H. Manlkowskl, A, R. McIntosh, 0. R. Bolton TEMPERATURE DEPENDENCE OF THE CHEMICALLY
IM9UCED DYNAMIC ELECTRON POLARIZATION IN GREEN PLAMTS AND ALGAE
The temperature dependence of the chemically induced dy- naalc electron polarization has been studied in the chloro- plasts of Anecystls nldulane and Scenedesmus obliquua. The spectrua of photosystea I observed Is different from that of P700 with respect to shape and the g value, and points to the presence of e non-chlorophyll organic radical in the rea-ction centre of photoeystsn I.
Research on photosynthetic organises has long provided a ne-sting ground for scientists froa e variety of disciplines. The photosynthetic systems has been able to challenge and Interest solid-state physicists, chemists and biologists by presenting thea with a aultltude of physical and biological processes rang-ing from exclton tranefer In pigment array to the growth of fo-rests. In this diverse field It Is therefore not surprising that a remarkable range of physical techniques has been employed to e- lucldate various aechanlstlc aspects of photosynthetic energy storage.
In tha past decade the application of magnetic resonance te-chniques has contributed much to our still imperfect understand-ing of those processes [4]. Its use in the study of photosynthe-sis la perhaps the aost rewarding application of electron parama-gnetic resonance (EPR) on material of biological relevance. Prac-tically all aspects of this technique are represented In one or another fora in the rapidly growing body of literature on this subject. Experiments exploiting the phenomenon of chemically
duced dynamic electron polarization (CXQEP) have been extraordi-narily uneful in photosynthetic research in the paet few year*
[3, 4, 5],
Spin polarized, non-Boltzmann, or historically «»lied CIOEP, EPR spectra result If a chemical reaction hea e pretere've #or one of the epln atatee of the product* of the reaction Two me-chanisms for the generation of the nonequlllbrlum epir distribu-tion have been identified In chemical systems, the radical pair, and triplet mechanisms [1, 9]. A radical pair le simply two ra-dicals whose electron spins are correlated with reepect to each other; i.e., the relative orientation of the two electron mag-netic moments 1« not random. This correlation can oxiet in a single colecule before radical formation or can be produced by spin-selective reactions between Independently generated . *diala. A single molecule with two unpaired electrons that Interest atio- ngly is usually called a triplet rather m e n a radical pair.
Photosynthesis begins *h»n a photon is s o e o r * b y s pigment molecule embedded in a biological membrane [.*] vtiie chic oplaat membrane of green plants or the cytoplasmic membtone of phoro- synthetic bacteria), promoting It from the ground state to an excited t*te. The energy la rapidly transferred to a chlorc- phyil (or bacteriochlorophyll in the caae of photoevntheUc
bac-teria ) in a specialized chlorophyll-protein complex called tr- reaction center. In this excited stato tha pigment moiecule is a; extremely strong reductent, and an electron Is lost to an accep-tor molecule strategically placed nearby. The oxidized chloro-phyll and reduced acceptor then rapidly react with eecondery ele-ctron donors end -coeptors to separate the chargee and stabili-ze the syetem against recombination losaee.
The first step In the spin polarization process is radical pair formation. In the case of photosynthetle system*, the ra-dicals ere oxidized and reduced species produced by the photoche-mical eleetron-transfer reaction. The two electron eplna were highly correlated Just before the reaction, and tha chemical re-action pre3ervos this correlation. Thus an excited singlet produ-ces a elngiet radical pair and an excited triplet producaa a tri-plet raolcal pair. Ir the radicals ere far enough apart,the indi- v tual spin vectors are free to precea* aout the field direction
et a frequency determined by the electron g factor and nuclear state of the radical. Zn the presence of a magnetic field and the absence of any exchange coupling between the two radicals, on-ly the singlet (S) and middle triplet (T ) levels are mixed. The spin Hamlltomlan can be divided Into two parts, one of which gi-ves the frequency of S — ■ T mixing [9],
wab " t’ 1 | - la> * - § X \ „ V - (l)
In Eq, 1 represents the difference In srgular proces-sion frequency of the two electrons, h Is Planck's constant divided by 2H, 0 is the Bohr magneton, N is the applied mag-netic field, gj^ and g2 are the electronic g factors of the
two radicals, and Aln^°r AZm^ 8r® t*1® isotropic hyperflne coup-ling constants of nucleus n (or m) on radical 1 (or 2) with mag-netic quantum number Mln (or M2ra^» '»"be simple S -*■ Tq mixing process described above can account for some of the CIDGP obser-vations In photosynthetic systems.
Doublet polarization requires both S ~*‘T0 «ixlng and an ex-change interaction between the two radicals. Tha S -** Tq mixing and exchange can either occur simultaneously on a single radical encounter or sequentially on two encounters. The doublet polari-zation (excess of upper spin state) of radical pair can generate in a single encounter [ 9 ]j
p * V * b < z >
“ !b * ^
where 20 ■ E„ - Er is the singlet-trlplot splitting. In dlffus-S *.
lng systems, due to the rapid Brownian notion of the molecules (a 10~3Zs) thla condition ie not satisfied for a long enough time for significant polarization to develop in this manner. The fixed geometry of the photosynthetic reaction center effectively precludes free diffusion and multiple encounters of the radi-cals but is idaal for development of single-encounter polariza-tion via alngle-trlplet mixing end exchange interacpolariza-tion, if the radical separation Is appropriate*
Materials and methods
Whole cells of the algae Anacystis ntdulans and Scenedeamus obliquus were Investigated, the culture» being maintained In our laboratory. Scenedaamua obllquua tvae cultivated In deuterated growing medium In a result 99% deuterated cells were used for ex-periments. Whole chloroplasta were prepared fro* market spinach by the procedure of W h a t l e y and A r n o n [11] and contain about 5 mg/ml of chlorophyll. Chloroplaste ’ In a medium of 50Tii glycerol and 50 mM Trie at pH 8 were oriented In a 2.4 T magnetic field and scored In the frozen state at 77 K for subse-quent EPR measurements, Ther> vere no exoganoous redox agents ad-ded to the samples. Flesh photolysis measurements were carried out as described previously [5, 6],
Results
A single deuteron gives a triplet (Instead of a doublet In proton case) hyperflni* pattern. However, If all other factors are identical, the deuteron epllttlng is only one-seventh that of the proton. Beceuso the EPR line widths are reduced in
deu-terated samples we have used deudeu-terated algae in order to ob-tain batter signal-to-nolse ratios for kinetics taken without magnetic field modulation.
Examples of time-resolved EPR spectra for deuterated Scene- desrau3 obliquus in 0°C are presented In Fig. 1. The shape of the field profile and g-factor region are Identical to those obta-ined at liquid nitrogen temperature [7] and room temperature also [12] but the rete of the decay of tranalent signal is remarkably faster in higher temperatures.
The shape of the field profile at the beginning of the de-cay is also similar to that obtained by spin echo method [10], In Fig. 1 a and b four stages of field profile in time span avai-lable for our direct detection system are shown. Changes of the shape of field profiles in time is an argument in favour of pre-sence of more than one component with different rate of tha de-cay. The last field profile (300 y.s) Is identical with steady
— - * --1 ' 1----' l~ { m T ) — --* — ' * 1 ‘“ It nT ) F i g , 1 . Ti me-r e sol ved E P R s p e c t r a o bser ve d f r o m f l a s h ph o toly sis ex peri me nt s w ith t h e di re ct a b -sor pt i on det ec ti on »y st ew f o r f * h o l e cel l9 o f t h « 9 9 ^ 5 de uter at e d a l g a e Sce n ed es mu9 o b l i q uu # * mi c ro wa ve pow er w a s 1 » W a t 9 .2 G H z , te m pe ra t u re 273 K . Fi el d profiles in par t v b / a r e 5 x ex-pe nd e d i n v erti ca l dir ection i n co m pa ris on t o p a rt (a ) Cz as ro zk ł a du wi dm a E P R ob se rw ow ane go ek s pery ment u z f o t ol i z g b ł y s kó w ? z be zpo śre dn i m sy st e m em de t ek cj i a bs or pc ji d l a ca ł ko wi t y ch k o m ó re k w 9 9 5 5 z dena turo wany ch a g Sc ene d es mus o b l i qu u e , t o c mi kr o fal ow a 1 mW przy 9, 2 G H z , t e m pe r a t u r a 2 73 K . Ob sz a r pr ofil u w c zęś ci ( b ) roz s zer z ony 5 x w ki er u n ku pi onow ym w p orównaniu d o cz ę śc i ( a )
•tato signal I In terms of the shape, g-factor value and half- -wldth of the spectrum.
Wo have previously reported [6, 8] that the apectrua of the CIDEP transients exhibit an orientation dependence for ••■plea of oriented whole chloroplasts at liquid nitrogen teaparature. Thi» anisotropy is remarkable specially for alcroaacond resolved spectra. In Fig. 2 are presented parallel »nd perpendicular com-ponents with respect to the thylakold meabren* of tiae-re»olved EPR spectra. In this figure we can obsarv* changes.of the field profiles In time and different ratee of th* decay of transient signal for this two orientation«. It la additional argument In favour of presence of more than ona component.
Fig. 2. Time-resolved EPR absorption spectra obeervad after flaah excitation (tlae is written in figure) with direct EPR absorp-tion detecabsorp-tion of transients observed from the fleeh photolysis of oriented whole chloroplests et 100 K with a alcrowave power of 10 ¿¿w. The upwsrd direction is that of alcrowave absorption and downward is microwave emission. The normal to tha thylakold membrane with respect to the magnetic field la in (a) parallel
and in (b) perpendicular
Czas rozkładu widma absorpcji EPR obserwowanego po przejściu bły-sku (czas wpisany na rysunku) z bezpośredni« detekcje chwilom« absorpcji EPR z fotollz? błysków« dla orientowanych
At higher temperatures rat» of the decoy 1# fatter, but the ehape of the field profile end g-factor epan are the aame [12]. Even at room temperature where rete of the decey 1® very feet, low field part of the CIOEP spectrum 1» etil present In microse-cond tin«.
Conclusion»
CIOEP spectrum of photosystem I appear» generally within the »aae *egnetlc~fleld profile between room end liquid helium tem-perature». Observed deviations are caused mainly by limited tl- <0«-re solution of the spectrometer and frozen state of seconder/ chemical reactions at lower temperatures. In deutereted samples rete of the decay of transient signal Is slower end temperature variations are minor.
Observed polarized spectrum in Its early stege Is completely different from thet of P700+ both In the shape and g-fector span. There must be contribution from the acceptor radical to this spectrum with g-factors between 2.00 and 2.01. Thus, there la evidence for an organic free radical (beside chlorophyll) in the photosystem I reaction center. These conclusions are consistent with our previous results obtained at low temperatures [6-8].
Correspondence of the results obtained in EPR experiments us-ing both modulated detection and direct detection supports rea-lity of CIDEP elgnal from photosystem I observed in shorter tines after the flash when eignai-to-nols* ratio is better for direct detection method.
Acknowledgement
This study waa supported by the Notional Sciences and Engi-neering Research Council of Canada and Project No R. III.13.4.3. coordinated by the Institute of Biochemistry and Biophysics of Lodz University.
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[12] Sent for publication.
Inatltute of Physics Poznan Technical University
Photochemistry Unit Unlvereity of W»at»rn Ontario
M. Manikowski, A. R. McIntosh, 0. R. Bolton
ZALEŻNOŚĆ TEMPERATUROWA OYNAMICZNEO POLARYZACJI ELEKTRONOWEJ INDUKOWANEJ CHEMICZNIE U ROŚLIN WYŻSZYCH I GLONOW
Badano zależność temperaturo»«® dynamicznej polaryzacji elek-tronowej Indukowanej chemicznie w chloroplastach Anacystls ntdu- lana 1 Scenedeamua obllquus. Obserwowane widmo fotoayatemu I Jeet różne od widmo P700* co do k&ztaltu 1 wartości g 1 wskazuje na obecność nlechlorofiłowego rodnika organicznego w centrum re-akcyjnym fotoayatemu I.