THE J O U R N A L O F
PHYSICAL CHEMISTRY Registered in US.Patent Office 0 Copyright, 1979, by the American Chemical Society
VOLUME 83, NUMBER 26
DECEMBER 27, 1979
Observations of Chemically Induced DynamlG Electron Polarization in Photosystem I of Green Plants and Algaet Alan R. McIntosh, Henryk Manlkowskl, and James
R. Bolton"
Photochemistry Unit, Department of Chemlstry, University of Western Ontarlo, London, Ontario, Canada N6A 587 (Received August 22, 1979) Publicatlon costs assisted by the Natural Sciences and Engineering Research Council of Canada
Flash photolysissxperimentswith electron paramagneticresunmix detection were carried out between 10 and 300 K on samples of green plant and whole algal species. Chemically induced dynamic electron polarization (CIDEP) was evident for the transient signals observed at low temperatures in the g = 2.0 region for 100-kHz and 2-MHz modulated detection as well as for a direct detection system with no magnetic field modulation. The light reversible signals decaying in about 0.6 ms are interpreted as arising from photosystem I of the green plant and algal samples. These transient signals exhibited orientation-dependent differences when samples of whole chloroplasts or whole algal cells were oriented in a strong magnetic field (2.4 T)and then frozen at 77 K. The algal species included both normal and -97% deuterated Scenedesmus obliquus and normal Amcystis nidulans. These results are considered in terms of the information which they reveal concerning the identity of the electron acceptors of photosystem I.
Introduction In the continuing study of the photoreactions occurring in photosynthetic reaction centers, the technique of flash photolysis with electron paramagnetic resonance (EPR) detection has been particularly usefu1.l The overall goal of such studies has been to identify the participants in the primary photochemistry occurring in bacterial or greenplant reaction centers. The bacterial reaction center has been studied intensively both in whole cells and in reaction-center preparations, and most of the components of certain bacterial reaction centers are relatively well characterized both by optical and EPR techniquesa2However, the two photosystems of green plant photosynthesis are much less well characterized partly because it has not been possible as yet to prepare pure reaction center proteins which exhibit normal photochemical activity. Thus, most Publication No. 236 of the Photochemistry Unit, Department of Chemistry, University of Western Ontario, London, Ontario, Canada N6A 5B7. 0022-365417912083-3309$0 1 .OO/O
~ t u d i e sof~ ?green-plant ~ materials have been carried out on whole chloroplasts, whole cells of algae, or on thylakoid membrane fragments which are relatively enriched in one of the two photosystems. The present study concerns itself with an interpretation of the flash photolysis-EPR results obtained from experiments on photosystem I of green plants. It is well established3 that the photochemical electron donor in photosystem I is a chlorophyll dimer species called P700 which probably5 has a midpoint potential (E,,') of +375 mV relative to the normal hydrogen electrode. It is also known that photooxidation of P700 at cryogenic temperatures is coupled to the reduction of two membrane-bound ferredoxin species A and B6with midpoint potentials of -540 and -590 mV, respectively. These species act as irreversible electron acceptors at low temperatures. Another membrane-bound component denoted as X with a midpoint potential more negative than -730 mV has been observed by EPR to exhibit reversible reduction kinetics at low temperatures coupled to the electron donor P700 0 1979 American Chemical Society
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The Journal of Physical Chemlstry, Vol. 83, No. 26, 1979
when centers A and B are r e d u ~ e d However, . ~ ~ ~ the phenomenon of chemically induced dynamic electron polarization (CIDEP) has not been reported directly for any of the EPR observations of the membrane bound ferredoxins or the component X. On the other hand, CIDEP signals have been observed from bacterial reaction center^.^ The phenomenon of CIDEP in green-plant photochemistry has been observed at room temperature1°-12and at low temperatures'*l6 in the g = 2.0 region of the EPR field profile. In our earlier work,13we erroneously assigned the low temperature CIDEP signals to photosystem I1 photochemistry. The present study was undertaken to demonstrate that P700 in normal protonated species is coupled to at least one electron acceptor in photosystem I which is independent of the membrane-bound ferredoxins and the component X. This new electron acceptor can be observed directly by flash photolysis-EPR in the g = 2.0 region to lower field from P700' over a range in temperature from 10 to 300 K. The field profiles in the g = 2.0 region are shown to reveal anisotropy upon membrane orientation which is also observed for other membranebound constituentsl6 of the thylakoid membrane.
Experimental Section The EPR detection system was a Varian E-12 spectrometer with a modulation frequency of 100 kHz and a concomitant l / e rise time of 20 psI7 for routine flash photolysis experiments. A 2-MHz magnetic-field modulation and phase-sensitive detection system was constructed by Photochemical Research Associates Inc. and utilized on the same EPR spectrometer to achieve a l / e rise time of 1.5 ps for field-modulated experiments. A modulation free or direct EPR absorption system was also employed by recording the output of the Varian E-101 microwave bridge in the absence of magnetic-field modulation. With a slight bandwidth modification to the existing preamplifier in the microwave bridge, we-obtained a l / e risetime of 0.4 ps in flash-photolysis experiments. The low level voltages from the output of the microwave bridge were measured directly by the transient recorder with no further amplification. Time constants longer than -10 ms cannot be measured with this system because the microwave bridge was ac coupled to the transient recorder. All EPR signals were digitized by a Nicolet 2090-111 digital oscilloscope, with either 9 or 12 bits of vertical resolution, which was interfaced with a Nicolet 1180 instrument computer for signal averaging and data analysis.14 For flash-photolysis experiments with 100-kHz modulated detection, the light source was a Photochemical Research Associates Model 610A flash system with a flash half-life of 25 ps filtered with a Corning CS-262 filter having a wavelength cutoff at about 600 nm. For 2-MHz fieldmodulated detection, the light source was either a Photochemical Research Associates Model 610C flash system with a flash half-life of 1.5 ps filtered with a Corning CS-262 filter, or a Phase-R 2100-D dye laser operating at 620 nm with a flash half-life of 0.2 ps. For direct EPR absorption detection, the light source was the same Phase-R 2100-D dye laser. None of the light sources was of saturating intensity, even the dye laser with about 20-50 mJ per light pulse. Great care was taken during direct EPR absorption measurements to ensure that virtually no flash artifacts originating from the dye laser were present in the observed kinetic profiles. Nearly all flash artifacts were removed by subtracting an off-resonance kinetic profile from each kinetic profile measured at a given magnetic field position. Typically, this correction was about 10% or less of the magnitude of the EPR absorption response. The small
McIntosh, Manikowski, and Bolton
magnitude of the correction for the laser pulse was achieved by performing the following operations: the dye laser head and power supply had radio frequency insulation (Phase-R Corp.) installed, the dye laser was in a Faraday cage in a room separate from the EPR spectrometer, and the EPR spectrometer was fitted with radio frequency insulation on most of the cables interconnecting the main console, magnet, and microwave bridge. Furthermore, the dye laser was operated at a low light output level so as to avoid very large time-dependent changes in concentrations of paramagnetic species. Such large changes in concentration would result in a significant time dependence of the Q factor and the resonant frequency of the cavity. We employed a Varian Associates E-238 EPR cavity which operates in the TMllo mode and has an unloaded Q factor of 12 000. We believe that we were operating below the saturating light intensities required to produce excessively large changes in the Q and resonant frequency of the cavity. Care was also taken to ensure that the EPR cavity had a negligible modulation offset when the 100-kHz modulation system was used to verify the magnetic-field positions for direct absorption detection (e.g., we have measured a modulation offset of less than 5.0 pT for the TMllo EPR cavity). Whole cells of the algae Anacystis nidulans and Scenedesmus obliquus were investigated, the cultures being maintained in our laboratory. Photosystem I enriched particles from spinach were prepared14with about 2 mg mL of total chlorophyll measured spectrophotometrically, All photosynthetic samples, including packed cells of intact algae, were buffered at pH 8, and there were no exogenous redox agents unless otherwise indicated.
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Results and Discussion We have established in our earlier work14 that magnetically palarized emissionand absorption EPR signals can be observed from whole algae, from spinach chloroplasts, and from photosystem I enriched preparations. The polarized signals which are associated with photosystem I appeared to decay in the time range 0-10 ps when direct-absorption detection of the signals from 97% deuterated whole cells of algae was used. It was also determined that the signals in the g = 2.0 region were probably due to short-lived radical species, both P700+ and at least one electron acceptor, with both components decaying together with a half-life of about 0.6 me at low temperatures below 100 K. We have continued these flash photolysis-EPR studies, and we now have been able to detect similar transient signals, with lower signal-to-noise ratios, from normal protonated green-plant materials including spinach chloroplasts and whole cells of the algae Anacystis nidutans and Scenedesmus obliquus. Typical kinetic profiles are shown for an algal species in Figure 1,where both direct EPR absorption and 2-MHz modulated kinetics are presented. A microwave power level of 1 mW was chosen for observations of the transients at 100 K to obtain the maximum signal-to-noise ratio. It was determined from the microwave power saturation curve of P700+that the P700' signal amplitude was at a maximum value for a 1 mW power level at 100 K for both 100-kHz and 2-MHz modulated detection with a -0.2-mT modulation amplitude in each case. There was no observable change in the line shape of the P700+ spectra for microwave power levels less than 3 mW. While there may be some microwave power saturation at 1mW, we consider it to be minor on the basis of the observed P700' line shape.
The Journal of Physical Chemistry, Vol. 83,
CIDEP of Photosystem I
No. 26, 1979 331 1
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Flgure 2. Time-resolved EPR absorption spectrum observed at 0.4 ps after flash excltation with direct EPR absorption detection of signals observed from the flash photolysis of whole cells of Anacystis nidulans at 100 K with a microwave power of 1 mW. 256 laser flashes were accumulated at each magnetic field position. The upward direction Is that of microwave absorption and downward is microwave emission.
Figure 1. Some typical kinetic profiles for flash photolysis with two kinds of EPR detection of signals observed from whole cells of the alga Anacystis nidulans at 100 K with a microwave power of 1 mW at 9.2 GHz. There were 256 laser flashes for each kinetic trace. (a) Direct EPR absorption at the magnetic field position -0.2 mT of Figure 2. (b) 2-MHz modulated EPR detectlon at the magnetic field position -0.35 mT of Figure 3. The modulation amplitude is about 0.1 mT.
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I t is to be expected that the observed transients have kinetics which show some microwave power saturation effects. These effects are mainly in the observation of a spin-lattice decay rate which is too short relative to a value measured at a sufficiently low nonsaturating microwave power. At this preliminary stage of these studies, we did not choose to measure the spin-lattice decay rates very accurately because of the necessity of obtaining the maximum signal-to-noiseratio in the transient kinetic profiles. Some studies were carried out at lower microwave power levels (particularly 0.1 mW) where it was determined that the observed spin-lattice decay rates were slower, but we did establish that the magnetic-field profiles of the transients were essentially independent of the microwave power level from 0.1 to 1 mW. It appears that the polarized enhanced-absorption signal of Figure 1decays in about 10 pus in both kinetic traces with the faster decay rate observed in the direct-absorption kinetic trace as would be expected from the faster response time. The longer decay time constant of about 0.6 ms from chemical decay can be seen by using both types of detection systems at a better signal-to-noise ratio by time averaging more kinetic traces from flash photolysis over a longer time span. A typical time-resolved absorption EPR spectrum obtained from the flash photolysis of whole cells of Anacystis nidulans with direct detection is presented in Figure 2. This is essentially the spectrum which is limited by the time response of 0.4 p s of the detection system. It is interesting to note that this spectrum has a central region of enhanced absorption and outer wings of enhanced emission as did a similar spectrum14 obtained from a deuterated algal species also at 100 K. The spectrum of Figure 2 covers a wider magnetic field domain than that of the deuterated algae. A significant common feature of the protonated spectrum of Figure 2 as compared with the deuterated spectrum14 is that both have detectable magnetic-field EPR responses as much as 1.2 mT to lower field than the center of the P700' spectrum at g = 2.0025. The
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Figure 3. Time-resolved first derivative EPR spectrum observed 2 ps after flash excitation with 2-MHz modulated EPR detection of signals obsswedfrom the flash photolysis of whole cells of Anacystis nkhkns at 100 K with a microwave power of 1 mW and a modulationamplttude of 0.1 mT. 256 laser flashes were accumulated at each magnetic field position.
EPR spectrum of P700' should not exhibit any significant intensity below -0.6 mT downfield from the center of P700'. A time-resolved EPR spectrum obtained from the flash photolysis of whole cells of Anacystis nidulans with 2-MHz EPR detection is presented in Figure 3. In this case, the spectrum is obtained about 2 ps after the flash because this is the time response of the 2-MHz detection system. The essential observation to be made from Figure 3 is that there are EPR responses over the same magnetic field domain as observed in Figure 2, the direct EPR absorption result. It is useful to make this observation because it indicates that the presence or absence of magnetic-field modulation in the detection system does not alter the basic physical observation (neglecting phase information) of an EPR response at a given magnetic field position. However, a more careful examination of the 2-MHz modulated result revealed a discrepancy in the phase information: an analytical integration of the first derivative result of Figure 3 only roughly agrees with the absorption spectrum of Figure 2 with as much as 25% deviation at higher fields. These deviations are significant, but do not distort the main features of the spectra. There are two explanations for the observed deviations. First, the 2-MHz detection system has a slower time response than the direct absorption system; and for the detection of more than one radical species (such as an electron donor and an acceptor), one would expect dif-
The Journal of Physical Chemlstty, Vol. 83, No. 26, 7979
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Flgure 4. Orientation dependence of the time-resolved EPR spectrum observed 30 ps after flash excitation wlth 100-kHz modulated EPR detection of signals observed from the flash photoiysls at 100 K of magnetically oriented whole chloroplasts with a microwave power of 1 mW and a modulation amplitude of 0.2 mT. 258 flashes were accumulated at each magnetic field position. The indlcated parallel and perpendicular orientatlons refer to the angle between the strong magnetic fieid and the normal to the thylakoid membrane. The chloroplasts were oriented and frozen In a 1:l mixture of 50 mM tris buffer and ethylene glycol.
ferent spin-lattice relaxation rates for these species which would lead to distortions in the slower time response spectrum of these species from the 2-MHz detection spectrum. Secondly, it was apparent that rapid passage effects were present in the 100-kHz modulated detection;lJ4 and they are certainly still present at the faster modulation frequency of 2 MHz used to obtain the spectrum of Figure 3. Furthermore, we believe that the kinetic curves themselves suffer from distorting influences resulting from rapid-passage effects. Distorted kinetic profiles were probably seen most clearly in the enhanced absorption region of the deuterated spectrum when using 100-kHzmodulated dete~ti0n.l~ With modulated detection in this spectral region, one could be led to conclude erroneously1J3that only enhanced emission signals are to be observed from the deuterated species of algae. We have also performed some membrane orientation studieslBon whole chloroplasts and on whole cells of algae. These samples were oriented in a 2.4-T field where they were frozen in liquid nitrogen; they were subsequently examined at 100 K to measure the flash photolysis timeresolved spectra. Such spectra for whole chloroplasts are presented in Figure 4 where there is a clear orientation dependence on turning the frozen sample in a 4-mm tube through an angle of 90°. For convenience, these spectra were measured with 100-kHzmodulated detection, and the field profile of Figure 4a is very similar to the nonoriented 100-kHz field profile measured in the previous work.14 While it is true that the 100-kHz kinetic profiles exhibit rapid-passage effects, it is highly improbable that they are
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Flgure 5. Orientation dependence of the steady state spectra obtained from oriented samples of whole cells of Anacystis nidukns at 12 K with a microwave power of 10 mW, a modulatlon amplitude of 1.25 mT at 100 kHz, and a receiver gain of 1.25 X lo3. The indlcated mutually parallel arid perpendlcular dlrections were deflnd in an arbitrary manner. The parallel minus perpendicular difference spectrum Is presented on the same vertical scale as the two spectra above it. The algae was suspended in a 1:l mixture of 50 mM phosphate and ethylene glycol.
the source of the orientation effects. However, we have not yet been successful in performing this experiment with direct absorption detection. It is interesting to point out that very similar spectra can be obtained from whole cells of Anacystis nidulans and of Scenedesmus obliquus which were oriented in a similar manner. The signal-to-noise ratio is not as good for the latter as for the spectra from whole chloroplasts. However, it is remarkable that whole algal cells of the blue-green alga Anacystis nidulans, containing no discrete chloroplasts, can be oriented. This observation also demonstrates that the orientation dependence of the photosystem I transients is a general result, as suggested by Sauer and co-workers.1° We conclude that the orientation dependence of the flash-induced transients points to the assignment of membrane-bound species with fixed orientations relative to the thylakoid membrane as has been observed12Jafor other electron-transport components involved in photosystem I photochemistry. For interest, we have included one more orientationdependent EPR spectrum, shown in Figure 5, obtained from whole cells of Anacystis nidulans. This is a steady-state spectrum measured at 12 K, and it almost certainly represents a manganese species from the photosystem I1 portion of the thylakoid membrane. The orientation dependence at 12 K of this spectrum is probably indicative of a sensitive power saturation phenomenon. For instance, the orientation dependence was less marked at lower microwave power levels at 12 K; and the orientation dependence was much less evident at temperatures close to 100 K. As Mn2+ itself could not be
CIDEP of Photosystem I
expected to have an anisotropic power saturation dependence, it must be the Mn2+environment in the thylakoid membrane which is anisotropic. However, it should be pointed out that the spectra of Figure 5 possibly do not represent an active water-splitting enzyme. Usually, the presence of Mn2+spectralDfrom the thylakoid membrane is indicative of a loss of membrane “bound” manganese eventually to become “free” manganese. Such “free” Mn2+ may be associated with membrane binding sites for divalent ions such as phosphate groups which would give rise to orientation effects. In conclusion, we interpret our results for the flash photolysis of protonated spinach chloroplasts and algal species. More support for the photosystem I assignment of these transient signals comes from a redox titration with ferricyanide and ascorbate performed in the course of these studies. We have previously reported14that the transients have a midpoint potential of +500 mV when observed at 100 K. This measurement was performed on whole algal cells, and the observed redox potential was probably too positive because of the necessity of saturating the thylakoid membrane with -10 mM ferricyanide which results in the oxidation of antenna ~hlorophyll.~ We have repeated this measurement by using photosystem I enriched particles from spinach, and we obtained a midpoint potential of +370 i 10 mV for the disappearance of the EPR transients at reasonable ferrocyanide/ferricyanide concentrations
