1-s2.0-0005272880901954-main
TRANSCRIPT
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B i o c h i m i c a e t B io p h y s i c a A c t a , 592 1980) 349--363
Elsevier/North-Holland Biomedical Press
BBA 47898
E N E R G Y D I S T R I B U T I O N IN T H E P H O T O C H E M I C A L A P P A R A T U S O F
PORPHYR IDIUM R UENTUM
I N S T A T E I A N D S T A T E I I
A.C. LEY and W.L. BUTLER
D epa r t m en t o f B i o l ogy , Un i ver s i t y o f C al if or ni a San Di ego , L a Jo ll a, C A 92093 U .S .A . )
Received January 31st, 1980)
K e y w o r d s : E n e r g y d i s tr i b u ti o n ; P h o t o s y s t e m I ; P h o t o s y s t e m I I ; F l u o r e sc e n c e ;
O x y g e n e v o l u t i o n
S u m m a r y
F l u o r e s c e n c e o f
Porphyridium cruentum
i n s t a t e I c e l ls e q u i l i b r a t e d i n li g h t
a b s o r b e d p r e d o m i n a n t l y b y P h o t o s y s t e m I ) a n d in s t a t e I I c e ll s e q u i l i b ra t e d
i n li g h t a b s o r b e d a p p r e c i a b l y b y P h o t o s y s t e m I I) w a s e x a m i n e d t o d e t e r m i n e
h o w t h e d i s t r i b u t i o n o f e x c i t a ti o n e n e r g y w a s a l t e re d i n t h e t ra n s it i o n s b e t w e e n
s t a t e I a n d s t a t e I I . L o w t e m p e r a t u r e e m i s s i o n s p e c t r a o f c e ll s f r o z e n i n s t a t e I
a n d s t a t e I I c o n f i r m e d t h a t a l a rg e r f r a c t i o n o f t h e e x c i t a t i o n e n e r g y i s
d e l iv e r e d t o P h o t o s y s t e m I I i n s ta t e I . L o w t e m p e r a t u r e m e a s u r e m e n t s s h o w e d
t h a t t h e y i e l d o f e n e r g y tr a n s f e r f r o m P h o t o s y s t e m I I t o P h o t o s y s t e m I w a s
g r e a t e r i n s t a t e I I a n d c a l c u l a t i o n s i n d i c a t e d t h a t t h e p h o t o c h e m i c a l r a t e c o n -
s t a n t f o r s u c h e n e r g y t r a n s f e r w a s a p p r o x i m a t e l y t w i c e a s l a rg e in s t a t e I I.
M e a s u r e m e n t s a t l o w t e m p e r a t u r e a l so s h o w e d t h a t t h e c r o s s s e c t io n s a n d t h e
s p e c tr a l p r o p e r t i e s o f t h e p h o t o s y s t e m s d i d n o t c h a n g e i n t h e t r a n si t io n s
b e t w e e n s t a t e I a n d s t a t e II . I n a g r e e m e n t w i t h p r e d ic t i o n s m a d e f r o m t h e
p a r a m e t e r s m e a s u r e d a t l o w t e m p e r a t u r e , t h e a c t i o n s p e c t r a f o r o x y g e n ev o l u -
t i o n m e a s u r e d a t r o o m t e m p e r a t u r e w e r e f o u n d t o b e th e s a m e in s ta t e I a n d
s t a t e I I .
n t r o d u c t i o n
W h e n p h o t o s y n t h e t i c o r g a n i s m s a r e f i r st i r r a d i a t e d t h e r e i s a n i n d u c t i o n
p e r i o d f o r p h o t o s y n t h e s i s t o r e a c h i ts f i n al s te a d y ~ s t a t e r a t e . T h i s t i m e , w h i c h
i s g e n e ra l ly i n t h e o r d e r o f a f e w m i n u t e s f o r t h o r o u g h l y d a r k - a d a p t e d
o r g a n is m s , i s t h e t i m e r e q u i r e d f o r t h e p h o t o s y n t h e t i c m a c h i n e r y t o r e a c h a
A b b r e v i a ti o n : D C M U 3- 3,4-d/chlorophenyl)-l,l-dimethylurea.
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s t e a d y - s t a t e r a t e o f t u r n o v e r . D u r i n g t h is p e r i o d t h e y i e l d o f f l u o r e s c e n c e
i n c re a s e s r a p i d l y t o a n e a r l y m a x i m u m a n d t h e n d e c r e a s e s s l o w l y t o a f i na l
s t e a d y - s t a t e l e v el a s t h e s t e a d y - s t a t e r a t e o f p h o t o s y n t h e s i s i s a p p r o a c h e d [ 1 ] .
A c c o r d i n g t o t h e t e r m i n o l o g y w h i c h h a s b u i l t u p a r o u n d t h e s e p h e n o m e n a
[ 2 ] , c e l l s w h i c h a r e d a r k a d a p t e d o r e q u i l i b r a t e d i n l i gh t a b s o r b e d p r i m a r i l y
b y P h o t o s y s t e m I ar e s a id t o b e i n s t a t e I w h i l e c e ll s a t s t e a d y - s t a t e p h o t o -
s y n t h e s is i n l ig h t a b s o r b e d t o a n a p p r e c i a b le e x t e n t b y P h o t o s y s t e m I I a r e
s a i d t o b e i n s t a t e I I . M u r a t a [ 3 ] , w o r k i n g w i t h Porphyridium cruentum and
B o n a v e n t u r a a n d M y e r s [ 4 ] , w o r k i n g w i t h hlorella pyrenoidosa p r o p o s e d
i n d e p e n d e n t l y t h a t t h e t r a n si t io n f r o m s t a t e I t o s t a t e I I is a c c o m p a n i e d b y a
r e a d j u s t m e n t i n t h e d i s t r i b u t i o n o f e x c i t a t i o n e n e rg y b e t w e e n 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 a n d t h a t t h e d e c r e a s e in f l u o r e s c e n c e f r o m t h e i n it ia l
m a x i m u m t o t h e f i na l s t e a d y - s t a t e l e ve l i s d u e t o r e d i s t r i b u t i o n o f e x c i t a t i o n
e n e r g y t o w a r d t h e w e a k l y f l u o r e s c e n t P h o t o s y s t e m I . T h a t p r o p o s i t i o n w a s
c h a l l e n g e d r e c e n t l y b y B r i a n t a is e t al . [ 5 ] w h o c o n c l u d e d , o n t h e b a s is t h a t t h e
f l u o r e s c e n c e e m i s s io n s p e c t r u m o f c h lo r o p l a s t s a t - - 1 9 6 C w a s t h e s a m e r eg a rd -
l e ss o f w h e t h e r t h e c h l o r o p l a s t s w e r e f r o z e n i n s t a t e I o r i n s t a t e I I, t h a t t h e
d i s t r ib u t i o n o f e n e rg y b e t w e e n t h e p h o t o s y s t e m s d i d n o t c h a n g e i n t h e t ra n si -
t i o n s b e t w e e n s t a t e I a n d s t a te I I . T h e w o r k r e p o r t e d h e r e w i ll s h o w t h a t lo w
t e m p e r a t u r e f l u o r e s c e n c e e m i s s i o n s p e c t r a a r e d i f f e r e n t i n s t a t e s I a n d I I , t h a t
a g r e a t e r f r a c t i o n o f t h e e x c i t a t i o n e n e r g y i s d e l i v e r e d t o P h o t o s y s t e m I i n s t a t e
I I a n d t h a t t h i s c h a n g e i n e n e r g y d i s t r i b u t i o n i s d u e p r i m a r i l y t o a c h a n g e i n t h e
r a t e c o n s t a n t f o r e n e r g y t r a n s fe r f r o m P h o t o s y s t e m I I t o P h o t o s y s t e m I . T h e
r e a s o n w h y B r i a n t a i s e t a l . r e a c h e d a d i f f e r e n t c o n c l u s i o n w i l l a l s o b e a p p a r e n t .
a t e r ia l s a n d e t h o d s
U n i a l g a l c u l t u r e s o f
Porphyridium cruentum
w e r e g r o w n i n a n a r t i f i c i a l s e a
w a t e r m e d i u m [ 6 ] a t 1 9 C i n 1 2 5 m l E r l e n m e y e r f la s ks e a c h c o n t a i n in g 5 0 m l
o f m e d i u m . T h e c u l t u r e s w e r e k e p t o n a r o t a r y s h a k e r a n d w e r e e x p o s e d t o c o n -
t i n u o u s d i f f u s e l a te r ia l i l lu m i n a t i o n ( 8 0 / z W / c m 2) f r o m c o o l - w h i t e f l u o r e s c e n t
l a m p s . C e ll s f r o m s u c h c u l t u r e s c o r r e s p o n d b o t h i n p i g m e n t c o n t e n t a n d in t h e
c h a r a ct e r is t ic s o f th e i r p h o t o s y n t h e t i c a p p a r a t u s t o c el ls w e h av e p r e v io u s l y
d e s c r i b e d a s L - c el ls [ 7 ] . C u l t u r e s t y p i c a l l y e x h i b i t e d l o g a r i t h m i c g r o w t h f o r
t w o t o t h r e e w e e k s f o l l o w i n g i n o c u l a t i o n . F o r a ll e x p e r i m e n t s d e s c r i b e d h e re
c e ll s f r o m 7 t o 1 0 d a y o l d c u l t u r e s w e r e c o l l e c t e d b y c e n t r i f u g a t i o n a n d w e r e
r e s u s p e n d e d i n fr e sh g r o w t h m e d i u m p r i o r t o us e .
T h e k i n e ti c s o f f l u o r e s c e n c e y i e ld c h a n g e s at r o o m t e m p e r a t u r e w e r e
m e a s u r e d o n 0 . 5 m l s u s p e n s i o n s o f d a r k - a d a p t e d c e ll s ( 1 1 0 s - - 2 1 0 s c e l ls / m l ) .
G r e e n ( 5 6 0 n m , 8 0 0 ~ W / c m 2) o r b l u e ( 4 4 0 n m , 3 0 0 ~ W / c m 2) e x c i ti n g l ig h t
w a s p r o v i d e d b y a t u n g s t e n - i o d i d e l a m p f i l te r e d t h r o u g h 7 c m o f 2 C u S O 4
s o l u t i o n a n d f o c u s e d t h r o u g h a p p r o p r i a t e b l o c k i n g a n d i n t e r f e re n c e f i l te r s o n t o
o n e a r m o f a t h r e e - a r m e d f i b e r - o p t i c s li g h t, p i p e a s s e m b l y [ 8 ] . T h e i n t e n s i ti e s
o f a c t i n i c i r r a d ia t i o n s d e l i v e r e d v i a a l i g h t- p i p e w e r e m e a s u r e d a t t h e e x i t e n d
o f t h e l ig h t - p ip e a s s e m b l y . T h e e n d o f t h e l i g h t - p ip e a s s e m b l y w h e r e t h e f ib e r
o p t i c s o f t h e t h r e e a r m s w e r e j o i n e d t o g e t h e r b o t h i l l u m i n a t e d t h e s a m p l e a n d
c o l l e c t e d l ig h t f l u o r e s c e d f r o m t h e f r o n t s u r f a c e o f t h e s a m p l e. F l u o r e s c e n c e
f r o m t h e s a m p l e w a s d i r e c t e d b y t h e r e m a i n in g t w o a rm s o f th e li g h t- p ip e
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assembly ont o a photomu ltipli er EMI 9558) protected with a red cut-off filter
Toshiba VR-65) and a 692 nm interference filter. The outpu t of the photo-
multipl ier was stored in a small com put er Fabrit ech 1024) for subsequ ent
analysis.
Fluorescence emission spectra were measured on 0.5 ml suspensions of cells
5 106--10 106 cells/ml) whic h had been fro zen to - -196 C duri ng illumina-
tion. Th e samples were irradiated for 5 min wi th blue 440 nm, 450 pW/ cm 2)
or green 560 nm, 1 mW/cm 2) light at room tem pera tur e to establish state I or
II, respectively, and then were frozen to -- 19 6 C by pouring liquid N2 around
the vertical cylindrial cuvette in the Dewar [9] while the irradiation continued.
In experiments where a multiple series of irradiations were given, the sample
was frozen during continuation of the final irradiation. The end of the light-
pipe assembly rested directly on the frozen sample which was immersed in
liquid N2. Excitation at 560, 500 or 435 nm was directed onto the sample
through one arm of the light-pipe assembly while fluorescence from the front
surface was carried by the othe r two arms to the entra nce slit of a Bausch and
Lomb 500 mm mon och rom ato r 1.5 nm passband). Light at the exit slit of the
mon och rom ato r was measured with a GaAs phot otub e Hamamatsu R666)
and the signal was digitized and stored as a function of wavelength in a small
comp uter . The emission spectra, difference spectra and ratio spectra shown
were calculated were calculated by the computer and plotted directly with an
X Y recorder.
Fluorescence excitation spectra were measured at --196C with the com-
puter-link ed, singie-beam spe ctr oph oto met er as described previously [ 7,10,11].
Excitation from the scanning monochromator was incident on the top surface
of t he frozen sample and fluorescence at 693 or 730 nm was measured from
the bot tom surface with a pho tot ube EMI 9558) protected by appropriate
filters. In some experiments fluorescence excitation spectra were measured on
frozen samples at the m ini mum Fo level. In these cases the mono ch ro mat or
slits were set to 0.28 mm which gave light intensities of 0.1 ~uW/cm2 or less at
the sample. These light intensities did n ot alter the Fo level of fluorescence
measurably during the course of a scan. Absorption spectra were also mea-
sured with the same spectrophotomet er.
Most of the fluorescence measurements were made on cells which had been
frozen to --196C during irradiation and thus were at the maximum Fm level
of fluorescence. For some experiments, however, it was necessary to compare
cells in state I and state II at the minimum Fo level which occurs when all
of the Photosystem II reaction centers are open. For reasons we do not fully
unde rsta nd, we were unable to freeze cells in state II wi th our usual procedures
i.e. pouring liquid nitro gen arou nd t he cuvette) if the cells, after reaching state
II, were placed in darkness for a short period to allow the Photosystem II reac-
tion centers to reopen. In order to freeze cells in state II in darkness it was
necessary to freeze th em mu ch mor e rap idly. 1 . 107--5 107 cells were
collected o nt o Millipore filters discs 16 mm diameter) by gentle suction filtra-
tion. The cells were moistened with a drop o f growth medium and illuminated
for 5 min at roo m temp erat ure w ith t he blue or green light sources used to
establish state I and state II. The cells were then placed in the dark for 45 s,
a time sufficient to completel y restore the Phot osyst em II reaction centers but
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t o o s h o r t f o r a n y s i g n i f i c a n t c h a n g e s i n s t a t e I I , a n d t h e n w e r e f r o z e n r a p i d l y
b y p l u n g i n g t h e f i l te r d i s cs d i r e c t l y i n t o l i q u i d n i t r o g e n . T h e f r o z e n d i sc s w e r e
m o u n t e d i n d ar k n e s s in p re c h i ll e d c u v e t t e s .
A c t i o n s p e c t r a f o r o x y g e n e v o l u t i o n w e r e m e a s u r e d p o l a r ig r a p h ic a l ly i n
m o d u l a t e d l i gh t u s i n g a n a p p a r a t u s d e s c r i b e d p r e v i o u s l y [ 1 1 , 1 2 ] . A s in g l e
l a y e r o f c e ll s o n a b a r e p l a t i n u m e l e c t r o d e w a s i l lu m i n a t e d s i m u l t a n e o u s l y
w i t h a r e la t iv e l y s t r o n g c o n t i n u o u s b a c k g r o u n d b e a m a n d a w e a k m o d u l a t e d
m o n o c h r o m a t i c b e a m o f v a r ia b l e w a v e l e n g t h . T h e b a c k g r o u n d i l l u m i n a ti o n
o b t a i n e d f r o m a 5 0 0 W x e n o n l a m p f i lt e r e d t h r o u g h 2 c m o f 3 CuS04 a n d
a p p r o p r i a t e i n t e r f e r e n c e f i lt e r s w a s e i t h e r b l u e ( 4 4 0 r i m , 3 5 0 W / c m 2 ) o r g r e e n
( 5 6 0 , 3 0 0 W / c m 2 ) . T h e m o d u l a t e d l i g ht p r o v i d e d b y a 1 5 0 W x e n o n l a m p
a n d a s c an n in g m o n o c h r o m a t o r ( 1 0 n m p a s s b an d ) w as c h o p p e d a t 1 7 H z b y
a r o t a t i n g s e c t o r. T h e m o d u l a t e d b e a m w a s m a i n t a i n e d a t a c o n s t a n t i n t e n s it y
( 5 / ~ W /c m 2 ) b y a t h e r m o p i l e d e t e c t o r i n a s e r v o s y s t e m w h i c h a d j u s t e d a n
o p t i ca l d e n s i t y w e d g e a t t h e e x i t s li t o f t h e m o n o c h r o m a t o r . T h e m o d u l a t e d
c o m p o n e n t o f t h e o x y g e n e v o l u t i o n w a s m e a s u r e d w i t h a l o c k -i n am p l i fi e r.
T h e a m p l i t u d e o f t h e m o d u l a t e d s ig n al w a s f o u n d t o b e li n ea r w i t h t h e in t en s i-
t y o f t h e m o d u l a t e d b e a m u p t o 1 0 W / c m 2 i n t h e p r e s e n c e o f t h e s t r o n g b ac k -
g r o u n d i l l u m i n a t i o n .
esults
T y p i c a l f l u o r e s c e n c e y i e l d c h a n g e s w h i c h o c c u r d u ri n g t h e ir r a d ia t i on o f
P cruentum w i t h g r e e n l i g h t ( 8 0 0 / ~W / c m 2 a t 5 6 0 n m ) a r e s h o w n i n F ig . 1 .
T h e f l u o r e s c e n c e i n c r e a se s r a p i d l y d u r i n g t h e f i r s t f e w s e c o n d s t o a n i n i ti a l
m a x i m u m a n d t h e n d e c a y s m o r e s l o w l y t o a f in a l s t e a d y - s t a te le v el w h i c h is
c h a r a ct e r is t ic o f t h e s t a te p r o d u c e d b y l ig h t a b s o r b e d b y P h o t o s y s t e m I I. ( W e
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e r u e n t u m
cel ls a t 690 nm durLng i~a dia t ion with 5 60 nm l ight
(800 l ~W / em2 a t r o o m
t e m p e r a t u r e T h e s e c o n d
i r radi a t ion wl th 560 n m l ight fon ows th e f~wst af ter
a period of 3 ra in . Dashed l ine , 3 ra in of d a r k n e s s ; s o l i d l i ne , 2 . 5 n~n i r rad ia t ion wi th 440 nm l ight (800
W/cm 2) in the midd le of the 3 ra in period.
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w i ll n o t c o n s i d e r t h e i n it i al f a s t t r a n s i e n t s p i k e in t h e f l u o r e s c e n c e c u r v e w h i c h
o f t e n p r e c ed e s t h e i ni ti al m a x i m u m o r t h e s e c o n d a r y m a x i m u m w h i c h s o m e -
t i m e s f o l l o w s t h e i n it ia l m a x i m u m i n t h e a p p r o a c h t o s t a t e I I. ) W i t h d ar k -
a d a p t e d c e ll s t h e r a t i o o f t h e i n t e n s i t y o f t h e f l u o r e s c e n c e a t t h e i n it i al
m a x i m u m t o t h a t a t t h e f in a l s t e a d y - s t a t e le v e l is t y p i c a l l y 1 . 5 t o 1 . 6 .
S t a t e I I p e r s is t s f o r s o m e t i m e i n t h e d a r k f o l l o w i n g a n i r r a d i a ti o n . T h e
d o t t e d c u r v e i n F ig . 1 s h o w s f l u o r e s c e n c e c h a n g e s d u r i n g a s e c o n d i r r a d i a t i o n
w i t h 5 6 0 n m l i g h t f o l l o w i n g a d a r k p e r i o d o f 3 m i n . T h e s m a ll in i ti a l m a x i m u m
i n d i c a t e s a r a t h e r s m a l l r e t u r n t o t h e d a r k - a d a p t e d s t a t e . T h e h a l f t i m e t o
r e g e n e r a t e t h e d a r k - a d a p t e d s t a t e i s 5 t o 1 0 m i n . H o w e v e r , i f t h e s e c o n d i r r ad i a-
t i o n i s p r e c e d e d b y a 2 . 5 m i n i r r a d ia t i o n w i t h 3 0 0 W / c m 2 o f b l u e l i gh t ( 4 4 0
n m ) , w h i c h h a s v e r y l it t le a c t i o n f o r p h o t o s y n t h e s i s i n t h e s e c e ll s, t h e c o n d i t i o n
f o r t h e h i g h i n i ti a l m a x i m u m is r e g e n e r a t e d e v e n b e y o n d t h e o r ig i na l d a rk -
a d a p t e d s t a t e ( s e e s o l id c u r v e in F i g . 1 ). T h i s s t a t e , r e f e r r e d t o a s s t a t e I b e c a u s e
i t r e s u lt s f r o m l i g h t a b s o r b e d b y P h o t o s y s t e m I , s h o w s a r a t i o o f t h e i n t e n s i ti e s
a t t h e i n it ia l m a x i m u m t o t h e f i n al l e ve l o f a b o u t 1 . 8 a n d a l o n g e r i n d u c t i o n
p e r i o d d u r i n g a s u b s e q u e n t i rr a d i a t io n t o r e a c h s t a t e II . S t a t e I d e c a y s t o t h e
o r ig i n al d a r k - a d a p t e d s t a t e w i t h a h a l f - t im e o f a b o u t 3 m i n . T h u s , t h e d a r k-
a d a p t e d s t a t e i s i n t e r m e d i a t e b e t w e e n s t a t e I a n d s t a t e II b u t i s c o n s i d e r a b l y
c l o s e r t o s t a t e I t h a n t o s t a t e I I. M o s t o f t h e s e c h a r a c t e r is t i c s o f st a t e s I a n d I I
h a v e b e e n d e s c r i b e d p r e v i o u s l y [ 2 - - 4 ] . O u r p u r p o s e h e r e is t o d e t e r m i n e h o w
t h e p h o t o c h e m i c a l p r o p e r t i e s o f t h e c e ll s c h a n g e d u ri n g th e tr a n s i ti o n b e t w e e n
t h o s e s t a t e s .
C e l ls w e r e p l a c e d i n s t a t e I o r s t a t e I I b y i r r a d i a t io n f o r 5 m i n w i t h b l u e o r
g r e en l ig h t a t r o o m t e m p e r a t u r e a n d t h a t c o n d i t i o n w a s fr o z e n i n b y f r e e zi n g
t h e c e ll s t o - - 1 9 6 C d u r i n g c o n t i n u e d i r r a d ia t i o n . E m i s s i o n s p e c t r a o f t h e ce l ls
a t - - 1 9 6 C i n s t a t e s I a n d I I a r e s h o w n i n F i g. 2 w i t h f l u o r e s c e n c e e x c i t a t i o n a t
5 6 0 , 5 0 0 a n d 4 3 5 n m . 5 6 0 n m l ig h t i s a b s o r b e d p r e d o m i n a n t l y b y t h e p h y c o -
b i li s o m e s ; 4 3 5 n m l ig h t is a b s o r b e d p r e d o m i n a n t l y b y c h l o r o p h y l l a n d 5 0 0 n m
l ig h t i s a b s o r b e d b y b o t h s e ts o f t h e p i g m e n t s . T h e e m i s s io n b a n d s a t 6 4 2 , 6 6 2 ,
a n d 6 8 3 n m a re d u e t o p h y c o c y a n i n , a l l o p h y c o c y a n i n a n d a l l o p h y c o c y a n in B
o f t h e p h y c o b i l i s o m e s , r e sp e c t iv e l y , t h e b a n d s a t 6 9 4 a n d 7 5 5 n m a r e d u e t o
t h e a n t e n n a c h l o r o p h y l l o f P h o t o s y s t e m I I a n d t h e b a n d a t 7 1 6 n m i s d u e t o
t h e a n t e n n a c h l o r o p h y l l o f P h o t o s y s t e m I [ 1 1 , 1 3 ] .
I n e a c h o f t h e c a s e s i n F i g . 2 , t h e s t a t e I m i n u s s t a t e I I d i f f e r e n c e s p e c t r u m
( p l o t t e d j u s t a b o v e t h e e m i s s io n s p e c t ra ) s h o w s a g r e at e r y i e l d o f f l u o r e s c e n c e
f r o m P h o t o s y s t e m I I ch l o r o p h y ll ( 6 9 4 n m ) a n d a l lo p h y c o c y a n i n B ( 6 8 3 n m )
i n s t at e I b u t n o d i f f e r e n c e in t h e y i e l d s o f fl u o r e s c e n c e f r o m p h y c o c y a n i n o r
a l l o p h y c o c y a n i n . T h e f l u o r e s c e n c e y i e l d c h a n g e s o f a l l o p h y c o c y a n i n B a r e
k n o w n t o f o l l o w t h o s e o f P h o t o s y s t e m I I c h l o r o p h y ll b e c a u s e o f t h e t ig h t
e n e r g y c o u p l i n g . T h e d a t a i n F i g . 2 s h o w c l e a r l y t h a t t h e f l u o r e s c e n c e y i e l d o f
t h e P h o t o s y s t e m I I p i g m e n t s i s g r e a t e r in s t a t e I t h a n i n s t a t e I I .
Y i e ld s o f e n e r g y t r a n s f er f r o m P h o t o s y s t e m I I to P h o t o s y s t e m I w e r e de t e r-
m i n e d b y m e t h o d s d e v e l o p e d p r e v i o u s l y [ 1 3 ] f o r c e ll s f r o z e n i n s t a te s I a n d I I.
I n b r i ef , m e a s u r e m e n t s w e r e m a d e o f t h e e x c i t a ti o n s p e c t r u m f o r 7 3 0 n m f l u o-
r e s c e n ce a n d o f t h e a b s o r p t i o n s p e c t r u m f o r a d i l u te s u s p e n s io n o f ce ll s ( s ee
F i gs . 3 B a n d 3 E ) . T h e r a t i o s p e c t r a
F73o A
i n F i gs . 3 A a n d 3 D i n d i c a t e t h e
w a 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 la t iv e y i e l d o f t h e 7 3 0 n m f l u o r e sc e n c e . I f t h e
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Fig. 2. L o w t e m p e r a t t w e e m i s s i o n s p e c t r a O f P cruentum c e l l s f r o z e n t o --19 6C in sta te I SI) during
i r r a d l a t i o n w i t h b l u e l i g h t o r f r o z e n i n s t a t e I I ( S I I ) d u r i n g i l T a d i a t io n w i t h g r e e n l ight. A, e x c i t a t i o n a t
5 6 0 nra; B, 500 nm; C, 4 3 5 n m . D i f f e r e n c e s p e c t r a s t a te I - - s t a t e I I a n d r a t i o s p e c t r a s t a t e I / s t a t e II
a r e p l o t t e d a b o v e t h e e r a i s s i o n s p e c t ra ,
7 2 0 n m f l u o r e s c e n c e w e r e a p u r e P h o t o s y s t e m I e m i s s io n w e w o u l d e x p e c t
t h a t t h e m a x i m u m y i e ld o f f lu o r e s c e n c e w o u l d o c c u r a t 6 9 0 n m w h i c h i s
a b s o r b e d s o l e ly b y P h o t o s y s t e m I c h l o r o p h y l l a n d t h a t sh o r te r w a v e le n g t h s
w h i c h e x c i t e P h o t o s y s t e m I a t l e a s t i n p a r t v ia en e r g y t r a n s fe r f r o m P h o t o -
s y s t e m I I s h o u l d s h o w l o w e r y i e ld s o f f lu o r e s c e n c e . O n t h a t b a s is t h e r a t io
s p e c t r a
F 73o A
p r e s e n t s o m e t h i n g o f a n a n o m a l y i n t h a t th e y ie l d o f t h e 7 3 0
n m f l u o r e s c e n c e i s g r e at e r in t h e 5 6 0 n m r e g i o n t h a n i t i s a t 6 9 0 n m b u t t h a t
a n o m a l y i s r e a d il y r e so l v e d b y a n i n s p e c t i o n o f t h e e m i s s io n sp e c t r u m e x c i t e d
a t 5 6 0 n m . I t i s a p p a r e n t t h a t t h e 7 3 0 n m f l u o r e s ce n c e e x c i t e d b y 5 6 0 n m l i g h t
i s n o t a p u r e P h o t o s y s t e m I e m i s s io n b u t a l so i n c l u d e s a s i g n if i ca n t c o n t r i b u -
t i o n f r o m t h e l o n g w a v e l e n g t h t a il o f t h e P h o t o s y s t e m I I e m i s s io n . T h u s in
o r d e r t o p r o c e e d w i t h t h e a n a ly s is w e n e e d t o d e t e r m i n e t h e fr a c t io n o f t h e
5 6 0 n m - e x ~ t e d 7 3 0 n m f l u o r e s c e n c e w h i c h i s d u e s o le l y t o P h o t o s y s t e m I .
T h e 5 6 0 - e x c i t e d e m i s s io n s p e c t r u m c a n b e r es o lv e d i n t o it 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 p a r ts a s w a s d o n e p r e v i o u s ly [ 1 3 ] . T h e l o w
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F i g . 3 . W a v e l e n g t h d e p e n d e n c e o f th e r e la t iv e y i e ld 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 cruentum 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 m i s s io 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 ra
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 m i s s i o n s p e c t ru m e x c i t e d a t 5 6 0 r im , ~ 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 . C e l ls i n s t a t e I ( S I ) o r s t a t e II ( S I I) a s i n d i c a t e d .
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temperature emission spectrum of cells in state II excited at 435 nm can be
taken as a virtually pure Photosystem I emission spectrum. We have deter-
mined that the ratio F716/F693for a pure Photosystem II emission spectrum is
approx. 0.14 [7]. Thus, the d econvoluti on requires that the proper amount of
the pure Photosystem I emission spectrum ZFI be subtracted from the 560
nm-exci ted emission spectru m ZF s6 so tha t the resulting difference spectrum
ZFII has a ratio
FTI6/F693
of 0.14. Figs. 3C and 3F show such deconvolutions
for cells in SI and SII. The relative contributions of Photosystem I and Photo-
system II to the 730 nm fluorescence were then calcul ated by integrating
the product of the deconvoluted emission spectra times the spectral trans-
mission curve of the 730 nm filter used in the measurement over the spectral
passband of th e filter (see Ref. 7). These calculations indicated that 76 of the
730 n m fluorescence was due to Pho tos yst em I in state I and that 85 was due
to Photosystem I in state II. These values allow us to correct the ratio F73o/A
at 560 nm to include only the Photosystem I component of the 730 nm fluo-
rescence. The pro duc t of the values in the first two colum ns in Table I gives
the corrected value for the ratio o f the yields of the Photos ystem I fluo-
rescence at 730 nm excited at 560 and 690 nm (given in the third colu mn) and
this corrected value of the ratio is taken as the maximum yield of energy trans-
fer from Photo system II to Ph otosys tem I [7].
The excitation spectra of the 730 nm fluorescence were measured at the
maximal M level where the Photosystem II reaction centers are all closed
(recall that the cells were frozen to --196C during irradiation) so that 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 no t signi ficant ly dif fer ent f or cells in state I or
state II; both are close to uni ty. However, it is more meaning ful to consider
values of ~T(II-*I)(o} which are obtained when the Photosystem II reaction
centers are all open since that is the condition for optional photosynthesis.
The ratio of
~ T I I - . . I ) M ) /~ 0T I I - ~ I ) o )
will be the same as the ratio of FM/Fo for
Ph otos ys te m II fluo rescen ce so th at the value of ~0T(ii-,i)(o) Can be deter min ed
fr om the value of ~0T(II~I)(M) and t he ra tio F69S M)/Fs93 o). The ratio deter-
min ati on requires t hat cells be frozen in states I and II in t h e dark with the
Photosystem II reaction centers fully open in order to measure the increase of
fluorescence at 693 nm which occurs when the Photosystem II centers are
closed p hoto chemi call y. Cells can be frozen in state II qui te readily if the freez-
ing occurs in the light b ut some dif ficu lty was experienced in tryi ng to freeze
cells in state II in the dark after a short dark period to allow the regeneration
TABLE I
COMPARISON OF FLUORESCENCE AND ENERG Y TRAN SFER PR OPERTIES OF CELLS OF
P C RU EN TU M
AT --196C IN STATE I AND STATE II
560 ~T n --~) o)
F730 FI 730) ~T II--~ I) M) FS93 M)
~F690 F730 F693 o)
7a o
t a t e
I 1.28 0.76 0.97 2.04 0.48
State n 1.10 0.85 0.94 1.54 0.61
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o f o p e n P h o t o s y s t e m I I r e a c t i o n c e n t e rs . S im i l ar p r o b l e m s w e r e r e p o r t e d
p r e v i o u s l y w i t h g r e e n le a v e s [ 1 4 ] . E v e n t h o u g h s t a t e I I i s r e a s o n a b l y s t a b l e i n
t h e d a r k a t r o o m t e m p e r a t u r e , i t a p p e a r s t o e sc a p e q u i t e r a p i d l y to s t a t e I
d u r i n g t h e f r e e z i n g p r o c e s s . T h i s e s c a p e f r o m s t a t e II w h i c h o c c u r s w h e n t h e
t e m p e r a t u r e is l o w e r e d is t h e r e a s o n w h y B r i an t a is e t a l. [ 5 ] c o n c l u d e d t h a t
t h e r e w e r e n o d i f f e r e n c e s i n e n e r g y d i s t r i b u t i o n b e t w e e n s t a t e I a n d s t a t e I I;
t h e y a s s u m e d t h a t t h e y h a d f r o z e n c h l o r o p l a s t s to - - 1 9 6 C i n s t a t e I I w h i l e ,
i n f a c t , t h e c h l o r o p l a s t s w e r e f r o z e n i n s t a t e I. T h e e s c a p e f r o m s t a t e I I m a y b e
e v e n m o r e r a p i d i n c h l o r o p l a s t s t h a n i n w h o l e c e ll s. H o w e v e r , s ta t e I I c a n b e
t r a p p e d i n s p in a c h c h l o r o p l a s t s a t - - 1 9 6 C i f t h e c h l o r o p l a s t s a r e f r o z e n d u r i n g
t h e a c t i n i c i r r a d i a t io n ( K i t a j i m a , M . a n d B u t l e r , W . L . , u n p u b l i s h e d d a t a ) . W i t h
o u r u s u a l f r e e zi n g p r o c e d u r e s w e w o u l d i n v a r ia b l y o b t a i n
P. cruentum
c e l l s i n
s t a t e I w h e n w e t r i e d t o f r e e z e t h e ce l ls i n s t a t e I I i n t h e d a r k . T o o v e r c o m e
t h e s e p r o b l e m s s a m p l e s w e r e p r e p a r e d b y f i l te r i n g ce ll s o f
P. cruentum
o n t o
M i l l i p o r e f i l t e r di s cs t o p r o v i d e v e r y th i n s a m p l e s w h i c h c o u l d b e f r o z e n r a p i d l y
b y p l u n g i n g t h e m d i r e c t l y i n t o l i q u i d n i t r o g e n . W i t h t h e s e r a p i d f r e e z in g p r o c e -
d u r e s w e w e r e a b l e t o f r e e z e i n s t a t e I I a f t e r a s h o r t d a r k p e r i o d ( 4 5 s w a s s u f fi -
c i e n t f o r al l o f th e P h o t o s y s t e m I I c e n t e r s t o o p e n ) s o t h a t t h e r a t i o o f
F693 M)/F693 o)
c o u l d b e d e t e r m i n e d f o r c e ll s i n b o t h s t a t e I a n d s t a t e I I
( T a b l e I ). T h e v a l u e s o f ~ T(II-* X) (o ) w e r e t h e n c a l c u l a t e d b y d i v i d i n g t h e v a l u e s
of ~T II---~I) M) b y t h e r a t io s o f F69S M)/F693 o) ( T a b l e I ). I t is a p p a r e n t t h a t
~ W (.I I- *I )( o) i s s i g n i f i c a n t l y l a r g e r i n s t a t e I I ( 0 . 6 1 ) t h a n i n s t a t e I ( 0 . 4 8 ) . I t w i l l
b e s h o w n i n t h e d i s c u s s i o n ( v i d e i n f r a ) t h a t t h e v a lu e s o f ~ T ( II ~ I) i n T a b l e I
a r e c o n s i s t e n t w i t h a d o u b l i n g o f t h e r a t e c o n s t a n t f o r e n e r g y t ra n s f e r ,
k T ( i X ~ x ) , i n t h e t r a n s i t i o n b e t w e e n s t a t e I a n d s t a t e I I .
W e c a n a l s o d e t e r m i n e t h e r e l a ti v e c r os s s e c ti o n s o f 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, a a n d f i ( w h e r e a + fi = 1. 0 ) , f o r a n y w a v e l e n g t h o f e x c i t a t i o n
[ 1 1 ] . C e ll s w h i c h h a d b e e n b r o u g h t t o s t a t e I o r I I b y i r r a d i a t io n w i t h 4 4 0 o r
5 6 0 n m l ig h t a t r o o m t e m p e r a t u r e w e r e f r o z e n r a p i d l y t o - -1 9 6 C a f t e r a 4 5 - s
d a r k p e r i o d . E x c i t a t i o n s p e c t r a w e r e m e a s u r e d f o r f l u o r e s c e n c e a t 6 9 3 a n d
7 3 0 n m b e f o r e a n y a c t i n ic i r r a d i a t io n ( F o ) , a f t e r a ct i n ic i r r a d ia t i o n w i t h f a r - re d
l ig h t w h i c h c l o s e d t h e P h o t o s y s t e m I r e a c t i o n c e n t e r s ( i. e. , p h o t o o x i d i z e d
P - 7 0 0 ) b u t h a d n o i n f l u e n c e o n t h e P h o t o s y s t e m I I c e n t e r s ( F o ') a n d a f te r
a s a t u r a t i n g i r r a d i a t i o n w i t h w h i t e l i g h t w h i c h c l o s e d a ll o f t h e P h o t o s y s t e m I I
c e n t e r s ( FM ) . P h o t o o x i d a t i o n o f P - 7 0 0 c a u s e d t h 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 t o i n c r e as e 1 5 t o 2 0 % b u t h a d n o e f f e c t o n t h e 6 9 3 n m f l u o r e sc e n c e .
T h e w a v e l e n g t h d i s tr i b u t i o n o f a w a s t h e n c a l c u la t e d b y t h e c o m p u t e r f r o m t h e
e x c i t a t i o n s p e c t r a f o r t h e 6 9 3 a n d 7 3 0 n m f l u o r e s c e n c e m e a s u r e d a t t h e
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a n d M l e v e l s a n d t h e v a l u e o f ~ T(II--~ I)(M ) a c c o r d i n g t o e q u a t i o n s p r e s e n t e d
p r e v i o u s l y [ 7 , 1 1 ] . W e c o n c l u d e f r o m t h e s im i l a ri t y b e t w e e n t h e s p e c t r a o f a
i n s t a t e I a n d s t a t e I I p r e s e n t e d i n Fi g . 4 t h a t t h e c r o s s s e c t i o n s o f 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 d o n o t c h a n g e d u r i n g t r a n s it i o n s b e t w e e n s t a t e s I a n d I I .
T h e a b o v e r e s u l t s o n t h e s p e c t r a l d i s t r i b u t i o n o f a l e a d t o c e r t a i n p r e d i c t i o n s
f o r t h e a c t i o n s p e c t r a o f o x y g e n e v o l u t i o n m e a s u r e d i n s ta t e I an d s t a t e I I.
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e v o l u t io n w a s m e a s u r e d w i t h a w e a k m o n o c h r o m a t i c m o d u l a t e d b e a m . A c ti o n
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c e l ls i n s t a t e I S I ) a n d s t a t e n s I I ) a t - - 1 9 6 C .
F i g . 5 . R e l a ti v e r a t e o f m o 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 w a v e l e n g t h w i 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 . M e a s t ~ e m e n t s w e r e m a d e w i t h a v e r y w e a k 3 - - 5 / ~ W / c m 2 ) m o d u l a t e d m o n o c h r o m a t i c
b e a m s u p e r i m p 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 rc l es ) 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 c e l l s i n s t a t e II c l o s e d c i r c l e s ) .
s p e c t r a f o r th e m o d u l a t e d o x y g e n e v o l u t i o n c o r r e c t e d f o r e q u a l i n c i d e n t
q u a n t u m f l u x a n d n o r m a l i z e d a r e p r e s e n t e d i n F ig . 5 f o r c e ll s h e l d in s ta t e I
a n d s t a t e I I . T h e t w o s p e c t r a w h i c h a r e , i n e s s e n c e , a c t i o n s p e c t r a f o r P h o t o -
s y s t e m I I s h o w n o s ig n i fi c an t d i f f e re n c e s . E n e r g y d i s t r i b u t i o n i n t h e p h o t o -
c h e m i c a l a p p a r a t u s c a n b e d e s c r i b e d in t e r m s o f t w o p a r a m e t e r s , a , w h i c h is a
f u n c t i o n o f w a v e l e n g t h a n d ~ w n -* x), w h i c h is i n d e p e n d e n t o f w a v e l e n g t h . T h e
o b s e r v a t i o n t h a t ~ T I I- ,~ ) o ) c h a n g e s b e t w e e n s t a t e s I a n d I I , w h i l e a d o e s n o t ,
l e ad s t o th e p r e d i c t i o n t h a t t h e a c t io n s p e c t r u m f o r P h o t o s y s t e m I I w h i c h
f o l l o w s t h e w a v e l e n g t h d e p e n d e n c e o f ~ ) s h o u l d b e t h e s a m e in b o t h s t a t e s.
T h a t p r e d i c t i o n is b o r n o u t i n t h e r e s u l ts o f F ig . 5 . F u r t h e r m o r e , w e n o t e t h a t
t h e p r e d i c t i o n , w h i c h w a s b a s e d o n m e a s u r e m e n t s m a d e a t l o w t e m p e r a t u r e ,
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t o s t a t e I I b u t n o t t h e r e v e r s e t r a n s it i o n f r o m s t a t e II to s t a t e I. F i g . 6 s h o w s
t h e r e su l ts o f a s i m il ar s t u d y i n w h i c h l o w t e m p e r a t u r e e m i s s io n s p e c t r a a t t h e
F M le v e l w e r e u s e d a s t h e a s s a y f o r t h e t w o s t a t e s . F i g . 6 A s h o w s t h e
n o r m a l s t a t e I a n d s t a t e I I e m i s s i o n s p e c t r a a s w e l l a s t h e s t a t e I - s t a te I I d i f f e r-
e n c e s p e c t r u m f o r c e ll s w h i c h h a d b e e n i r r a d i a t e d f o r 5 r a in w i t h b l u e l i g h t
s t a t e I) a n d g r e e n l i g h t s t a t e I I) b e f o r e b e i n g f r o z e n t o - - 1 9 6 C w h i l e t h e
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F i g . 6 . E f f e c t s o f D C M U o n t r a n s i t i o n s b e t w e e n s t a t e s I S I ) e n d II S I I ) i n P c r uen t um c e ll s . A , e m i s s i o n
s p e c t r a o f c e ll s i r r a d i a t e d 5 i n i n w i 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 ls i r r a d i a t e d 5 r a i n w i 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 ls i r r a d i a t e d 5 r a i n w i t h g r e e n l i g h t , t h e n 5 r a i n
w i 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 ls i n s t a t e I a n d s t a t e I I . C , e m i s s i o n s p e c t r u m o f c e l ls i r z a d i a t e d 5 r a i n w i t h b l u e l i g h t , t h e n 5 r a i n w i 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 s p ee t~ c a v e r s u s c e l ls i n s t a t e I
e n d s t a t e I I . D , s a m e a s C b u t w i t h 5 p M D C M U a d d e d b e t w e e n t h e b l u e e n d g r e e n ~ m - r a d ia t to n . E , s a m e
a s B b u t w i t h 5 /~M D C M U a d d e d b e t w e 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 m i s s io n s p e c t r u m o f c e ll s
i r r a d i a t e d 5 r a i n w i t h g r e e n l i g h t w i t h 5 p M D C M U 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
w e r e t h e n f r o z e n t o - - 1 9 6 C i n th e g r e e n li g h t.
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i r ra d i a ti o n c o n t i n u e d . T h e t w o s t a te s a r e f u l ly re v e rs ib l e a t r o o m t e m p e r a t u r e
i n t h a t c e ll s w h i c h w e r e i r r a d i a t e d f o r 5 m i n w i t h g r e e n l i gh t a n d t h e n f o r 5 r a in
w i t h b l u e l i g h t b e f o r e b e i n g f r o z e n i n b l u e l i g ht w e r e in s t a t e I d u e t o t h e f i n a l
i r r a d i a ti o n w i t h b l u e l i g h t ( F ig . 6 B ) w h i l e c e ll s w h i c h w e r e i rr a d i a t e d f o r 5 ra i n
w i t h b l u e a n d t h e n 5 m i n w i t h g r e e n b e f o r e b e i n g f r o z e n i n g r e e n l i g h t w e r e in
s t a t e I I ( F ig . 6 C ) . H o w e v e r , i f D C M U w a s a d d e d a f t e r t h e i n i t ia l b l u e i r r a d i a ti o n
b u t b e f o r e t h e g r e e n , t h e c e l ls r e m a i n e d i n s ta t e I ( F ig . 6 D ) . D C M U p r e v e n t e d
t h e t r a n si t io n f r o m s t a t e I t o s t a te I I . O n t h e o t h e r h a n d , i f D C M U w a s a d d e d
a f t e r a n i n i t ia l g r e e n b u t b e f o r e a s u b s e q u e n t b l u e , t h e c e l ls w e r e a l s o i n s t a t e I
( F ig . 6 E ) . D C M U d i d n o t p r e v e n t t h e s t a t e I I t o s ta t e I t r a ns i t io n m e d i a t e d b y
b l u e l ig h t . F u r t h e r m o r e , i f D C M U w a s a d d e d d u r i n g a g r e e n i r r a d i a ti o n o n e
m i n u t e b e f o r e t h e c e ll s w e r e f r o z e n t o - - 1 9 6 C i n t h e g r e e n li g h t, t h e c e ll s
r e v e r t e d f r o m s t a t e I I t o s t a t e I d u r i n g t h e f i n a l m i n u t e o f i r r a d ia t i o n ( F i g. 6 F ) .
I n t h e p r e s e n c e o f D C M U e v e n g r e e n l i g h t a c t s a s P h o t o s y s t e m I l ig h t . A d d i n g
D C M U d u r i n g a b l u e i r r a d i a ti o n h a d n o e f f e c t o n t h e fi n a l s t a t e ( d a t a n o t
s h o w n ) . T h u s , i t is c l ea r t h a t D C M U b l o c k s th e P h o t o s y s t e m I I m e d i a t e d s t a t e I
t o s t a t e I I t ra n s i t io n b u t h a s n o e f f e c t o n t h e P h o t o s y s t e m I m e d i a t e d s t a te I I
t o s t a t e I t r a n s i ti o n .
i s c u s s i o n
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t h e c o n t e x t o f t h e t r i p a rt i t e m o d e l in d i c a t e d t h a t t h e p h o t o -
c h e m i c a l a p p a r a t u s w a s c o m p r i s e d o f r e l at i ve l y l ar g e P h o t o s y s t e m I u n i t s w h i c h
c o n t a i n e d a p p r o x i m a t e l y 9 5 o f t h e c h l o r o p h y l l a n d s m a ll P h o t o s y s t e m I I
u n i t s w h i c h c o n t a i n e d t h e r e m a i n in g 5 o f t h e c h l o r o p h y l l an d t h a t t h e l ar g e
p h y c o b i l i s o m e s t r a n s f er r e d t h e ir e x c i t a t i o n e n e r g y a l m o s t e x c l u s iv e l y t o t h e
s m a ll P h o t o s y s t e m I I u n i t s [ 1 1 ] . I n a d d i t i o n , t h e y i e l d o f e n e r g y t r a n s fe r f r o m
P h o t o s y s t e m I I t o P h o t o s y s t e m I w a s f o u n d t o b e q u i t e hi g h r a ng i ng f r o m
v a lu e s o f a b o u t 0 . 5 0 w h e n t h e P h o t o s y s t e m I I r e a c ti o n c e n t er s w e r e o p e n t o
0 . 9 5 w h e n t h e c e n t e r s w e r e c l o s e d [ 1 3 ] . R e c e n t l y , i t w a s s h o w n i n a s t u d y o f
c h r o m a t i c a d a p t a t i o n i n P .
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[ 7 ] t h a t t h e s e p h o t o c h e m i c a l p r o p e r t i e s
a r e c h a r a c t e r i s t i c o f c e ll s g r o w n i n l ig h t a b s o r b e d p r i m a r i l y b y t h e p h y c o -
b i l i s o m e s w h i l e c e ll s g r o w n i n l i gh t a b s o r b e d p r i m a r i l y b y c h l o r o p h y l l m a k e
m u c h l ar g er P h o t o s y s t e m I I u n i t s w h i c h c o n t a i n a p p r o x . 4 0 o f t h e c h l o r o -
p h y l l a n d w h i c h t r a n s f e r e n e r g y l e ss e f f i c i e n t l y t o P h o t o s y s t e m I . T h e c e l ls u s e d
i n t h e p r e s e n t s t u d y w e r e o f t h e o ri g in a l t y p e w h i c h s h o w e d v e r y s m a ll P h o t o -
s y s t e m I I u n i t s w i t h h i g h p r o b a b i l i ti e s f o r e n e r g y t r a n s f e r t o P h o t o s y s t e m I .
T h o s e c h a r a c t er i st i c s a re c o n f i r m e d b y t h e l a rg e va l u e s o f ~ ( a p p r o a c h i n g u n i t y )
a t 4 3 5 n m a n d t h e l o w v a l u e s o f ~ ( a p p r o a c h in g z e r o ) in t h e 5 6 0 n m r e g io n
( s ee F ig . 4 ) a n d b y t h e h i g h y i e l d s f o r e n e r g y t r a n s f e r f r o m P h o t o s y s t e m I I
t o P h o t o s y s t e m I ( T a b l e I ) .
M e a s u r e m e n t s o f th e w a v e l e n g t h d i s t r i b u t i o n o f a , d i d n o t i n d i c a t e a n y
s i g n i fi c a n t c h a n g e s i n t h e c r o s s s e c t i o n s o f t h e p h o t o s y n t h e s i s i n t h e t r a n s i t i o n
b e t w e e n s t a t e I a n d s t a t e I I. H o w e v e r , t h e t r a n s it i o n f r o m s t a t e I t o s t a te I I
w a s f o u n d t o b e a c c o m p a n i e d b y a d e c r e a s e i n t h e f l u o r e s c e n c e y i e l d o f P h o t o -
s y s t e m I I , a n i nc r e as e i n t h e m i n i m u m y i e l d o f e n e r g y t ra n s f e r f r o m P h o t o -
s y s t e m I I t o P h o t o s y s t e m I , ~ T ~H ~ I) Co ), a n d a d e c r e a s e in t h e r a t i o o f FM/Fo
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f o r P h o t o s y s t e m I I f l u o r e s c e n c e. I n o r d e r t o r e la t e t h o s e o b s e r v a ti o n s to
c h a n g e s i n f u n d a m e n t a l p h o t o c h e m i c a l p a r a m e t e r s w e w i l l a d o p t t h e s i m p l i -
f ie d b i p a rt i te f o r m u l a t i o n o f t h e t ri p a r t it e m o d e l [ 1 6 ] . A c c o r d i n g t o t h a t
f o r m u l a t i o n :
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is t h e p r o b a b i l i t y t h a t e x c i t a t i o n e n e r g y i n a P h o t o s y s t e m I I u n i t
w i ll b e t r a p p e d b y t h e r e a c t i o n c e n t e r c h l o r o p h y l l a n d ~ t x i i s t h e p r o b a b i l i t y
t h a t t h e e n e r g y t r a p p e d b y t h e c h l o r o p h y l l o f a c l o se d P h o t o s y s t e m I I r e a c t i o n
c e n t e r w i ll b e r e t u r n e d t o t h e a n t e n n a c h l o r o p h y l l o f t h a t u n i t . W e w ill as s u m e
f o r s i m p l i c i t y t h a t t h e r e a r e n o f l u o r e s c e n t o r n o n r a d i a t i v e d e c a y l o s se s a t t h e
r e a c t i o n c e n t e r c h l o r o p h y l l s o t h a t ~ t i i = 1 .0 . T h e s u m o f t h e p r o b a b i l it i e s o f
a ll o f t h e d e e x c i t a t i o n p r o c e s se s in t h e a n t e n n a c h l o r o p h y l l o f P h o t o s y s t e m I I
m u s t a d d u p t o u n i t y ; i .e . , ~ i + ~ D I I + ~ Z~ I + S T ( I I ~ I ) = 1 . 0 w h e r e t h e s u b -
s c r ip t s i n d i c a t e f l u o r e s c e n c e , n o n r a d i a t i v e d e c a y , tr a p p i n g b y t h e r e a c t i o n
c e n t e r c h l o r o p h y l l a n d e n e r g y t r a n s f e r t o P h o t o s y s t e m I , r e sp e c ti v e ly . T h u s ,
F M 1 SFII 4 ~ / D I I 4 ~ T I I + ~ T ( I I ' - ~ I )
F o 1 - - ~ / T I i ~ F I I [ ~ ) D I I + ~ JT ( I I' -* 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 )
F o k F l I 4 k D l l 4 -
k T l l . . ~ l )
I f w e a s s ig n r el at iv e v a l u e s o f k F i i + k m i ) : k T i x : k z i x- ~l ) o f 5 : I 0 0 : 9 0 f o r
cells in state I we calculate theoretical valu es wh ic h are in reasonably go od
a g r e e m e n t w i t h t h e e x p e r i m e n t a l d a t a o b t a i n e d i n s t a te I : F M F o = 2.05 vs
the me as ur ed value of 2.04 in Table I), ~0T II~I) o) 0.46 VS. 0.48) and
~0T IX~I) M) = 0.95 VS. 0.97). M os t of t he exper iment al data for the cells in
s t at e I I c a n b e a c c o m o d a t e d q u i te w e l l if w e a s s u m e o n l y t h a t t h e re la ti ve a l u e
of kz ii-.~) increases fr om 90 to 18 0 in the transition fr om state I to state If.
In that ca se in st ate If, F M F o = 1 . 5 4 v s . t h e m e a s u r e d v a l ue o f 1 . 54 ) ,
~r H-*i) o) = 0.63 vs. 0.61) an d ~T II-~i) M)= 0.97 VS. 0.94) . Ho we ve r, the
s t at e I / st a te I I r a t io o f t h e 6 9 3 n m f l u or e s ce n c e a t t h e F M l e ve l m a y n o t b e
p r e di c t ed a d e q u a t e l y b y a s s u m i n g t h a t o n l y kT i I- *i ) c h a ng e s . T h e m e a s u r e d
value of that ratio wa s 1.5 + 1.0 while the value predicted f ro m the relative
v a l ue s o f r a t e c o n st a n ts i s 1 . 9 5 . T h e m e a s u r e d v a l ue c o u l d b e t o o l o w b e c a u s e
o f o ve rl ap f r o m t he 6 8 0 a n d 7 1 6 n m b a n d s in t he m e a s u r e m e n t a t 6 9 3 n m
but such effects are pro bab ly no t sufficient to ac co unt for the discrepancy. It
m a y b e t h a t o n e o f t h e o t h e r r a t e c o n st a n ts i n t h e s y s t e m s u c h a s k T i i a l s o
c h a n g e s d u r i n g t h e t r a n si t io n f r o m s t at e I t o s t a t e If. l l o f t h e e x p e r i m e n t a l
d a t a c a n b e p r e di c t ed q u i te c lo s e ly b y a s s u m i n g c h a n g e s i n v a r io u s p a i rs o f ra t e
c o n s t a n t s b u t i n e a c h c a s e o n e m e m b e r o f t h e p a ir m u s t b e k T ( i i . _ . i ) . W e c a n
s t a t e u n e q u i v o c a l l y f r o m o u r d a t a t h a t t h e t r a n s i ti o n f r o m s t a t e I t o s t a t e I I
is a c c o m p a n i e d b y a m a j o r i n c r e a s e ( 6 0 - - 1 0 0 ) i n k T (i i- *i ) b u t w e c a n n o t a s s er t
t h a t t h is i s t h e o n l y p h o t o c h e m i a l p a r a m e t e r t o c h a n g e . W e a s s u m e t h a t t h e
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increase in
k T i i - + l )
reflects a closer physical association betwe en Photosystem I
and Pho tosyst em II units.
It has been suggested tha t 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 thylakoi ds 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 that such correlations are probably over-
simplifications. In the presence of DCMU, we would expe ct that Photosystem I
could p roduce ion gradients by cyclic electron transport th at were smaller but
in the same direction as those prod uced 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 b ut we would not expec t Photosystem I to
actively stimulate that conversion. The fact that Photosystem I activity does
stimulate the transition f rom 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]) that some component or components
between Photosystem II and Photosystem I must be reduced in order for
state II t o occur and th at state I prevails when those compone nts 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 impo rtant role in these phenomena.
cknowledgements
This work was supported by a National Science Foundation grant, PCM
79-03987.
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