Análisis De Resultados

A ttributed to A.

In order to confirm the id en tity of the g = 2.00 photo-accum ulated E P R signal a ttrib u te d to the reduction of th is signal w as m onitored u n d e r conditions which would lead to the gradual double reduction of th e quinone. Double reduction was a ttem p ted in D igitonin photosystem -I poised a t pH 8.0 in th e presence of dithionite a t 4°C b u t it w as found th a t th ese conditions would only allow p a rtia l double reduction an d so th e sam ples h a d to be poised a t pH 10.0. I t would have been preferable to carry o u t th is experim ent a t pH 8.0 as the resu lts in section 3.6.1. indicate th a t only one of each and Aq is photo-accum ulated, th ereb y sim plifying th e situ atio n .

Sam ples were illu m in ated a t 4°C for gradually longer periods of tim e in order to progressively double reduce th e quinone. They w ere th e n briefly d a rk ad ap ted an d frozen u n d er liquid nitrogen. A fter sh o rt periods of illu m in atio n a t 4°C the redox sta te of th e sam ples w as P700, Aq, A^, Fe- Sx', F e - S / an d Fe-Sg' an d the sam ples had no g = 2.00 E PR signal. A sh o rt period of su b seq u en t 205K illum ination a t th is stage induced th e g = 2.00 E P R sp ectru m consistent w ith the photo-accum ulation of A / (fig. 3.32). How ever, a fte r longer periods of 4°C illum ination th e su b seq u en t 205K photo-induced signal became m uch broader. A fter 90 m in u tes of 4°C illu m in atio n th e 205K photo-inducible A f E PR signal h a d been to tally replaced by a signal w ith a g-value of 2.0037 and a lin ew id th of 1.75 m T sim ila r to a signal previously assigned to Aq' (fig. 3.32) (M ansfield a n d

E v an s, 1988). This indicated th a t illum ination of D igitonin photosystem -I a t pH 10.0 in dithionite a t 4°C for 90 m in u tes doubly reduced A^, ren d erin g it E P R silent, an d th e subsequent 205K illum ination led only to th e photo­ accum ulation of Ag'.

These experim ents were repeated w ith T riton X-100 photosystem -I sam ples and th e sam e resu lts obtained w ith th e exception th a t full double red u ctio n w as achieved w ith a sh o rter 4°C illum ination tim e of 60 m inutes.

Palace et al. (1987) and Ziegler et at. (1987) previously rep o rted th a t prolonged u.v. illu m in atio n of photosystem -I re su lts in in activ atio n of th e quinone. To confirm th a t th e loss of the 205K photo-induced A f signal w as n o t a re s u lt of quinone photo-dam age the sam ples w ere dialysed a g a in st T ris-H C l pH 8.0 in the d ark a t 4°C in order to re-oxidise th e quinol to quinone. Such sam ples were th en frozen a t pH 8.0 in th e presence of d ith io n ite th e n illu m in ated for 2 m in u tes a t 205K to look for th e re- em ergence of th e A f E PR signal. A fter a 24 hour period of dialysis th e E P R signal a ttrib u te d to the reduced quinone re tu rn e d alth o u g h in some D igitonin photosystem -I p rep aratio n s longer periods of dialysis w ere required.

Prolonged 230K illum ination of D igitonin particles a t pH 10.0 in th e presence of d ithionite suggested th e presence of two quinone molecules. To in v estig ate th e effect of th e double reduction procedure on each of th ese quinones, sam ples were selected a t various points d u rin g th e tim e course of Aj double reduction an d illu m in ated for 90 m in u tes a t 230K in o rd er to achieve th e m axim um g = 2.00 E PR signal (fig. 3.33).

g = 2 0 0 3

319.7 322

Field mT

F ig u re 3.32

T h e E ffe c t o f Illu m in a tio n a t 4®C fo r P r o g r e s s iv e ly L o n g e r P e r io d s

o n th e 205K P h o to a c c u m u la te d E P R S p e c tr a o f in D ig ito n in

P h o to sy stem -1 a t pH 10.0.

Sam ples, p rep ared as described in m ateria ls and m ethods sections 2.1.4 an d 2.9.2, were illu m in ated a t 4°C for: l)D ark , 2)2 m in, 3)15 m in, 4)30 m in, 5)60 m in and 6)90 m in. A fter freezing sam ples w ere su b seq u en tly illu m in ated for 2 m in u tes a t 205K .

E PR conditions: Microwave pow er 5 pW, m odulation w id th 0.2 mT, recording tem p era tu re 75K.

322

3197

Field mT

F ig u re 3.33

The E ffect of Illum ination at 4®C for P rogressively Longer P eriods on the 230K Photoaccum ulated EPR Spectra o f A^’ and A^' plus in D igiton in Photosystem -I at pH 10.0.

Sam ples, p rep a red as described in m aterials an d m ethods sections 2.1.4 an d 2.9.2, w ere illum inated a t 4°C for: l)D ark , 2)2 m in, 3)15 m in, 4)30 m in, 5)60 m in and 6)90 m in. A fter freezing sam ples w ere su b seq u en tly illu m in ated a t 230K u n til no fu rth e r increase in signal size w as observed.

E P R conditions: Microwave power 5 pW, m odulation w id th 0.1 mT, recording te m p e ra tu re 75K.

P eriod of Illu m in atio n a t 4° Spin In ten sity R elative to P700^ 0 3.9 2 3.642 15 3.412 30 3.352 60 3.315 90 3.057 T able 3.6

Spin in ten sity o f the Maximum g = 2.00 sign als accum ulated follow in g 230K Illum ination o f D igitonin PS-I P articles at pH 10 P reviou sly Illum inated at 4°C in order to Double R educe A^.

Sam ples, p rep ared as described in m aterials an d m ethods sections 2,1.4 a n d 2.9.2, w ere illu m in ated a t 4° in order to double reduce Aj th e n fu rth e r illu m in a ted a t 230K u n til th e m axim um g = 2.00 E P R signal h a d been a tta in e d . The spin in te n sity w as estim ated by double in te g ratio n of th e sp ectra in figure 3.33 an d divided by the spin in te n sity in corresponding P700^ sam ples.

T he acquired spectra were th en double in te g ra te d in o rd er to com pare th e photo-inducible spin in ten sity w ith th a t of a m axim um P700^ signal (table 3.6). As th e 205K photo-accum ulated Aq' signal replaced th e A^' signal, indicating quinone double reduction, th e spin in te n sity dropped from 3.9 p er P700^ to 3.0 spins per P700^. The sp ectra show th a t th e com ponent a ttrib u te d to A / is decreasing on double red u ctio n b u t it is n o t com pletely abolished (fig. 3.33).

The re su lts indicate th a t one of the quinone species is read ily double reduced by prolonged illum ination a t 4°C in th e presence of d ith io n ite a t pH 10.0, w hilst th e o th er is not so easily double-reduced. T he re su lts also in d icate th a t th e A / E PR signal, seen afte r b rie f illu m in atio n a t 205K, is a risin g from th e quinone th a t is more easily double-reduced as th e o th er quinone req u ires longer periods of 205K illum ination to be photo­ accum ulated.