349

Biochimica et Biophysica Acta, 592 (1980) 349--363

Elsevier/North-Holland Biomedical Press
BBA 47898
ENERGY DI STRI BUTI ON IN THE PHOTOCHEMI CAL APPARATUS OF
PORPHYRIDIUM CR UENTUM IN STATE I AND STATE II
A.C. LEY and W.L. BUTLER
Depart ment of Biology, University of California San Diego, La Jolla, CA 92093 (U.S.A.)
(Received January 31st, 1980)
Key words: Energy distribution; Phot osyst em I; Phot osyst em II; Fluorescence;
Oxygen evolution
Summa r y
Fl uor es cence of Porphyridium cruentum in st at e I (cells equi l i br at ed in l i ght
abs or bed pr edomi nant l y by Phot os ys t e m I) and in st at e II (cells equi l i br at ed
in l i ght abs or bed appr eci abl y by Phot os ys t e m II) was exami ned t o det er mi ne
how t he di st r i but i on of exci t at i on ener gy was al t er ed in t he t ransi t i ons be t we e n
st at e I and st at e II. Low t e mpe r a t ur e emi ssi on spect r a of cells f r ozen in st at e I
and st at e II conf i r med t hat a larger f r act i on of t he exci t at i on ener gy is
del i vered t o Phot os ys t e m II in st at e I. Low t e mpe r a t ur e meas ur ement s s howed
t hat t he yi el d of ener gy t r ansf er f r om Phot os ys t e m II t o Phot os ys t e m I was
great er in st at e II and cal cul at i ons i ndi cat ed t hat t he phot ochemi cal r at e con-
st ant f or such ener gy t r ansf er was appr oxi mat el y t wi ce as large in st at e II.
Measur ement s at l ow t e mpe r a t ur e al so s howed t hat t he cr oss sect i ons and t he
spect ral pr oper t i es of t he phot os ys t e ms di d not change in t he t ransi t i ons
be t we e n st at e I and st at e II. In agr eement wi t h pr edi ct i ons made f r om t he
par amet er s meas ur ed at l ow t emper at ur e, t he act i on spect r a f or oxygen evol u-
t i on meas ur ed at r oom t e mpe r a t ur e wer e f ound t o be t he same i n st at e I and
st at e II.
Int roduct i on
When phot os ynt he t i c organi sms are fi rst i r r adi at ed t her e is an i nduct i on
per i od f or phot os ynt he s i s t o r each its final st eady~st at e rat e. Thi s t i me, whi ch
is general l y in t he or der of a f ew mi nut es f or t hor oughl y dar k- adapt ed
organi sms, is t he t i me r equi r ed f or t he phot os ynt he t i c machi ner y t o r each a
Abbr e vi at i on: DCMU, 3-(3,4-d/chlorophenyl)-l,l-dimethylurea.
350
st eady- st at e rat e of t ur nover . Dur i ng t hi s per i od t he yi el d of f l uor escence
i ncreases r api dl y t o an earl y ma xi mum and t hen decreases sl owl y t o a final
st eady- st at e level as t he st eady- st at e r at e of phot os ynt hes i s is appr oached [ 1 ].
Accor di ng t o t he t er mi nol ogy whi ch has bui l t up ar ound t hese phe nome na
[ 2] , cells whi ch are dar k adapt ed or equi l i br at ed in light abs or bed pri mari l y
by Phot os ys t e m I are sai d t o be in st at e I whi l e cells at st eady- st at e phot o-
synt hesi s in light abs or bed t o an appr eci abl e ext ent by Phot os ys t e m II are
sai d t o be in st at e II. Mur at a [ 3] , wor ki ng wi t h Porphyridium cruentum and
Bonavent ur a and Myer s [ 4] , wor ki ng wi t h Chlorella pyrenoidosa pr opos ed
i ndependent l y t hat t he t r ansi t i on f r om st at e I t o st at e II is accompani ed by a
r eadj us t ment in t he di st r i but i on of exci t at i on ener gy bet ween Phot os ys t e m I
and Phot os ys t e m II and t hat t he decr ease in f l uor escence f r om t he initial
ma xi mum t o t he final st eady- st at e level is due t o r edi st r i but i on of exci t at i on
ener gy t owa r d t he weakl y f l uor es cent Phot os ys t e m I. That pr opos i t i on was
chal l enged r ecent l y by Bri ant ai s et al. [ 5] who concl uded, on t he basis t hat t he
f l uor escence emi ssi on s pect r um of chl or opl ast s at - - 196 C was t he same regard-
less of whet her t he chl or opl ast s wer e f r ozen i n st at e I or in st at e II, t hat t he
di st r i but i on of ener gy bet ween t he p h o t o s y s t e ms di d not change in t he transi-
t i ons bet ween st at e I and st at e II. The wor k r epor t ed her e will s how t hat l ow
t emper at ur e f l uor escence emi ssi on spect r a are di f f er ent in st at es I and II, t hat
a great er f r act i on of t he exci t at i on ener gy is del i vered t o Phot os ys t e m I in st at e
II and t hat t hi s change in ener gy di st r i but i on is due pri mari l y t o a change in t he
r at e cons t ant f or ener gy t r ansf er f r om Phot os ys t e m II t o Phot os ys t e m I. The
r eason wh y Bri ant ai s et al. r eached a di f f er ent concl usi on will al so be appar ent .
Materials and Methods
Unialgal cul t ur es of Porphyridium cruentum wer e gr own i n an artificial sea
wat er me di um [ 6] at 19C in 125 ml Er l enmeyer flasks each cont ai ni ng 50 ml
of medi um. The cul t ur es wer e kept on a r ot ar y shaker and wer e expos ed t o con-
t i nuous di f f use l at eri al i l l umi nat i on ( 80/ z W/ c m 2) f r om cool - whi t e f l uor es cent
l amps. Cells f r om such cul t ur es cor r es pond bot h in pi gment c ont e nt and in t he
char act er i st i cs of t hei r phot os ynt he t i c appar at us t o cells we have pr evi ousl y
descr i bed as L-cells [ 7] . Cul t ur es t ypi cal l y exhi bi t ed l ogar i t hmi c gr owt h f or
t wo t o t hr ee weeks f ol l owi ng i nocul at i on. For all exper i ment s descr i bed her e
cells f r om 7 t o 10 da y ol d cul t ur es wer e col l ect ed b y cent r i f ugat i on and wer e
r es us pended in fresh gr owt h me di um pr i or t o use.
The ki net i cs of f l uor escence yi el d changes at r oom t e mpe r a t ur e wer e
meas ur ed on 0. 5 ml suspensi ons of dar k- adapt ed cells (1 10s- - 2 10 s cel l s/ ml ).
Gr een ( 560 nm, 800 ~W/ cm 2) or bl ue ( 440 nm, 300 ~W/ cm 2) exci t i ng l i ght
was pr ovi ded by a t ungst en- i odi de l amp f i l t er ed t hr ough 7 cm of 2% CuSO4
s ol ut i on and f ocus ed t hr ough appr opr i at e bl ocki ng and i nt er f er ence fi l t ers ont o
one arm of a t hr ee- ar med f i ber - opt i cs l i ght , pi pe assembl y [ 8] . The i nt ensi t i es
of act i ni c i rradi at i ons del i vered vi a a l i ght -pi pe wer e meas ur ed at t he exi t end
of t he l i ght -pi pe assembl y. The end of t he l i ght -pi pe assembl y wher e t he f i ber
opt i cs of t he t hr ee arms wer e j oi ned t oget her bot h i l l umi nat ed t he sampl e and
col l ect ed l i ght f l uor esced f r om t he f r ont sur f ace of t he sampl e. Fl uor es cence
f r om t he sampl e was di r ect ed by t he r emai ni ng t wo arms of t he l i ght -pi pe
351
assembly ont o a phot omul t i pl i er (EMI 9558) prot ect ed wi t h a red cut -off filter
(Toshiba VR-65) and a 692 nm interference filter. The out put of t he phot o-
multiplier was stored in a small comput er (Fabritech 1024) for subsequent
analysis.
Fluorescence emission spectra were measured on 0.5 ml suspensions of cells
(5 106--10 106 cells/ml) which had been frozen t o --196C during illumina-
tion. The samples were irradiated for 5 min wi t h blue (440 nm, 450 pW/cm 2)
or green (560 nm, 1 mW/ cm 2) light at room t emperat ure t o establish state I or
II, respectively, and t hen were frozen t o- - 196 C by pouring liquid N2 around
the vertical cylindrial cuvette in t he Dewar [9] while t he irradiation cont i nued.
In experiments where a multiple series of irradiations were given, t he sample
was frozen during cont i nuat i on of t he final irradiation. The end of the light-
pipe assembly rested directly on t he frozen sample which was immersed in
liquid N2. Exci t at i on at 560, 500 or 435 nm was directed ont o t he sample
t hrough one arm of t he light-pipe assembly while fluorescence from the front
surface was carried by t he ot her t wo arms t o t he entrance slit of a Bausch and
Lomb 500 mm monochr omat or (1.5 nm passband). Light at t he exit slit of the
monochr omat or was measured wi t h a GaAs phot ot ube (Hamamat su R666)
and t he signal was digitized and stored as a funct i on of wavelength in a small
comput er. The emission spectra, difference spectra and ratio spectra shown
were calculated were calculated by t he comput er and pl ot t ed directly with an
X- Y recorder.
Fluorescence exci t at i on spectra were measured at --196C wi t h the com-
puter-linked, singie-beam spect rophot omet er as described previously [ 7, 10, 11].
Exci t at i on from t he scanning monochr omat or was incident on t he t op surface
of t he frozen sample and fluorescence at 693 or 730 nm was measured from
t he bot t om surface with a phot ot ube (EMI 9558) prot ect ed by appropriate
filters. In some experi ment s fluorescence excitation spectra were measured on
frozen samples at the mi ni mum Fo level. In these cases the monochr omat or
slits were set to 0.28 mm which gave light intensities of 0.1 ~uW/cm 2 or less at
the sample. These light intensities did not alter the Fo level of fluorescence
measurably during the course of a scan. Absorption spectra were also mea-
sured wi t h t he same spect rophot omet er.
Most of t he fluorescence measurement s were made on cells which had been
frozen to --196C during irradiation and t hus were at t he maxi mum Fm level
of fluorescence. For some experiments, however, it was necessary t o compare
cells in state I and state II at t he mi ni mum Fo level which occurs when all
of t he Phot osyst em II reaction centers are open. For reasons we do not fully
underst and, we were unable t o freeze cells in state II wi t h our usual procedures
(i.e. pouring liquid nitrogen around t he cuvette) if t he cells, after reaching state
II, were placed in darkness for a short period t o allow t he Phot osyst em II reac-
tion centers to reopen. In order t o freeze cells in state II in darkness it was
necessary to freeze t hem much more rapidly. 1. 107--5 107 cells were
collected ont o Millipore filters discs (16 mm diameter) by gentle suction filtra-
tion. The cells were moi st ened wi t h a drop of growt h medi um and illuminated
for 5 mi n at room t emperat ure wi t h t he blue or green light sources used t o
establish state I and state II. The cells were t hen placed in t he dark for 45 s,
a t i me sufficient t o compl et el y restore t he Phot osyst em II react i on centers but
352
t oo s hor t f or any si gni fi cant changes in st at e II, and t hen wer e f r ozen r api dl y
by pl ungi ng t he fi l t er discs di r ect l y i nt o l i qui d ni t r ogen. The f r ozen discs wer e
mo u n t e d in dar kness in pr echi l l ed cuvet t es.
Act i on spect r a f or oxygen evol ut i on wer e measur ed pol ar i gr aphi cal l y in
modul a t e d light usi ng an appar at us descr i bed pr evi ousl y [ 11, 12] . A single
l ayer of cells on a bar e pl at i num el ect r ode was i l l umi nat ed s i mul t aneous l y
wi t h a rel at i vel y st r ong c ont i nuous backgr ound beam and a weak modul a t e d
monoc hr oma t i c beam of vari abl e wavel engt h. The backgr ound i l l umi nat i on
obt ai ned f r om a 500 W xe non l amp f i l t er ed t hr ough 2 cm of 3% CuS04 and
appr opr i at e i nt er f er ence fi l t ers was ei t her bl ue ( 440 rim, 350 #W/ cm 2) or green
( 560, 300 #W/ cm2). The modul a t e d light pr ovi ded b y a 150 W xe non l amp
and a scanni ng mo n o c h r o ma t o r ( 10 nm passband) was c hoppe d at 17 Hz by
a r ot at i ng sect or . The modul a t e d beam was mai nt ai ned at a cons t ant i nt ensi t y
(5 /~W/cm 2) b y a t her mopi l e de t e c t or in a servo s ys t em whi ch adj ust ed an
opt i cal dens i t y wedge at t he exi t slit of t he monoc hr oma t or . The modul a t e d
c o mp o n e n t of t he oxygen evol ut i on was measur ed wi t h a l ock-i n ampl i fi er.
The ampl i t ude of t he modul a t e d signal was f ound t o be l i near wi t h t he intensi-
t y of t he modul a t e d beam up t o 10 #W/ cm 2 in t he pr esence of t he st r ong back-
gr ound i l l umi nat i on.
Results
Typi cal f l uor escence yi el d changes whi ch occur dur i ng t he i rradi at i on of
P. cruent um wi t h green l i ght ( 800 /~W/cm 2 at 560 nm) are s hown i n Fig. 1.
The f l uor escence i ncreases r api dl y dur i ng t he fi rst f ew seconds t o an initial
ma xi mum and t hen decays mor e sl owl y t o a final st eady- st at e level whi ch is
char act er i st i c of t he st at e pr oduc e d by l i ght abs or bed b y Phot os ys t e m II. (We
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Fi g. 1. Ti me c ~ of f l uor es cence ot P. eruent um cells at 690 nm durLng i ~a di a t i on wi t h 560 nm l i ght
( 800 l~W/em 2) at r oom t e mpe r a t ur e . The s e c o n d i r r adi at i on wl t h 560 nm l i ght f onows t he f~wst af t er
a per i od of 3 rai n. Das hed l i ne, 3 rai n of darknes s ; s ol i d l i ne, 2. 5 n~n i r r adi at i on wi t h 440 nm l i ght ( 800
#W/ c m 2) i n t he mi ddl e of t he 3 r ai n per i od.
353
will not consi der t he initial f ast t r ansi ent spi ke in t he f l uor escence cur ve whi ch
of t e n pr ecedes t he initial ma xi mum or t he s econdar y ma xi mum whi ch some-
t i mes f ol l ows t he initial ma xi mum in t he appr oach t o st at e II. ) Wi t h dark-
adapt ed cells t he r at i o of t he i nt ensi t y of t he f l uor escence at t he initial
ma xi mum t o t ha t at t he final st eady- st at e level is t ypi cal l y 1. 5 t o 1.6.
St at e II persi st s f or s ome t i me in t he dar k f ol l owi ng an i rradi at i on. The
dot t e d cur ve in Fig. 1 shows f l uor escence changes duri ng a second i rradi at i on
wi t h 560 nm l i ght f ol l owi ng a dar k per i od of 3 mi n. The small initial ma xi mum
i ndi cat es a r at her small r et ur n t o t he dar k- adapt ed st at e. The hal f t i me t o
r egener at e t he dar k- adapt ed st at e is 5 t o 10 mi n. However , i f t he s econd irradia-
t i on is pr eceded by a 2. 5 mi n i rradi at i on wi t h 300 #W/ cm 2 of bl ue light ( 440
nm) , whi ch has ver y l i t t l e act i on f or phot os ynt hes i s in t hese cells, t he condi t i on
f or t he high initial ma xi mum is r egener at ed even b e y o n d t he original dark-
adapt ed st at e (see sol i d cur ve in Fig. 1). Thi s st at e, r ef er r ed t o as st at e I because
i t resul t s f r om l i ght abs or bed b y Phot os ys t e m I, shows a r at i o of t he i nt ensi t i es
at t he initial ma xi mum t o t he final level of a bout 1. 8 and a l onger i nduct i on
per i od dur i ng a s ubs equent i rradi at i on t o r each st at e II. St at e I decays t o t he
original dar k- adapt ed st at e wi t h a hal f-t i me of a b o u t 3 mi n. Thus, t he dark-
adapt ed s t at e is i nt er medi at e be t we e n st at e I and st at e II but is consi der abl y
cl oser t o st at e I t han t o st at e II. Mos t of t hese char act er i st i cs of st at es I and II
have been descr i bed pr evi ousl y [ 2- - 4] . Our pur pos e her e is t o det er mi ne how
t he phot ochemi cal pr oper t i es of t he cells change dur i ng t he t r ansi t i on bet ween
t hos e st at es.
Cells wer e pl aced in st at e I or st at e II b y i rradi at i on f or 5 mi n wi t h bl ue or
green light at r oom t emper at ur e and t hat condi t i on was f r ozen in by freezi ng
t he cells t o - - 196 C dur i ng cont i nued i rradi at i on. Emi ssi on spect r a of t he cells
at - - 196 C in st at es I and II are s hown in Fig. 2 wi t h f l uor escence exci t at i on at
560, 500 and 435 nm. 560 nm l i ght is abs or bed pr edomi nant l y by t he phyco-
bi l i somes; 435 nm light is abs or bed pr edomi nant l y by chl or ophyl l and 500 nm
light is abs or bed b y bot h set s of t he pi gment s. The emi ssi on bands at 642, 662,
and 683 nm are due t o phyc oc ya ni n, al l ophycocyani n and al l ophycocyani n B
of t he phycobi l i s omes , r espect i vel y, t he bands at 694 and 755 nm are due t o
t he ant enna chl or ophyl l of Phot os ys t e m II and t he band at 716 nm is due t o
t he ant enna chl or ophyl l of Phot os ys t e m I [ 11, 13] .
In each of t he cases in Fig. 2, t he st at e I mi nus s t at e II di f f er ence s pect r um
( pl ot t ed j us t above t he emi ssi on spect r a) s hows a gr eat er yi el d of f l uor escence
f r om Phot os ys t e m II chl or ophyl l ( 694 nm) and al l ophycocyani n B ( 683 nm)
in st at e I but no di f f er ence in t he yi el ds of f l uor escence f r om phyc oc ya ni n or
al l ophycocyani n. The f l uor escence yi el d changes of al l ophycocyani n B are
known t o f ol l ow t hos e of Phot os ys t e m II chl or ophyl l because of t he t i ght
ener gy coupl i ng. The dat a in Fig. 2 s how cl earl y t hat t he f l uor escence yi el d of
t he Phot os ys t e m II pi gment s is gr eat er in st at e I t han in s t at e II.
Yi el ds of ener gy t r ansf er f r om Phot os ys t e m II t o Phot os ys t e m I wer e det er-
mi ned by me t hods devel oped pr evi ousl y [ 13] f or cells f r ozen in st at es I and II.
In bri ef, meas ur ement s wer e made of t he exci t at i on s pect r um f or 730 nm fl uo-
r escence and of t he abs or pt i on s pect r um f or a di l ut e suspensi on of cells (see
Figs. 3B and 3E). The r at i o spect r a F73o/A in Figs. 3A and 3D i ndi cat e t he
wavel engt h de pe nde nc e of t he rel at i ve yi el d of t he 730 nm f l uor escence. I f t he
354
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Fig. 2. L o w t e mp e r a t t we e mi s s i o n s pe c t r a Of P. cruentum c e l l s f r o z e n t o - - 196 C in st at e I (SI) dur i ng
i r r adl at i on wi t h b l u e l i ght or f r o z e n i n s t at e I I ( S I I ) dur i ng i l Tadi at i on wi t h gr e e n l i ght . A, e x c i t a t i o n at
5 6 0 nr a; B, 500 nm; C, 4 3 5 n m. Di f f e r e n c e s pe c t r a s t at e I - - s t a t e I I a nd r at i o s pe c t r a s t at e I / s t a t e I I
are p l o t t e d a b o v e t he e r ai s s i on s pe c t r a,
7 2 0 nm f l uo r e s c e nc e we r e a pur e P h o t o s y s t e m I e mi s s i o n, we wo u l d e x p e c t
t ha t t he ma x i mu m y i e l d o f f l uo r e s c e nc e wo u l d o c c ur at 6 9 0 nm whi c h i s
a bs o r be d s o l e l y by P h o t o s y s t e m I c h l o r o p h y l l and t hat s hort er wave l e ngt hs ,
whi c h e x c i t e P h o t o s y s t e m I at l e as t i n part vi a e ne r gy t ransf er f r o m Ph o t o -
s y s t e m II, s h o u l d s h o w l o we r yi e l ds o f f l uo r e s c e nc e . On t hat basi s, t he rat i o
s pe c t r a F73o/A pr e s e nt s o me t h i n g o f an a n o ma l y i n t hat t he y i e l d o f t he 7 3 0
n m f l uo r e s c e nc e i s great er i n t h e 5 6 0 n m r e gi on t han i t i s at 6 9 0 n m b u t t hat
a n o ma l y i s readi l y r e s ol ve d by an i ns pe c t i o n o f t he e mi s s i o n s pe c t r um e x c i t e d
at 5 6 0 nm. I t i s appar e nt t hat t h e 7 3 0 n m f l uo r e s c e nc e e x c i t e d b y 5 6 0 n m l i ght
i s n o t a pur e P h o t o s y s t e m I e mi s s i o n b u t al s o i nc l ude s a s i gni f i cant c ont r i bu-
t i o n f r o m t he l o n g wa v e l e ng t h t ai l o f t h e P h o t o s y s t e m II e mi s s i on. Thus , i n
order t o pr o c e e d wi t h t h e anal ys i s , we n e e d t o de t e r mi ne t he f r ac t i on o f t he
5 6 0 n m- e x ~ t e d 7 3 0 n m f l uo r e s c e nc e whi c h i s d u e s o l e l y t o P h o t o s y s t e m I.
The 5 6 0 - e x c i t e d e mi s s i o n s pe c t r um c an be r e s ol ve d i n t o i t s P h o t o s y s t e m I
and P h o t o s y s t e m II c o mp o n e n t part s as was d o n e pr e v i o us l y [ 1 3 ] . Th e l o w
355
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7 0 0 8 0 0
WAVELENGTH
S ~
. . / ~ F s s
z
i%:
j ' ,
r o e 8oo
(nm)
Fi g . 3. Wa v e l e n g t h d e p e n d e n c e o f t h e r e l a t i v e y i e l d o f t h e 7 3 0 n m f l u o r e s c e n c e o f P. cruent um c e l l s a t
- - 1 9 6 C a n d d e c o n v o l u t i o n o f t h e e mi s s i o n s p e c t r u m i n t o P h o t o s y s t e m I a n d P h o t o s y s t e m I I c o m-
p o n e n t s . A a n d D, r a t i o s p e c t r a F730/ A; B a n d E, f l u o r e s c e n c e e x c i t a t i o n s p e c t r a F 7 3 0 , c o r r e c t e d f o r a
c o n s t a n t q u a n t u m f l u x o f e x c i t a t i o n , a n d a b s o r p t i o n s p e c t r a A. C a n d F , d e c o n v o l u t i o n o f t h e f l u o -
r e s c e n c e e mi s s i o n s p e c t r u m e x c i t e d a t 5 6 0 r i m, ~ F $ 6 0 , i n t o i t s P h o t o s y s t e m I a n d P h o t o s y s t e m I I c o m-
p o n e n t s , ~ F I a n d ~ F I I . Ce l l s i n s t a t e I ( SI ) o r s t a t e I I ( SI I ) a s i n d i c a t e d .
356
t emperat ure emission spectrum of cells in state II excited at 435 nm can be
t aken as a virtually pure Phot osyst em I emission spectrum. We have deter-
mi ned t hat the ratio F716/F693 for a pure Phot osyst em II emission spectrum is
approx. 0.14 [7]. Thus, t he deconvol ut i on requires t hat the proper amount of
the pure Phot osyst em I emission spectrum ZFI be subtracted from t he 560
nm-excited emission spect rum ZF s6 so t hat t he resulting difference spectrum
ZFII has a ratio FTI6/F693 of 0.14. Figs. 3C and 3F show such deconvol ut i ons
for cells in SI and SII. The relative cont ri but i ons of Phot osyst em I and Photo-
system II t o the 730 nm fluorescence were t hen calculated by integrating
the product of t he deconvol ut ed emission spectra times the spectral trans-
mission curve of t he 730 nm filter used in t he measurement over t he spectral
passband of t he filter (see Ref. 7). These calculations indicated t hat 76% of t he
730 nm fluorescence was due to Phot osyst em I in state I and t hat 85% was due
t o Phot osyst em I in state II. These values allow us t o correct the ratio F73o/A
at 560 nm t o include onl y t he Phot osyst em I component of t he 730 nm fluo-
rescence. The product of the values in t he first t wo columns in Table I gives
the corrected value for t he ratio of the yields of t he Phot osyst em I fluo-
rescence at 730 nm excited at 560 and 690 nm (given in t he t hi rd col umn) and
this corrected value of t he ratio is taken as t he maxi mum yield of energy trans-
fer from Phot osyst em II t o Phot osyst em I [7].
The exci t at i on spectra of t he 730 nm fluorescence were measured at the
maximal F M level where t he Phot osyst em II reaction centers are all closed
(recall t hat the cells were frozen t o --196C during irradiation) so t hat the yield
of energy transfer will be the maximal ~0T(II-+I)(M ) value. The values of
~T(II~I)(M) in Table I are not significantly di fferent for cells in state I or
state II; bot h are close to uni t y. However, it is more meaningful t o consider
values of ~T(II-*I)(o} which are obt ai ned when t he Phot osyst em II reaction
centers are all open since t hat is t he condi t i on for opt i onal phot osynt hesi s.
The ratio of ~T( I I - . . I ) ( M) / ~0T( I I - ~I ) ( o) will be the same as t he ratio of FM/Fo for
Phot osyst em II fluorescence so t hat t he value of ~0T(ii-,i)(o) Can be det ermi ned
from the value of ~0T(II~I)(M) and t he ratio F69S(M)/Fs93(o). The ratio deter-
mi nat i on requires t hat cells be frozen in states I and II in t he dark wi t h the
Phot osyst em II reaction centers fully open in order t o measure the increase of
fluorescence at 693 nm which occurs when the Phot osyst em II centers are
closed phot ochemi cal l y. Cells can be frozen in state II quite readily if the freez-
ing occurs in the light but some di ffi cul t y was experienced in t ryi ng to freeze
cells in state II in t he dark after a short dark period t o allow t he regeneration
TABLE I
COMPARISON OF FLUORESCENCE AND ENERGY TRANSFER PROPERTIES OF CELLS OF
P. CRUENTUM AT - - 196C IN STATE I AND STATE II
560 ~T ( n --~) (o)
F730 FI ( 730) ~T (II--~ I) (M) FS93(M)
~F 690 F730 F693( o)
7a'o
St a t e I 1.28 0. 76 0. 97 2. 04 0. 48
St at e n 1. 10 0. 85 0. 94 1.54 0.61
357
of open Phot os ys t e m II r eact i on cent er s. Similar pr obl ems wer e r epor t ed
pr evi ousl y wi t h green l eaves [ 14] . Even t hough st at e II is r easonabl y st abl e in
t he dar k at r oom t emper at ur e, i t appear s t o escape qui t e r api dl y t o st at e I
dur i ng t he freezi ng pr ocess. Thi s escape f r om st at e II whi ch occur s when t he
t e mpe r a t ur e is l ower ed is t he r eason wh y Bri ant ai s et al. [ 5] concl uded t hat
t her e wer e no di f f er ences in ener gy di s t r i but i on bet ween st at e I and st at e II;
t he y assumed t hat t he y had f r ozen chl or opl ast s t o - - 196 C in st at e II whi l e,
in f act , t he chl or opl ast s wer e f r ozen in st at e I. The escape f r om st at e II ma y be
even mor e r api d in chl or opl ast s t han in whol e cells. However , st at e II can be
t r apped in spi nach chl or opl ast s at - - 196 C i f t he chl or opl ast s are f r ozen dur i ng
t he act i ni c i rradi at i on (Ki t aj i ma, M. and But l er, W. L. , unpubl i s hed dat a). Wi t h
our usual freezi ng pr ocedur es we woul d i nvari abl y obt ai n P. cruentum cells in
st at e I when we t r i ed t o f r eeze t he cells in st at e II in t he dark. To over come
t hese pr obl ems sampl es wer e pr epar ed by fi l t eri ng cells of P. cruentum ont o
Mi l l i pore fi l t er discs t o pr ovi de ver y t hi n sampl es whi ch coul d be f r ozen r api dl y
by pl ungi ng t he m di r ect l y i nt o l i qui d ni t r ogen. Wi t h t hese r api d freezi ng pr oce-
dur es we wer e abl e t o f r eeze in st at e II af t er a s hor t dar k per i od (45 s was suffi-
ci ent f or all of t he Phot os ys t e m II cent er s t o open) so t hat t he r at i o of
F693(M)/F693(o) coul d be det er mi ned f or cells in bot h st at e I and st at e II
( Tabl e I). The val ues of ~T(II-*X)(o) wer e t hen cal cul at ed by di vi di ng t he val ues
of ~T(II---~I)(M) b y t he r at i os of F69S(M)/F693(o) ( Tabl e I). It is appar ent t hat
~W(.II-*I)(o) is si gni fi cant l y larger in st at e II ( 0. 61) t han i n st at e I ( 0. 48) . It will
be s hown in t he di scussi on (vi de i nfra) t ha t t he val ues of ~T(II~I) in Tabl e I
are cons i s t ent wi t h a doubl i ng of t he r at e cons t ant f or ener gy t ransfer,
kT(iX~x), in t he t r ansi t i on be t we e n st at e I and st at e II.
We can also det er mi ne t he rel at i ve cross sect i ons of Phot os ys t e m I and
Phot os ys t e m II, a and fi ( wher e a + fi = 1. 0), f or any wavel engt h of exci t at i on
[ 11] . Cells whi ch had been br ought t o st at e I or II b y i rradi at i on wi t h 440 or
560 nm l i ght at r oom t e mpe r a t ur e wer e f r ozen r api dl y t o - - 196 C af t er a 45- s
dar k per i od. Exci t at i on spect r a wer e meas ur ed f or f l uor escence at 693 and
730 nm bef or e any act i ni c i rradi at i on (Fo), af t er act i ni c i rradi at i on wi t h far-red
l i ght whi ch cl osed t he Phot os ys t e m I r eact i on cent er s (i.e., phot ooxi di z e d
P- 700) b u t had no i nf l uence on t he Phot os ys t e m II cent er s ( Fo' ) and af t er
a sat ur at i ng i r r adi at i on wi t h whi t e l i ght whi ch cl osed all of t he Phot os ys t e m II
cent er s (FM). Phot ooxi da t i on of P- 700 caused t he yi el d of t he 730 nm fl uo-
r escence t o i ncrease 15 t o 20% b u t had no ef f ect on t he 693 nm f l uor escence.
The wavel engt h di st r i but i on of a was t hen cal cul at ed b y t he c o mp u t e r f r om t he
exci t at i on s pect r a f or t he 693 and 730 nm f l uor escence meas ur ed at t he Fo'
and F M levels and t he val ue of ~T(II--~I)(M) accor di ng t o equat i ons pr es ent ed
pr evi ousl y [ 7, 11] . We concl ude f r om t he si mi l ari t y be t we e n t he spect r a of a
in st at e I and st at e II pr es ent ed in Fig. 4 t hat t he cross sect i ons of Phot os ys t e m
I and Phot os ys t e m II do not change dur i ng t r ansi t i ons be t we e n st at es I and II.
The above r esul t s on t he spect ral di st r i but i on of a l ead t o cer t ai n pr edi ct i ons
f or t he act i on s pect r a of oxygen evol ut i on meas ur ed i n st at e I and st at e II.
To make t hese meas ur ement s t he phot ochemi cal st at e of t he cells was
est abl i shed wi t h a cons t ant backgr ound i rradi at i on of 300 #W/ cm 2 at 560 nm
( f or st at e II) or 350 gW/ cm 2 at 440 nm ( f or st at e I) and t he r at e of oxygen
evol ut i on was meas ur ed wi t h a we a k monoc hr oma t i c modul a t e d beam. Act i on
358
l ' I ' I I
1 . 0
0 . 1 5
1.0
I , I , I , I
o.5
0
I , I , I i I
4 0 0 5 0 0 6 0 0 7 0 0
WAVELENGTH (nm)
1.2
,.o
bJ
o.s
wO, 4
~ 0 . 2
X
I I I I
o
o o , P , D
4 0 0 5 0 0 6 0 0 7 0 0
WAVELENGTH ( nm)
F i g . 4 . Wa v e l e n g t h d e p e n d e n c e o f a f o r P. c r ue nt um c e l l s i n s t a t e I ( SI ) a n d s t a t e n ( s I I ) a t - - 1 9 6 C .
F i g . 5 . Re l a t i v e r a t e o f mo d u l a t e d o x y g e n e v o l u t i o n a s a f u n c t i o n o f wa v e l e n g t h wi t h a c o n s t a n t q u a n t u m
f l u x o f e x c i t a t i o n . Me a s t ~ e me n t s we r e ma d e wi t h a v e r y we a k ( 3 - - 5 / ~ W/ c m 2 ) mo d u l a t e d mo n o c h r o ma t i c
b e a m s u p e r i mp o s e d o n a s t r o n g c o n s t a n t b e a m o f 4 4 0 n m l i g h t ( 3 5 0 / ~ W/ e m 2) t o h o l d t h e c e l l s i n s t a t e I
( o p e n c i r c l e s ) o r 5 6 0 n m l i g h t ( 3 0 0 / ~ W/ c m 2 ) t o h o l d t h e cel l s i n s t at e I I ( c l o s e d c i r c l e s ) .
spect r a f or t he modul a t e d oxygen evol ut i on cor r ect ed f or equal i nci dent
qua nt um f l ux and nor mal i zed are pr es ent ed i n Fig. 5 f or cells hel d in st at e I
and s t at e II. The t wo spect r a whi ch are, in essence, act i on spect r a f or Phot o-
s ys t em II s how no si gni fi cant di f f er ences. Ener gy di st r i but i on in t he phot o-
chemi cal appar at us can be descr i bed in t er ms of t wo par amet er s, a, whi ch is a
f unct i on of wavel engt h and ~w(n-*x), whi ch is i nde pe nde nt of wavel engt h. The
obser vat i on t hat ~T(II-,~)(o) changes be t we e n st at es I and II, whi l e a does not ,
l eads t o t he pr edi ct i on t ha t t he act i on s pect r um f or Phot os ys t e m II ( whi ch
f ol l ows t he wavel engt h de pe nde nc e of ~) s houl d be t he same in bot h st at es.
That pr edi ct i on is bor n o u t in t he resul t s of Fig. 5. Fur t her mor e, we not e t hat
t he pr edi ct i on, whi ch was based on meas ur ement s made at l ow t emper at ur e,
was t e s t e d b y meas ur ement s ma de at r oom t emper at ur e under physi ol ogi cal
condi t i ons.
Earl i er wor k [ 15] est abl i shed t ha t DCMU bl ocks t he t r ansi t i on f r om s t at e I
t o s t at e II but not t he reverse t r ansi t i on f r om st at e II t o st at e I. Fi g . 6 shows
t he resul t s of a similar s t udy in whi ch l ow t e mpe r a t ur e emi ssi on spect r a at t he
FM level wer e us ed as t he assay f or t he t wo st at es. Fig. 6A shows t he
nor mal st at e I and st at e II emi ssi on s pect r a as wel l as t he st at e I-st at e II di ffer-
ence s pect r um f or cells whi ch had been i r r adi at ed f or 5 rain wi t h bl ue l i ght
( st at e I) and green l i ght ( st at e II) be f or e bei ng f r ozen t o - - 196 C whi l e t he
359
LU
0
Z
h i
0
Or)
LU
n~
0
/
I.l_
w
k -
_1
w
I i i I I I ! , | I
A.
/ s X
/ s ] z
D.
, , T " I - - | " ' 1 - - , , , I
B . S Z - ( G - - B )
G --*'B
, , , , l l , l r l
C . ~ e ~ G )
( S- - G) - s' n"
B --'~G
i i i | I i I i i I i I I I | I I I I l i i i i I i , l i I
'E F
SX-- (B"~ DCMU". G } $ I - (G'-" DCMU~ B ) S I - ( G DCMU)
( B " D C M U ' * G ) - S 1T { G " D C M U ' * B ) - $ T/ ,~(G+ D C M U ) - S l l
G.-* DC MU-,P B
. . . . a , - j , , I i , i 1 7 1 " 0 0 i i i I . . . . I . . . . I
7 0 0 8 0 0 8 0 0 700 8 0 0
WAVELENGTH (nm)
Fi g . 6. E f f e c t s o f DC MU o n t r a n s i t i o n s b e t we e n s t a t e s I ( SI ) e n d I I ( SI I ) i n P. c r ue nt um c e l l s . A, e mi s s i o n
s p e c t r a o f c e l l s i r r a d i a t e d 5 i n i n wi t h b l u e l i g h t e n d f r o z e n t o - - 1 9 6 C i n t h e b l u e l i g h t , s t a t e I , a n d o f
c e l l s i r r a d i a t e d 5 r a i n wi t h g r e e n l i g h t e n d f r o z e n t o - - 1 9 6 C i n t h e g r e e n l i g h t , s t a t e I I , e n d t h e d i f f e r e n c e
s p e c t r u m s t a t e I - - s t a t e I I . B, e . , n t ~ o n s p e c t r u m o f c e l l s i r r a d i a t e d 5 r a i n wi t h g r e e n l i g h t , t h e n 5 r a i n
wi t h b l u e l i g h t e n d f r o z e n t o - - 1 9 6 C i n t h e b l u e l i g h t , G --~ B, a n d d i f f e g e n c e s p e c t r a o f t h e s e c e l l s v e r s u s
c e l l s i n s t a t e I a n d s t a t e I I . C, e mi s s i o n s p e c t r u m o f c e l l s i r z a d i a t e d 5 r a i n wi t h b l u e l i g h t , t h e n 5 r a i n wi t h
g r e e n l i g h t e n d f r o z e n t o - - 1 9 6 C i n t h e g r e e n l i g h t , B -+ G, a n d d i f f e r e n c e speet ~ca v e r s u s c e l l s i n s t a t e I
e n d s t a t e I I . D, s a me a s C b u t wi t h 5 p M DC MU a d d e d b e t we e n t h e b l u e e n d g r e e n ~ m- r a d i a t t o n . E , s a me
a s B b u t wi t h 5 /~M DC MU a d d e d b e t we e n t h e g r e e n e n d b l u e i r r a d i a t i o n . F , e mi s s i o n s p e c t r u m o f c e l l s
i r r a d i a t e d 5 r a i n wi t h g r e e n l i g h t wi t h 5 p M DC MU a d d e d d u r i n g t h e i r r a d i a t i o n a f t e r 4 r a i n . T h e c e l l s
we r e t h e n f r o z e n t o - - 1 9 6 C i n t h e g r e e n l i g h t .
360
i rradi at i on cont i nued. The t wo st at es are f ul l y reversi bl e at r oom t emper at ur e
in t hat cells whi ch wer e i r r adi at ed f or 5 mi n wi t h green light and t hen f or 5 rain
wi t h bl ue light bef or e bei ng f r ozen in bl ue light wer e in st at e I due t o t he final
i rradi at i on wi t h bl ue l i ght (Fig. 6B) whi l e cells whi ch wer e i rradi at ed f or 5 rain
wi t h bl ue and t hen 5 mi n wi t h green bef or e bei ng f r ozen in green l i ght wer e in
st at e II (Fig. 6C). However , i f DCMU was added af t er t he initial bl ue i rradi at i on
but bef or e t he green, t he cells r emai ned in st at e I (Fig. 6D) . DCMU pr event ed
t he t r ansi t i on f r om st at e I t o st at e II. On t he ot her hand, i f DCMU was added
af t er an initial green but bef or e a s ubs equent bl ue, t he cells wer e al so in st at e I
(Fig. 6E). DCMU di d n o t pr event t he st at e II t o st at e I t r ansi t i on medi at ed by
bl ue light. Fur t her mor e, i f DCMU was added dur i ng a green i rradi at i on one
mi nut e bef or e t he cells wer e f r ozen t o - - 196 C in t he green l i ght , t he cells
r ever t ed f r om st at e II t o s t at e I dur i ng t he final mi nut e of i rradi at i on (Fig. 6F) .
In t he pr esence of DCMU even green l i ght act s as Phot os ys t e m I light. Addi ng
DCMU duri ng a bl ue i rradi at i on had no ef f ect on t he final st at e ( dat a not
shown) . Thus, i t is cl ear t hat DCMU bl ocks t he Phot os ys t e m II medi at ed st at e I
t o st at e II t r ansi t i on but has no ef f ect on t he Phot os ys t e m I medi at ed st at e II
t o st at e I t r ansi t i on.
Di scussi on
Our original i nvest i gat i on of t he l ow t e mpe r a t ur e f l uor escence pr oper t i es of
P. cruentum in t he c ont e xt of t he t r i par t i t e mode l i ndi cat ed t hat t he phot o-
chemi cal appar at us was compr i s ed of rel at i vel y large Phot os ys t e m I uni t s whi ch
cont ai ned appr oxi mat el y 95% of t he chl or ophyl l and small Phot os ys t e m II
uni t s whi ch cont ai ned t he r emai ni ng 5% of t he chl or ophyl l and t hat t he large
phycobi l i s omes t r ansf er r ed t hei r exci t at i on ener gy al mos t excl usi vel y t o t he
small Phot os ys t e m II uni t s [ 11] . I n addi t i on, t he yi el d of ener gy t r ansf er f r om
Phot os ys t e m II t o Phot os ys t e m I was f ound t o be qui t e high ranging f r om
val ues of a b o u t 0. 50 when t he Phot os ys t e m II r eact i on cent er s wer e open t o
0. 95 when t he cent er s wer e cl osed [ 13] . Recent l y, i t was s hown i n a s t udy of
chr omat i c adapt at i on i n P. cruentum [7] t hat t hese phot ochemi cal pr oper t i es
are char act er i st i c of cells gr own in l i ght abs or bed pr i mar i l y b y t he phyco-
bi l i somes whi l e cells gr own in light abs or bed pr i mar i l y b y chl or ophyl l make
much larger Phot os ys t e m II uni t s whi ch cont ai n appr ox. 40% of t he chl or o-
phyl l and whi ch t r ansf er ener gy less ef f i ci ent l y t o Phot os ys t e m I. The cells used
in t he pr esent s t udy wer e of t he original t y p e whi ch s howed ver y small Phot o-
s ys t em II uni t s wi t h high pr obabi l i t i es f or ener gy t r ansf er t o Phot os ys t e m I.
Thos e char act er i st i cs are conf i r med b y t he large val ues of ~ ( appr oachi ng uni t y)
at 435 nm and t he l ow val ues of ~ ( appr oachi ng zer o) i n t he 560 nm regi on
(see Fig. 4) and b y t he high yi el ds f or ener gy t r ansf er f r om Phot os ys t e m II
t o Phot os ys t e m I ( Tabl e I).
Measur ement s of t he wavel engt h di st r i but i on of a, di d n o t i ndi cat e any
si gni fi cant changes in t he cr oss sect i ons of t he phot os ynt hes i s in t he t r ansi t i on
be t we e n s t at e I and st at e II. However , t he t r ansi t i on f r om s t at e I t o st at e II
was f ound t o be accompani ed by a decr ease i n t he f l uor es cence yi el d of Phot o-
syst em II, an i ncrease in t he mi ni mum yi el d of ener gy t r ansf er f r om Phot o-
s ys t em II t o Phot os ys t e m I, ~T~H~I)Co), and a decr ease in t he r at i o of FM/Fo
3 6 1
f or Phot os ys t e m II f l uor escence. In or der t o r el at e t hos e observat i ons t o
changes in f unda me nt a l phot ochemi cal par amet er s we will adopt t he simpli-
fi ed bi par t i t e f or mul at i on of t he t r i par t i t e model [ 16] . Accor di ng t o t hat
f or mul at i on:
F M _ 1
Fo 1 - - ~ T I I ~ t l I
wher e ~ / T I I is t he pr obabi l i t y t hat exci t at i on ener gy in a Phot os ys t em II uni t
will be t r apped by t he r eact i on cent er chl or ophyl l and ~t xi is t he pr obabi l i t y
t hat t he ener gy t r apped by t he chl or ophyl l of a cl osed Phot os ys t em II r eact i on
cent er will be r et ur ned t o t he ant enna chl or ophyl l of t hat uni t . We will assume
f or si mpl i ci t y t hat t her e are no f l uor es cent or nonr adi at i ve decay losses at t he
r eact i on cent er chl or ophyl l so t hat ~t i i = 1.0. The sum of t he probabi l i t i es of
all of t he deexci t at i on processes in t he a nt e nna chl or ophyl l of Phot os ys t em II
mus t add up t o uni t y; i.e., ~ i + ~DII + ~Z~I + ST(II~I) = 1.0 wher e t he sub-
scripts i ndi cat e f l uor escence, nonr adi at i ve decay, t r appi ng by t he r eact i on
cent er chl or ophyl l and ener gy t r ansf er t o Phot os ys t em I, respect i vel y. Thus,
F M 1 SFII "4" ~/DII 4" ~ T I I + ~T( I I ' - ~I )
F o 1 - - ~/ TIi ~ F I I "[" ~)DII + ~JT(II'-*I)
a n d s i n c e ~ X I I = k X I I / Z k I I :
F M _ k F I I + k D i I + k T i I + k T ( i i ~ i )
Fo k F l I 4" k D l l 4- k T ( l l . . ~ l )
If w e assign relative values o f (kFii + k m i ) : kTix : kz(ix-~l) o f 5 : I 0 0 : 9 0 for
cells in state I w e calculate theoretical v a l u e s w h i c h are in reasonably g o o d
a g r e e m e n t w i t h the e x p e r i m e n t a l d a t a obtained in state I : F M / F o = 2 . 0 5 (vs
the m e a s u r e d value o f 2 . 0 4 in T a b l e I), ~0T(II~I)(o) = 0.46 (VS. 0.48) a n d
~0T(IX~I)(M) = 0 . 9 5 (VS. 0.97). M o s t o f the e x p e r i m e n t a l data for the cells in
state II c a n b e a c c o m o d a t e d quite well if w e a s s u m e o n l y that the relative value
of kz(ii-.~) increases f r o m 9 0 to 1 8 0 in the transition f r o m state I to state If.
In that c a s e in s t a t e If, F M / F o = 1.54 (vs. the m e a s u r e d value o f 1.54),
~r(H-*i)(o) = 0 . 6 3 (vs. 0.61) a n d ~T(II-~i)(M)= 0 . 9 7 (VS. 0.94). H o w e v e r , the
state I/state II ratio of the 6 9 3 n m fluorescence at the F M level m a y n o t b e
predicted a d e q u a t e l y b y a s s u m i n g that o n l y kT(iI-*i) changes. T h e m e a s u r e d
value of that ratio w a s 1.5 + 1.0 while the value predicted f r o m the relative
values o f rate constants is 1.95. T h e m e a s u r e d value c o u l d b e t o o l o w b e c a u s e
of overlap f r o m the 6 8 0 a n d 7 1 6 n m b a n d s in the m e a s u r e m e n t at 6 9 3 n m
b u t s u c h effects are p r o b a b l y n o t sufficient to a c c o u n t for the discrepancy. It
m a y b e that o n e of the other r a t e constants in the s y s t e m s u c h as k T i i also
c h a n g e s d u r i n g the transition f r o m state I to state If. A l l o f the e x p e r i m e n t a l
d a t a c a n b e predicted quite closely b y a s s u m i n g c h a n g e s in various pairs of rate
const ant s but in each case one me mb e r of t he pai r mus t be k T ( i i . _ . i ) . We can
st at e unequi vocal l y f r om our dat a t hat t he t r ansi t i on f r om st at e I t o st at e II
is accompani ed by a maj or i ncrease ( 60- - 100%) in kT(ii-*i) but we c a nnot assert
t hat t hi s is t he onl y phot ochemi al par amet er t o change. We assume t hat t he
362
increase in k T ( i i - + l ) reflects a closer physical association between Photosystem I
and Photosystem II units.
It has been suggested that the changes of energy distribution which occur
during transitions between states I and II are similar to those which are
produced in chloroplasts by the presence and absence of divalent cations. In
such correlations cells in state II are assumed to be analogous to chloroplasts
suspended in the absence of divalent cations in that both conditions induce an
increase in the distribution of excitation energy to Photosystem I. Mechanisti-
cally, it could be envisaged that outward movement of magnesium which
occurs in response to the inward pumping of H" into the thylakoids results in
a membrane conformational change which increases energy transfer from
Photosystem II to Photosystem I in state II and that these same changes occur
when isolated chloroplasts are depleted of divalent cations. However, the
experiments with DCMU indicate t hat such correlations are probably over-
simplifications. In the presence of DCMU, we would expect that Photosystem I
could produce ion gradients by cyclic electron transport t hat were smaller but
in the same direction as those produced by noncyclic electron transport in the
absence of DCMU. If states I and II were solely a question of ion gradients or
the concomitant energization of thylakoid membranes we would not expect
strong antagonistic effects between Photosystem I and Photosystem II. At best
the effect of Photosystem I light would be similar to darkness in causing the
conversion of state II to state I but we would not expect Photosystem I to
actively stimulate that conversion. The fact that Photosystem I activity does
stimulate the transition from state II to state I indicates additional factors must
be involved. The results with DCMU suggest (in agreement with the earlier
work and suggestions of Duysens [15]) t hat some component or components
between Photosystem II and Photosystem I must be reduced in order for
state II to occur and t hat state I prevails when those components are oxidized.
However, some cooperative effects between ion gradients and the redox state
of components in the electron transport system between Photosystem II and
Photosystem I may play an important role in these phenomena.
Acknowledgements
This work was supported by a National Science Foundation grant, PCM
79-03987.
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