The rapid component of electron paramagnetic resonance Signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts. Biochim Biophys Acta 376:329-344 Barry B.A. and Babcock G.T. (1987) Tyrosine radicals are involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA 84:709%7103 Boussac A. and Etienne A.L. (1982) Spectral and kinetic pH-dependence of fast and slow Signal II in tris-washed chloroplasts. FEBS Letters 148:113-116 Boussac A. and Etienne A.L. (1984) Midpoint potential of Signal II (slow) in tris-washed Photosystem II particles. Biochim Biophys Acta 766:576-581 Fajer J., Davis M.S., Forman A., Klimov V.V., Dolan, E. and Ke R.H. (1980) Primary electron acceptors in plant photosynthesis. J Am Chem Soc 102:7143-7145 Fujita I., Davis M.S. and Fajer J. (1978) Anion radicals of pheophytin and chlorophyll a: Their role in the primary charge separations of plant photosynthesis. J Am Chem Soc 100: 6280-6282 Gelder B.F. and Slater E.C. (1962) The extinction coefficient of cytochrome c. Biochim Biophys Acta 58:593-595 Hoff A.J. (1979) Applications of ESR in Photosynthesis. Phys Rep 54:75-200 McElroy J.D., Feher G. and Mauzerall D.C. (1969) On the nature of the free radical formed during the primary process of bacterial photosynthesis. Biochim Biophys Acta 172:180-183 Nanba O. and Satoh K. (1987) Isolation of a photosystem II reaction center consisting of D-1 and D-2 polypeptides and cytochrome b-559. Proc Natl Acad Sci USA 84:10%112 Okamura M.Y., Feher G. and Nelson N. (1982) Reaction centers. In: Govindjee ed. Photosynthesis 1, 195-272. Academic Press, New York Okamura M.Y., Satoh K., Issacson R.A. and Feher G. (1987) Evidence of the primary charge separation in the D-1/D-2 complex of Photosystem II from spinach: EPR of the triplet state. In: Biggins J. ed. progress in Photosynthesis Research 1:379-381 Martinus Nijhoff, Dordrecht Rutherford A.W., Paterson D.R. and Mullet J.E. (1981) A light-induced spin-polarized triplet detected by EPR in Photosystem II reaction centers. Biochim Biophys Acta 635:205-214 Schepler K.L., Dunham W.R., Sands R.H., Fee J.A. and Abeles R.H. (1975) A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B12. Biochim Biophys Acta 397:510-518 Thurnauer M.C., Katz J.J. and Norris J.R. (1975) The triplet state in bacterial photosynthesis: Possible mechanisms of the primary photo-act. Proc Natl Acad Sci USA 72:3270-3274