Primary processes in isolated photosynthetic bacterial reaction centres from Chloroflexus aurantiacus studied by picosecond fluorescence spectroscopy

B i o c h i m i c a et Bioph)'~ica A c t a , 11198 ( 1901 ) 1- 12

]

~i 1991 Elsevier Science Publishers B.V. All right~ rc~c~,~'d 0 ( I Q 5 - 2 7 2 8 / 0 i /$tt3.50

B B A B I O 435(17

Primary processes in isolated photosynthetic bacterial reactior centres from Chloroflexus aurantiacus studied by picosecond fluorescence spectroscopy *'** Mare G. Miiller, Kai G r i e b e n o w and Alfred R. Holzwarth ,~lax-Planck-lnstitut f l i t S t r a h l e n c h e m i e . Miilh,'im a . d R u h r ~(;ermap~v J

(Received Ih May I q q l )

Key ~,.ords: Ph<-losynlhcsis; Bacterial reaction center: Picosecond spectroscopy: |:Iuorcs~:enc~.-: (('. auraettt~z( t,, )

The s t a t i o n a r y a n d time-resolved fluorescence emission spectra of reaction eentres (RCs) isolated from the thermophilJc phototrophic b a c t e r i u m Chloroflexus aurantiacus s t r a i n Ok-70-fl have been studied. Several lifetime c o m p o n e n t s in the picosecond a n d n a n o s e c o n d time range have been resolved at room temperature. A short-lived approx. 3 ps c o m p o n e n t is related to a n energy-transfer process from the excited accessory BChl-a L to the special p a i r P. This is d e m o n s t r a t e d by the existence o! positive a n d negative a m p l i t u d e s of this c o m p o n e n t , d e p e n d i n g on the emission wavelength. A c o n f o r m a t i o n a l relaxation of P * c a u s i n g a b a t h o c h r o m i e shift is excluded for this time c o n s t a n t due to the absence of this c o m p o n e n t a t a n excitation wavelength of 850 n m (P) and also by the t e m p e r a t u r e d e p e n d e n c e of the s t a t i o n a r y fluorescence. Two f u r t h e r short-lived c o m p o n e n t s with positive amplitades a n d lifetimes of a b o u t 7 ps a n d about 18 ps were also resolved. Their decay-associated spectra (DAS) a n d emission m a x i m a a t about 9 1 0 - 9 2 0 n m are similar, showing that their origin is fluorescence from P * . T h e i r relative a m p l i t a d e s change strongly, d e p e n d i n g on the excitation intensity. They represent the charge s e p a r a t i o n times of open RCs (7 ps from P * H Q A ) a n d closed RCs (18 ps from P*HQA-). The time c o n s t a n t for open RCs is in full a g r e e m e n t with the value reported recently from t r a n s i e n t a b s o r p t i o n m e a s u r e m e n t s (Becker et al. (1991) Biochim. Biophys. Acta 1057, 299-312). The presence of "heterogeneity" in the p r i m a r y rates of charge separation in RC p r e p a r a t i o n s proposed recently is discussed, it is suggested that the "heterogeneity" reported by other a u t h o r s is possibly a n o p e n / c l o s e d RC heterogeneity.

Introduction The primary processes of energy transfer and charge separation in isolated reaction centres (RCs) of photosynthetic bacteria have recently been the subject of intense investigation. With very tkzw exceptions, most

* This work will be part of the Ph.D. theses of M.G M. and K_(i. at lhc Heiarich-l:leine-Universit~it, Diisseldorf A p r e l i m i n a r y account of part of th¢,~ results has b e e n published in Ref. I. ** D e d i c a t e d to Professor Kurt Schaffner on the occasion of his 601h birthday. Abbreviations: SPT, single-photon-timing; 5 P h e o . b a c t e r i o p h e o phytin; BChl, bacteriochtorophyll: L D A O , lauo'ldimcthylamin¢-Noxide. DAS, decay-assa~Oated s p e c t r u m : PMS. phenaTine methosulfate: RC, reaction centre: P A G E . polyacq,'tamide gel d e c trophoresis; IE. ion-exchange. C o r r e s l ~ m d c ' l c c : A.R. t l o l z ~ a r t h . Max-Planck-lnstitut ffir Strahlcnchemic. Stiftslrasse 3 4 - 3 6 . D-4330 M i d h e i m a.d. Ruhr. G e r m a n y .

studies have c o n c e n t r a t e d on the R('s of purple bacteria, notably those of Rp~: ttridL-, and Rb. sptuwroides (t~r reviews see Refs. 2. 3). whose X-ray structures have been d e t e r m i n e d v,,ith high resolution [4-6]. in contrast, an X-ray structure is not available for RCs ol the green bacterium Cttlon~fl~:rt~ ,mranttacus. From compari'~m of the protein s e q u e n c e , as ~,'ell as from spectroscopic studies it ts believed, however, thai l~oth purple bacterial RCs and RCs from C. aurantiact/x have a very similar structure ( 7 - 9 k In both types of RC the primary electron d o n o r is the first excited singlet state of a BChi-a dimer, d e n o t e d P*. This similarity extends t o the pre.,~ence of two branches of pigments. d e n o t e d L and M. It is generally assumed that onty the L-branch is active in electron transfer [10,11]. There exists an important difference between purple bacterial RCs a n d C. aurannacus RCs with respect to pigment composition. While. in addition to P. the former contain one BChl-a m o n o m c r (aLso called accessor) BChl) and one BPheo each per branch, the latter contain a

B('hl-a m o n o m e r ,,nly in the L-branch. while in the M - b r a n c h the B('hl-a m o n o m e r is r e p l a c e d by a BPheo-a pigment in the c o r r e s p o n d i n g position [ 12,13]. Thus, C. a u r a n t i a c u s RCs contain a l t o g e t h e r t h r e e B P h c o molecules and only one BChl-a m o n o m e r . This difference r e n d e r s RCs o f C. a u r a n t i a c u s particularly interesting to study, because any c h a n g e s o c c u r r i n g in the BChl-a m o n o m e r b a n d d u r i n g e l e c t i o n transfer steps can be i n t e r p r e t e d more easily in view of the lack o f o v e r l a p with the a b s o r p t i o n o f the s e c o n d BChl-a m o n o m e r p r e s e n t in p u r p l e bacterial RCs [12]. Basically, all of the ultrafast studies on isolated RCs to d a t e have been carried out e m p l o y i n g f e m t o s e c o n d / p i c o s e c o n d transient a b s o r p t i o n techniques. F r o m these m e a s u r e m e n t s t h e r e exists g e n e r a l a g r e e m e n t that the p r i m a r y charge s e p a r a t i o n step in p u r p l e b a c t e r i a l RCs occurs with a lifetime of approx. 3 ps at r o o m t e m p e r a ture (for a review see Ref. 3), while this process is significantly slower, i.e., 7 ps, in RCs o f C. a u r a n t i a c u s [Ill]. in both types o f R C the p r i m a r y c h a r g e s e p a r a t i o n s p e e d s up significantly at low t e m p e r a t u r e s [10,14,15]. C o n s i d e r a b l e controversy exists in the l i t e r a t u r e as to the n a t u r e of this p r i m a r y e l e c t r o n - t r a n s t e r step. A c cording to o n e model, also known as the superexchange m e c h a n i s m [14,16,17], the e l e c t r o n is transferred in a single step directly from P* to BPheot.. In contrast, b a s e d on the o b s e r v a t i o n o f a w e a k ultrafast c o m p o n e n t o f about 1 ps in a d d i t i o n to the approx. 3 ps c o m p o n e n t , a consecutive two-step e l e c t r o n t r a n s f e r has b e e n invoked for p u r p l e bacterial R C s [18]. T h e first s t e p o f about 3 ps in this m o d e l was assigned to e l e c t r o n transfer from P* to m o n o m e r i c BChlL, while the ~ ' s t e r c o m p o n e n t was a t t r i b u t e d to a s e c o n d a r y elec~ ~n transfer s t e p from B C h I [ to B P h e o L. Such a mechanism, called the 'consecutive two-step m o d e l ' , had b e e n p r o p o s e d earlier, though b a s e d on experim e n t s with 32-ps-wide laser pulses [19,20], which were, however, c o n s i d e r e d to provide insufficient time resolution [18]. A third i n t e r p r e t a t i o n a s s u m e s a h e t e r o geneity in the rate constants o f the p r i m a r y electrontransfer step which would lead to a d i s t r i b u t i o n of lifetimes c e n t e r e d a r o u n d 3 ps [15]. A c c o l d i n g l y , the approx. 1 ps c o m p o n e n t would r e p r e s e n t p a r t o f this distribution for the direct P * - - - , B P h e o t electrontransfer step. T h e electron transfer kinetics for RCs o f C. a u r a n t i a c u s have been m e a s u r e d by several g r o u p s and in principle the s a m e controversy with respect to the a p p r o p r i a t e m o d e l s exists as for p u r p l e b a c t e r i a l RCs. Shuvalov et al. [21] p r o p o s e d the consecutive two-step m o d e l with a s e c o n d a r y e l e c t r o n t r a n s f e r s t e p o f a b o u t 3 ps, although again using pulses o f 32 ps width. In contrast, B e c k e t et el. [22] f o u n d no evidence for an i n t e r m e d i a t e B C h I [ state in transient a b s o r p t i o n , nor did they observe a 3 ps c o m p o n e n t . R a t h e r , they rep o r t e d a h e t e r o g e n e i t y in the p r i m a r y c h a r g e s e p a r a -

tion time which b e c a m e p r o m i n e n t at low t e m p e r a tures. A t rtx~m t e m p e r a t u r e , the p r i m a r y c h a r g e s e p a ration s t e p h a d a lifetime o f 7 ps and h e t e r o g e n e i t y was insignificant. A similar h e t e r o g e n e i t y in the r a t e of t h e p r i m a r y electron-translfer s t e p was also r e p o r t e d in two o t h e r studies at cryogenic t e m p e r a t u r e s and has b e e n r e l a t e d to a h e t e r o g e n e i t y in the R C p r e p a r a t i o n s found in various c h r o m a t o g r a p h i c s e p a r a t i o n p r o c e d u r e s [23,24]. in view of t h e o n - g o i n g c o n t r o v e r s y about t h e n a t u r e of the p r i m a r y e l e c t r o n t r a n s f e r step(s) and t h e possible h e t e r o g e n e i t y in its rate, it m a y b e useful to a p p l y an alternative m e a s u r i n g t e c h n i q u e in a d d i t i o n to t h e t r a n s i e n t a b s o r p t i o n t e c h n i q u e s used so far. T h e p r e s e n t study of p i c o s e c o n d f l u o r e s c e n c e kinetics of R C s f r o m C. a u r a n t i a c u s has two m a j o r aims. M e a s u r i n g the w a v e l e n g t h - r e s o l v e d f l u o r e s c e n c e decay u p o n selective excitation in two we!l-defined a b s o r p t i o n b a n d s (i) we a d d r e s s the possible origin(s) o f a h e t e r o g e n e i t y in the p r i m a r y e l e c t r o n t r a n s f e r rate a n d (if) we p r o vide f u r t h e r e v i d e n c e for an ultrafast e n e r g y t r a n s f e r p r o c e s s upon excitation of the BChl-at. m o n o m e r . M a t e r i a l s and Metl:ods

Cell growth and m e m b r a n e isolation have b e e n c a r r i e d out as previously d e s c r i b e d [25]. M e m b r a n e s w e r e a d j u s t e d to an a b s o r b a n c e of a b o u t 1 3 / c m a t 865 n m a n d w e r e s t o r e d at - 8 0 ° C . A f t e r i n c u b a t i n g t h e s e m e m b r a n e s with 0.7% L D A O (1 h at 4°C u n d e r slow stirring) d e b r i s and c h l o r o s o m e s w e r e r e m o v e d by c e n trifugation at 45 000 r p m for 1.5 h in a B e c k m a n n TiT0 rotor. T h e s u p e r n a t a n t , c o n t a i n i n g R C s , B 8 0 6 - 8 6 6 a n t e n n a c o m p l e x e s [26], o t h e r c o n t a m i n a t i n g p r o t e i n s , a n d free pigments, was s u b j e c t e d to a D E A E - S e p h a c e l ( P h a r m a c i a ) c o l u m n (300 x 26 m m ) e q u i l i b r a t e d with 20 m M Tris-HCI ( p H 9 . 0 ) / 0 . 3 % L D A O . F r e e p i g m e n t s were r e m o v e d by washing b u f f e r (20 m M T r i s - H C l b u f f e r ( p H 8 . 0 ) / ( I . 3 % L D A O ) . T h e c o l u m n was develo p e d with i n c r e a s i n g NaCI c o n c e n t r a t i o n s in the s a m e buffer. T h e R C s w e r e e l u t e d with 60 m M NaCI. T h i s s t e p was r e p e a t e d with a s m a l l e r c o l u m n u n d e r the s a m e c o n d i t i o n s except that 1% L D A O was p r e s e n t in the washing b u f f e r while the R C s were e l u t e d with buffer 0.1% L D A O . T h e A 2 ~ : A813 a b s o r b a n c e r a t i o o f isolated R C s was in all cases no g r e a t e r t h a n 1.7 a n d o f t e n b e l o w 1.5. C o l o u r e d c o n t a m i n a t i o n s w e r e a b s e n t a f t e r this purification. P A G E ( s a m e c o n d i t i o n s as d e scribed in Refs. 27, 28) s h o w e d t h e p r e s e n c e o f a n o n c o l o u r e d p r o t e i n with a m o l e c u l a r weight o f a b o u t 10000. T h e relative m o l e c u l a r weights o f t h e L a n d M subunits a n d the intact c o m p l e x w e r e d e t e r m i n e d to be a b o u t 27 000, 29 000 a n d 56 000, respectively. T h e s a m ple was c h e c k e d for c h e m i c a l h o m o g e n e i t y in a d d i t i o n by gel-filtration a n d analytical i o n - e x c h a n g e ( I E ) c h r o m a t o g r a p h y . T h e result o f the gel-filtration e x p e r i m e n t

(already described in (Ref. I) showed no heterogeneity of RCs samples, in a g r e e m e n t with native P A G E . Analytical I E - c h r o m a t o g r a p h y on a M o n o - Q 5 / 5 colu m n (Pharmacia) also gave n o indications for a R e heterogeneity. T h e activity tff the isolated RCs was tested by b l e a c h i n g of P as already dccribed [1]. For RCs used for fluorcscencc m e a s u r e m e n t s , more than 95% of the P ba~d was b l e a c h e d with potassium fcrricyanide (5 raM). T h i s excludes any significant contamin a t i o n with the B806-866 a n t e n n a complex. Q u i n o n e c o n t e n t u n d e r o u r isolation c o n d i t i o n s was > 90%, probably a p p r o a c h i n g i(X)% as m e a s u r e d by laser-ind u c e d optoacoustic e x p e r i m e n t s c o m p a r i n g closed (PHQ~,) a n d o p e n ( P H Q A) RCs ( u n p u b l i s h e d data). Prior to fluorescence m e a s u r e m e n t s the RCs were d i l u t e d with 20 m M Tris-HCI ( p H 8 . 0 ) / 0 . 1 % L D A O to an a b s o r b a n c e of 0 . 5 - 1 . 0 / c m at 813 nm. Samples c o n t a i n e d sodium ascorbate (10 m M ) a n d PMS (1(I p,M) for m e a s u r e m e n t . Higher ascorbate c o n c e n t r a tions (up to 50 m M ) had n o effect on the kinetics. T h e sample was purged with n i t r o g e n prior to a n d d u r i n g m e a s u r e m e n t s in o r d e r to keep oxygen c o n t e n t low. RCs were p u m p e d with a G i l s o n peristaltic p u m p (fast s p e e d a b o u t 20 m l / m i n ) from a reservoir that was kept in the dark to o b t a i n o p e n RCs (QA oxidizcd) and with m i n i m a l p u m p i n g speed ( a b o u t 2 m l / h ) for closed RCs ( q u i n o n e QA reduced). In the latter case the RCs we,e closed photochemically by the fluorescence e x c i t a t i o l b e a m . I n t e r m e d i a t e l a s e r i n t e n s i t i e s ( a b o u t 20 r o W / r a m z) at high p u m p i n g speed were used to achieve partially closed RCs. F l u o r e s c e n c e decays were m e a s u r e d by the singlep h o t o n - t i m i n g (SPT) t e c h n i q u e employing picosecond excitation pulses ( F W H M a b o u t 9 - 1 2 ps) from a Spectra Physics mode-locked and cavity-dumped dye lascr system (repetition rate 4 MHz). The fluorescence was d e t e c t e d by a c o m b i n a t i o n of a m o n o c h r o m a t o r ( D H 10 N I R , J o b i n - Y v o n , b a n d w i d t h a b o u t 12 n m ) a n d a fast m i c r o c h a n n e l plate d e t e c t o r (R2809U-05. H a m a matsu, 6 /xm c h a n n e l d i a m e t e r , S-! photocathode). The width of the a p p a r a t u s f u n c t i o n as m e a s u r e d by the S P T - t e c h n i q u e was a b o u t 30 ps ( F W H M ) using a resolution per c h a n n e l of 2.0 ps or 3.0 ps. G e n e r a l l y , 20000 and often up to 30000 c o u n t s were a c c u m u l a t e d in the peak c h a n n e l of each individual decay to achieve a high S / N ratio. T h e fluorescence decays were analyzed either by single-decay analysis or by global analysis m e t h o d s e m p l o y i n g a sum of e x p o n e n t i a l s as model f u n c t i o n in e i t h e r case [29]. Quality of the fits was j u d g e d by individual o r global gZ-values as well as by weighted residual plots. We have shown earlier [26] by m e a s u r e m e n t s o n various systems that lifetimes in the 2 - 3 ps r a n g e can be resolved u n d e r o u r c o n d i t i o n s with an a p p a r a t u s f u n c t i o n of about 30 ps. W e also verified by analysis of simulated decay d a t a which had the corn-

--4.1

|

3 exponentiata

X= = 1.170

I

,~ exponentiam

X z = 0.998

.3.2

_3.21

1"~: ~'2:

5

3.3 pll 13,2 pa

1"4: 974.0 pS

m

0

I

J

RI: 0.827 I R2: 0.153 I

R4:

-

Time, ns Fig. I. Single-dcca3,analysis ot a |luorescence decay (a ,,,, = gill) nm, At, m = 9()(1 rim) of R('s from Rh..~phaer~ide~. The rc.,,idual plots (upper and middle frame) are fi~r f;t~. v,ithout and with a 3.3 ps component. The main figure (lower frame) shows the ;Ipp[Ir[ltUS response function (dashed) and the superimposed measured and fitted fluorescence decay (full line) tm a semik~garithmicscale.

plexity a n d S / N ratio of o u r experiments that the resolution of close-lying lifetime c o m p o n e n t s is possible u n d e r conditions similar to those found for RCs. Fig. ! p r e s e n t s the results of a single-decay analysis on the m e a s u r e d fluorescence decay of isolated RCs from the purple b a c t e r i u m Rh. ~phaeroides. The wellcharacterized kinetics of purple bacterial RCs provides the closest possible analogous m e a s u r i n g c o n d i t i o n s for characterizing the resolution of o u r apparatus. It is clear from thc residuals plots that a lifetime of 3.3 ps is resolved which is -,cry close to the 3.5 ps lifetime observcd by Holzapfel et al. [18,30] by femtosccond time-resolved absorption spectroscopy. This d e m o n strates o u r ability to resolve an ultrafast c o m p o n e n t even within a complex kinetics. Fitting the decay without the short-lived 3.3 ps c o m p o n e n t le~ds to strong deviations in the residuals. F u r t h e r m o r e we m e a s u r e d a buffer solution without RCs but c o n t a i n i n g sodium ascorbate a n d P M S u n d e r the excitation a n d detection c o n d i t i o n s used for the m e a s u r e m e n t s with RCs. Only two counts were detected in the peak c h a n n e l after I h a c c u m u l a t i o n time. T h e kinetics reported for RCs in this m a n u s c r i p t requires analysis by five or six exponentials if analyzed over a long time-window. W c are well a ~ a r c of the problems related to such m u l t i c x p o n e n t i a l analysis. However, we should like to stress that we are using optimal global data analysis p r o c e d u r e on data of very high S / N ratio. F u r t h e r m o r e , as a general procedure

in critical cases, we regularly p e r f o r m analyses on simulated data of the same complexity and S / N ratio of the expcrimcntal data in o r d e r to assure ourselves thai the c o m p o n e n t resolution u n d e r the respective conditions is indeed possible. It should also bc n o t e d that five or six exponcntials are required only if the data arc analyzed over a long t i m e - r a n g e of several nanoseconds• In view of the fact that the lifetimes of most interest in this article are those in the range of 3 ps to about 55 ps, an analysis range of about 200 ps is sufficient. In this case the n u m b e r o f lifetimes n e e d e d for a good fit can be r e d u c e d to 3 or 4. Naturally the long lifetimes which have a very small a m p l i t u d e are not d e t e r m i n e d properly in this case• We thus perf o r m e d analyses over both short a n d long time windov,s and verified that we obtain the same results for the short-lived c o m p o n e n t s wilhin the e r r o r limits.

F"

25

h x~ =

7

o x v

20

Ic

15

811 nm

RT

20K

0

Io

"N c "E

.5 I

0 821

I

84-0 860

1

I

I

I

880 900 920 940 W o v e l e n g t h, n m

I

960

980

Fig. 2. Corrected fluorescence emis, n spectra of RCs from (7.

attrantiacus (,I.~,~ =811 nm) at room 'emperatt,re (RT) and 20 K. The two spectra are normalized to their maxima. The RCs were in the closed state. The maximal intensity in the low temperature spectrum is larger by a factor of more than 2 as compared to the r(vom temperature emission.

Results

Stationary fluorescence spectra Stationary fluorescence spectra of RCs have b e e n m e a s u r e d at room t e m p e r a t u r e (298 K) and at 20 K using a cw laserdiode (CQL-54, Philips, A =-811 nm), as the excitation source. T h e liquid sample was not p u m p e d and thus at the excitation p o w e r o f ab o u t 20 m W the R C s were closed both at r o o m t e m p e r a t u r e and at 20 K. Fig. 2 shows the n o r m a l i z e d c o r r e c t e d emission spectra a n d d e m o n s t r a t e s the p r o n o u n c e d t e m p e r a t u r e d e p e n d e n c e of t h e stationary emission. T h e total f l u o l e s c e n c e is substantially higher (by a factor of m o r e than 2) at low t e m p e r a t u r e as c o m p a r e d to r o o m t e m p e r a t u r e , despite the lower absorption caused by band shift and narrowing. W e have n e v e r t h e less n o r m a l i z e d o u r spectra b e c a u s e o f c h an g es in optical p r o p e r t i e s o f the s a m p l e at low t e m p e r a t u r e .

D u e to strong s c a t t e r i n g o f the s a m p l e s at low t e m p e r a t u r e s t h e f l u o r e s c e n c e s p e c t r u m co u l d be t a k e n only ab o v e 850 nm. T h e increase in emission intensity o n the blue e d g e (below 830 n m ) in t h e r o o m t e m p e r a t u r e s p e c t r u m arises d u e to the small a m o u n t o f f r e e B C h l which has a high f l u o r e s c e n c e q u a n t u m yield as has b e e n verfied previously b o t h by excitation s p e c t r a a n d lifetime m e a s u r e m e n t s ( d at a not shown; [26]).

Time-resoh~ed fluorescence spectra F l u o r e s c e n c e decays o f C. aurantiacus R C s h a v e b e e n m e a s u r e d at r o o m t e m p e r a t u r e varying e x c i t a t i o n w a v e l e n g t h , laser intensity, as well as s a m p l e p u m p i n g speed. F o r all t h ese c o n d i t i o n s t i m e - r e s o l v e d s p e c t r a have b e e n taken typically based on decay m e a s u r e m e n t s at up to seven d i f f e r e n t emission wavelengths.

B

60

.......

50

6

20

o

o--

o

4O

0

"0

~O. E

0 Lifetirne8

-20 --40

+

rz~

X

d

144

N .

[] 0

StationoP/ l

860

E --50

Spectrum I

880

I

-- 100

<~ I

I

900

I

920

Z~

157

[]

55

0

°N

~o o

•

Ufetlrnee (p=): + 1720

a.

(ps):

• Stettor~ry Spectrum t

I

940

860

I

880

I

I

900

I

I

920

I

940

Wovelength, nm Fig. 3. Decay-associated fluorescence emission spectra of RCs from C. attrantiacus calculated by global analysis from decays measured at rapid sample pumping speed and 812 nm excitation (conditions for "open RCs'). (A) Free-running parameters; (B) same data with the 18 ps lifetime fixed in the analysis. Essentially the same results as in (B) are obtained from a combined analysis of all time-resolved spectra taken under varying excitation intensities (c.f. Fig. 9). Wavelength, nm

O n e o f t h e aims was t o a c h i e v e c o n d i t i o n s w h e r e R C s w e r e e i t h e r in t h e o p e n s t a t e ( P H Q r , ) or in the c o m p l e t e l y c l o s e d s t a t e ( P H Q A). A l t o g e t h e r , 20 i n d e p e n dent time-resolved spectra have been measured under different conditions on several independently isolated R C p r e p a r a t i o n s . T h e d a t a h a v e b e e n a n a l y z e d b y the g l o b a l l i f e t i m e p r o c e d u r e . T h e r e s u l t s w e r e p l o t t e d as d e c a y - a s s o c i a t , . d s p e c t r a ( D A S ) . Fig. 3a s h o w s a typical e x a m p l e o f t h e D A S o f C. a u r a n t i a c u s R C s u p o n e x c i t a t i o n at 812 n m a n d fast p u m p i n g o f t h e s a m p l e in o r d e r to try to k e e p R C s o p e n . W i t h c o m p l e t e l y f r e e r u n n i n g p a r a m e t e r s a m i n i m u m o f five e x p o n e n t i a l c o m p o n e n t s was r e q u i r e d to a c h i e v e a g o o d fit. T h e l i f e t i m e s a r e r~ -~ 3 ps, ~2 ~ 13 ps, r~ ~ 30 ps, r4 ~ 144 ps a n d r s = 1.7 n s (for a c o l l e c t i o n o f all l i f e t i m e s a n d a s s o c i a t e d e r r o r s s e e T a b l e !; t h e s a m e n u m b e r i n g for l i f e t i m e s ~-~ is u s e d h e r e as in t h e table). T h e two l o n g e s t - l i v e d c o m p o n e n t s h a v e v e r y small b u t significant amplitudes. A characteristic feature of the fastest c o m p o n e n t ~-~ is a p o s i t i v e a m p l i t u d e at s h o r t e m i s s i o n wavelengths (decay term) and a negative amplitude (rise t e r m ) at l o n g e m i s s i o n w a v e l e n g t h s . T h i s c o m p o n e n t c o u l d be r e s o l v e d e v e n by s i n g l e - d e c a y analysis (c.f. Fig. 4; c.f. a l s o R e f . 1). A l l o t h e r c o m p o n e n t s s h o w o n l y p o s i t i v e a m p l i t u d e s in t h e i r D A S w i t h a m a x i m u m in t h e r a n g e 9 0 0 - 9 2 0 n m . W i t h o u t t h e 3 ps c o m p o n e n t a r e a s o n a b l e fit t o the d a t a c o u l d n o t be o b t a i n e d as j u d g e d f r o m the r e s i d u a l plots. H o w e v e r , e v e n w i t h five c o m p o n e n t s (Fig. 3a), t h e r e s i d u a l p l o t s w e r e n o t ideal in m a n y eases, b u t u p o n f u r t h e r i n c r e a s i n g t h e n u m b e r o f c o m p o n e n t s t o six, a r e a s o n a b l e fit c o u l d n o t b e o b t a i n e d u s i n g c o m p l e t e l y f r e e - r u n n i n g p a r a m e t e r s for a s i n g l e s p e c t r u m . F o r r e a s o n s t h a t will b e c o m e c l e a r in t h e f o l l o w i n g s e c t i o n , t w o f u r t h e r analysis r u n s w e r e p e r f o r m e d . In t h e first o n e w e fixed o n e l i f e t i m e c o m p o n e n t to 18 ps a n d left the o t h e r l i f e t i m e s f r e e - r u n n i n g in a six c o m p o n e n t fit (Fig. 3b). T h e m a j o r d i f f e r e n c e to t h e f i v e - e x p o n e n t i a l fit (Fig. 3a) c o n s i s t s in the

a p p e a r a n c e of a n e w 6 - 7 ps c o m p o n c n ! which rcstdls f r o m thc splitting of t h e 13 ps c o m p o n e n t p r e s e n t in t h e f i v e - e x p o n e n t i a l fit. It is i m p o r t a n t that the approx. 3 ps componcn*, was also r e s o l v e d in this case, a g a i n with p o s i t i v e / n e g a t i v e a m p l i t u d e s . A l t e r n a t i v e l y , all t i m e r e s o l v e d s p e c t r a t a k e n at d i f f e r e n t e x c i t a t i o n i n t e n s i t i c s a n d e x c i t a t i o n w a v e l e n g t h s (see b e l o w ) w e r e a n a l y z e d in a single g l o b a l analysis run. T h i s p r o c e d u r e also r e s o l v e d t h e a p p r o x . 7 ps a n d a p p r o x . 18 ps comr~on e n t s w i t h D A S very s i m i l a r to t h o s e s h o w n in Fig. 3b. In g e n e r a l , n o p o l a r i z e r has b e e n u s e d in t h e c m i s s i o n p a t h in o r d e r to avoid r e d u c t i o n in f l u o r e s c e n c e signal r e l a t e d to s u c h a d e v i c e a n d t h u s allow f o r s h o r t e r m e a s u r i n g times. W e d i d c h e c k , h o w e v e r , w h e t h e r a p o l a r i z e r in t h e m a g i c - a n g l e p o s i t i o n w o u l d i n f l u e n c e in p a r t i c u l a r t h e 3 ps c o m p o n e n t . T h i s was n e c e s s a r y in o r d e r to m a k e s u r e t h a t t h e 3 ps c o m p o n e n t was n o t an a n i s o t r o p i c d e c a y c o m p o n e n t . T h e 3 ps c o m p o n e n t with p o s i t i v e / n e g a t i v e a m p l i t u d e s was also r e q u i r e d w i t h t h e p o l a r i z e r in the m a g i c - a n g l e p o s i t i o n . S l i g h t c h a n g e s in l h e s h a p e o f t h e D A S of t h e 3 ps component between these two types of measurement c a n not b e c o m p l e t e l y e x c l u d e d , i n d i c a t i n g p e r h a p s t h a t the s h o r t - a n d l o n g - w a v e l e n g t h e m i s s i o n c o u l d exhibit s o m e w h a t d i f f e r e n t p o l a r i z a t i o n . W e d o n o t w a n t to stress this p o i n t , h o w e v e r , s i n c e we c o l l e c t e d i n s u f f i c i e n t d a t a u n d e r m a g i c - a n g l e c o n d i t i o n s in o r d e r to p r e c i s e l y d e t e r m i n e p o s s i b l e small c h a n g e s in p o l a r ization. U p o n s w i t c h i n g o f f t h e p u m p t h e total f l u o r e s c e n c e i n t e n s i t y i n c r e a s e d by a f a c t o r o f 5 to 9 o v e r t h a t o b s e r v e d with fast p u m p i n g u n d e r the s a m e l a s e r excit a t i o n intensity. T h i s i n c r e a s e in f l u o r e s c e n c e i n t e n s i t y o c c u r s d u e to t h e c l o s u r e o f R C s by t h e e x c i t i n g l a s e r light, as w a s also c h e c k e d by r e d u c i n g QA c h e m i c a l l y by a d d i t i o n o f d i t h i o n i t e . In g e n e r a l , t i m e - r e s o l v e d f l u o r e s c e n c e m e a s u r e m e n t s w i t h R C s c l o s e d w e r e carr i e d o u t w i t h a v e r y slow s a m p l e p u m p i n g s p e e d e n s u r -

TABLE I Fluorescence lifetimes o f isolated RC from C. aurantiacl~" in open and closed state as ot tained by global analysis of the time-resoh'ed spectra

The analysis showed that not all RCs were in the open state; for exact details see Results and Discussion sections. Errors: The error range of the rt-component is 1.5-4 ps; other errors are r2::1:2 ps, ~r3:_-2-3ps. all other: + 10%. RC state Open Open Open Closed Open Open

Aexc (nm) 795 812 812 812 850 855

Tl (ps) 2-3 2-3 3 -

r 2 (ps) 7 13 b 6-7 7 5

r~ (ps) 21 _ 18 0 16 22 18

r 4 (ps) 30 c 55 44 -

rs (ps) 74 1,14 157 280 ! 10 78

% (ns) 1.9 ~ 1.7 a 1.7 ~ 5.3 1.1 ~ 1.2 ~

a Free BChl. b Mixed component; c.f. Discussion section. c This low value of about 30 ps is only obtained in "open" RCs if the r 2- and r~-components are not resolved. In all other cases this lifetime is > 44 ps. d Lifetime has been kept fixed during the fit.

O ~--O---_

u,_~.l[

eli

[I

3 exponemiom

•

X = 1.223 __J

6

~-~o~

..xpo~o,.

5 "~

3.2 pe m.e p, ~'~: 83.2 p~ ~'~: 1180.0 ps

4

_j

e~

Lifetimes (ps):

+

E

R,: 0.4"/3' Re: O485 R.~: 0.040 R~: 0.022

I",: ~:

• '

~3

x ' ° 10~

<-5

\

5300

z~ 0 o

\

28o 44

~"

\ \

~e

/

\

^

O~

•

$totionory Spectrum ~ 0 I I I I i I , 880 900 920 940 Wavelength, nm Fig. 5. Decay-associated fluorescence emission spectra of RCs from ( , auran#u(',.s calculated by global analysis from decays measured at slow sample pumping speed ('closed RCs') and 812 nm excitation. --15 860

0 c~

"--,,-"'--",,

2

0

~"~".'. I

0.0

0.2

0.4

0.6

0.8

T i m e , ns Fig. 4. Single decay analysis- - f the lluorescence kinetics *~l"R('s from C'. ,,ran/iac,.~ (h~.,~ = 812 rim). Lower frame: The dashed line sho~,s the semilogarithmic plot of the excitation function. The full lines sho'x the semilogarithmic plots of the fluorescence kinetics of open RCs (lower cup'c) and closed R('s (upper curve). The lifetimes given are those t'or closed RCs from single-decay analysis. The corresponding residual plots and ~" values (upper :wo frames) indicate that the 3 ps component can be re:~,)lved even in single-decay analysis.

ing that there was no significant decrease in total fluorescence intensity as compared to the intensity observed without pumping. Fig. 4 compares typical fluorescence decays for 812 nm excitation obtained with fast pumping and very. slow pumping. The increase in long lifetime components is directly visible in

the decays without any analysis. A typical DAS resulting from global analysis of a time-resolved spectrum taken with very slow pumping for 812 nm excitation is shown in Fig. 5. Five lifetimes are required in a fit with all parameters free running. These lifetimes are about 3 ps, 16 ps, 44 ps, 280 ps and 5.3 ns. The residual plots in this case do not show any significant deviations from a random distribution (Fig. 6a). In contrast, systematic deviations in the residuals are observed when the number of lifetimes is reduced to four (Fig. 6b). One of the essential features of these DAS is again an approx. 3 ps lifetime with positive/negative amplitudes as observed already under the conditions of fast pumping (Fig. 3). B

A 2"51

I

~Xem = 870 nm

•

3.3,

-

= 900 nm

._o, - I ~ 1 " UT'~I' r*]l~'~ lntr~v ' ~ ' '

'l

5 exponer~ioL~

2.8

/

~q'l ~~"'~"~ ~"~'~_ f~ I 5 expormntial$ X m 1 0 4 4

~~I~I/

_ 2 . 5 / ...............

U~_3.31

•

J '

1

x E 2.8 J "~' " r'Y'l F'~'v~r]w~l-v'~ If.~. w'.,~''''~ V~[ o

-

_2.8 ~

~

2.8

91o

-15 eqx)rmntlom X = 1.049 |

70 nm

=

4, e x p o n e n t l ~

il

~

I'

,~ =,.,w,~,.,ti.~,

3.0 L,~ °°F~r I ~,,,~,,~ -3"0 1

I!

I

r','r'' ~IM" ~v i

~_o,~.~F~l,r, ~ - "r v ' ~ ' v,_3.5 l

,,, 9 o o . m

,,

"14 o ~

x ' = t.244 I

.,,

~

~,s~,, ~ ~

z X

l

1.138 l

- 92 nm

~, ""[', I"lq~rtlL~ -"'~ ~-~"PVV'" r'v' "1 ^ ^ ~ --z.B

-

~' ~q"~'~ Ttif' "TrF "V"' r~,,v'v~ R 1"11 -'_2.81 ! . . . . I x= - !.o~o I 3"51

~v I

X t'~= 1.144

,x~

• z

, ' 0~1

s q ~ o , , , , ~ l ~ , x' - t.02~ I 0.2 0.3 0.4. T i m e , ns

.~, o.o[., ,-n~i~vV,rl q¢~ ~ V"K'W'" r"" ~'_&O l

/1, 0.1

* ~l,,r,o,',-~i°~, x' = t04~ 0.2 0.3 0.4T i m e , ns

Fig. 6. Residual plots and X 2 values of the DAS shown in Fig. 5 for five exponentials (A) and four exponentials (B). The deviations in residuals and the changes in the 4,2 values suggest that five components are required for a good fit.

..0 . . . . . • - .

Lifetimes (ps):

+

O~--O--C.'-.

60

/

"~

[]

22

0

="

•

O -

~00 7 Stationary Spectrum

1.7 + 1).2 n s c o m p o n e n t with a high relative amplitude ( a t ~ o u t 3(Y:; a t 8 2 0 n m ) a n d a m a x i m u m o ! its I ) A S b t ' l o w 8 2 0 n m w a s n e c e s s a r y . T h i s con~pOllCllt ; i r i s e s f r o m t h e s m a l l a m o u n t o f f r e c B ( ' h ! p r e s e n t in i s o l a t e d R C s (c.f. a l s o t h e s t a t i o n a r 3 ' e m i s s i o n s p e c t r a ) .

40

Dependence on excitation httensio" O.

E 20 <~

0

z~

A

9

I

I

I

860

880

,~ . . . . . . . . . . . .

,,x- ,__r.,

I

I

900 Wavelength,

I

I

920

940

nm

Fig. 7. D e c a y - a s s o c i a t e d 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 o f R C s f r o m C. attrantiacus as calculated by global analysis from decays measured

at rapid sample pumping speed ('open RCs') and "~e~, = 850 nm.

Fig. 7 shows the D A S resulting from a m e a s u r e m e n t with fast p u m p i n g using an excitation wavelength of 850 n m , i.e., selective excitation of the special pair to P*. F o u r lifetime c o m p o n e n t s are r e q u i r e d for a good fit with lifetimes of a b o u t 7 ps, 22 ps, 110 ps a n d 1.1 ns in a f r e e - r u n n i n g p a r a m e t e r fit. T h e r e are n o indications in the residuals necessitating a n a d d i t i o n a l comp o n e n t . In p a r t i c u l a r n o approx. 3 ps c o m p o n e n t is required. T h e residual plots for global three- a n d f o u r - c o m p o n e n t analyses of this D A S are shown in Figs. 8a a n d 8b, respectively. They indicate that three c o m p o n e n t s are not sufficient for a good fit. Excitation at 795 n m (see T a b l e I) resulted in short lifetime c o m p o n e n t s that were similar to those f o u n d with excitation at 850 nm. However, in addition a

The fluorescence kinetics with fast sample p u m p i n g speed has b e e n measured as a function of laser excitation intensities. In each case a n entire time-resolved emission spectrum has b e e n recorded. All data were subjected to a c o m b i n e d analysis by thc global lifetime analysis procedure. It was found in this analysis that the a m p l i t u d e s of two c o m p o n e n t s , i.e., the approx. 7 ps a n d the approx. 18 ps c o m p o n e n t , were d c p e n d c n t o n excitation energy in a reciprocal m a n n e r . While the relative a m p l i t u d e of the approx. 18 ps c o m p o n e n t increased with excitation intensity, the relative amplitude of the approx. 7 ps c o m p o n e n t decreased. This d e p e n d e n c e along with a linear fit to the data is shown in Fig. 9. Each data point r e p r e s e n t s the amplitude ratio A~sp~/AT~ averaged over the entire emission s p e c t r u m for a particular laser intensity, corresponds to a different sample and was generally m e a s u r e d at a different day. T h e lower range of absorbed laser intensities used was dictated by the necessity of still achieving a sufficient s i g n a l / n o i s e ratio ( n u m b e r of counts) in a r e a s o n a b l e time. At a typical average absorbed laser intensity of a b o u t 2 m W / m m 2 the recording of a n e n t i r e time-resolved spectrum took 10-12 h. These long m e a s u r i n g times are due to the low detector sensitivity in the n e a r - i n f r a r e d region. By a l t e r n a t i n g m e a s u r e m e n t s at short a n d long emission wavelengths we verified that the exposure of the sample to the laser B

A 2.6

-

o oi

,tli

-2"61 r

" •

70

4.2

m

'

4. .,xpm~entiol~ X ' '

x

-aao

1.o3s I

to --4.2 o

nm

II

~ ~xx~n~k~,

3.4-

X " 0-S00~l

to --3.0

I

3.4

"

~,_3.~ ~"l~T~l"l~Vl~WP1~'arq~V~_ ~' ~l~iI / , e~oentk=~ ~--1.057 x

3.3

- -

"

'~

' 0.2

"~W~rl r 0.4

= ~ooam

~'~"~ ~1~" 0.6

Time. ns Fig. 8. Residual plots and

Xz

0.8

3 exponentials

X t ~ 1.38~

I

3.0

. ~ 0.0

.-~ "~

~ 870 nm

.~_t~ 0.0 "'

2.8

_2.81

~.

~IHt'l~w~',,,~ 'I"" "I'll

qF'r~q~l'~'V/

~?~."x,

i

- 3 . 4 I' ~

= ,.o~

- 890 am

a e~mentl0b X " 1.2a8 1

3"71

J

x_

- 9oo am

I

.~ ~ - - ~ ~ ~ W r ~ 1~1',~rl, ,V~r,lll~l~,,r • I ~'_a.711 1 '. ] "~=~,~.~ = ~-m/ 0.2

0.4 Time,

0.6

0.8

ns

values of DAS shown in Fig. 7 for four exponentials (A) and three exponentials (B). The deviations in residuals and the changes in X: values suggest that four components are required for a good fit.

1.6

12

0.8

ape

~. '~

O.4

0.0

I¢"

0

t

t

t

I

I

1

2

3

4

5

6

Absorbed Loser-Intensity [mW/mm 2] Fig. 9. A m p l i t u d e r a t i o o f the l/g p~ a n d 7 ps c o m p o n c n l ~ o f R ( ' s as, a f u n c l i o n o f the a b s o r b e d la~er inlcnsiiy at r a p i d s a m p l e p u m p i n g speed. T h e d a t a w e r e c a l c u l a t e d from a c o m b i n e d globzd anal~.,,is o f all t i m e - r e s o l v e d s p e c t r a l u k c n at d i f f e r e n t e x c i t a t i o n inlensities.

light for the m e a s u r i n g time did not have any significant effect on the .~pcctra. As a further p r e c a u t i o n thc sample was also replaced by a fresh o n e after the first half of the m e a s u r i n g time in the case of long runs. We should like to note here that the sample p u m p i n g speed also has a p r o n o u n c e d effect on the a m p l i t u d e ratio of these two lifetime c o m p o n e n t s (see above). While we a t t e m p t e d to keep the p u m p i n g speed the same as much as possible for all the m e a s u r e m e n t s r e p r e s e n t e d in Fig. 9, variations in the p u m p i n g speed between different samples or days c o n t r i b u t e substantially to the scatter in the data shown in Fig. 9. A further source of u n c e r t a i n t y arises from the errors in resolving the approx. 7 ps and approx. 18 ps components, sometimes ) t ~ = 812 n m ) in the f u r t h e r presence of an approx. 3 ps c o m p o n e n t , although the global analysis procedure largely improves the resolvability of such components.

Discussion In the following, we shall first discuss the d e p e n dence of the various lifetime c o m p o n e n t s a n d their amplitudes on the excitation wavelength a n d intensity. These d e p e n d e n c i e s give a clue as to the origin of the different c o m p o n e n t s . The 3 ps component This lifetime c o m p o n e n t has b e e n consistently observed in all o u r data for ,'tcxc =" 812 nm, which excites almost exclusively the BChl-a L m o n o m e r excited state [13]. It was f o u n d to be i n d e p e n d e n t within the error limits from laser intensity a n d sample p u m p i n g speed. In contrast, this c o m p o n e n t did not show up for a~x, = 850 nm, which excites selectively the special pair, P. This indicates that for a¢~,. = 850 n m the relative amplitude of the 3 ps c o m p o n e n t is either zero or at least drastically reduced as c o m p a r e d to a¢~ = 812 nm. W e

conclude that the presence of this c o m p o n e n t is specifically coupled to BChl L m o n o m e r excitation. T h e D A S of the 3 ps c o m p o n e n t is f u r t h e r m o r e u n i q u e in the sense that it shows both positive and negative amplitudes (rise term). Such a D A S is characteristic for an energy-transfer process a n d / o r a relaxation process b e t w e e n excited states, its p r e s e n c e or a b s e n c e can be tested very sensitively by the global analysis p r o c e d u r e due to the distinct shape in the D A S T h e r e are several possibilities of i n t e r p r e t i n g this c o m p o n e n t . W e have excluded the possibility that it arises from an a n i s o t r o p y decay. T h e most straightforward model would involve energy transfer from the BChi~-state to the special pair P. T h e only a r g u m e n t that might speak against such an i n t e r p r e t a t i o n of the 3 ps c o m p o n e n t is the relatively large Stokes shift of u p to approx. 900 c m - ' for the BChI~. emission that would be implied. This value has b e e n e s t i m a t e d from the species-associated spectra of the two e m i t t i n g states which have their maxima a r o u n d 880 n m a n d 920 n m as p u b l i s h e d earlier [1]. T h e lower e m i t t i n g state, giving rise to the emission m a x i m u m at 9 1 0 - 9 2 0 nm, is clearly the P*-state, since its emission s p e c t r u m is in good a g r e e m e n t with that of purple bacteria a n d the stimulated emission s p e c t r u m of C. aurantiacus [10]. A n alternative i n t e r p r e t a t i o n for the 3 ps compon e n t - formally in a g r e e m e n t with our data for 812 n m excitation - would be a c o n f o r m a t i o n a l relaxation process of the P * state causing a t i m e - d e p e n d e n t b a t h o c h r o m i c shift of the P* emission spectrum. Such a shift has b e e n r e p o r t e d m the s t i m u l a t e d emission s p e c t r u m of C. aurantiacus RCs, a l t h o u g h with a somewhat shorter time c o n s t a n t of about 1.5 ps [10]. However, such a process can be excluded as the m a i n cause of the 3 ps c o m p o n e n t d u e to the fact that such a c o m p o n e n t was not observed by us for direct P* excitation at 850 n m (Fig. 7). If it were to r e p r e s e n t a n exciled state relaxation process of P*, it should also be p r e s e n t u p o n direct excitation of P*. The t r a n s i e n t a b s o r p t i o n data of Becker et al. [10] in this respect d o not contradict o u r data and i n t e r p r e t a t i o n , i n the exp e r i m e n t s of Becker et al. [10] the ratio of BChl~. to P* excitation is u n k n o w n due to a n u n k n o w n c o n t r i b u tion of the two excited states to the a b s o r p t i o n at their exciting wavelength of 605 nm. In the case that they preferentially had excited P * , the absence or low amplitude of such an e n e r g y - t r a n s f e r c o m p o n e n t in their data would be in full a g r e e m e n t with o u r observations. A further a r g u m e n t against a relaxation process involving a r e a r r a n g e m e n t of c h r o m o p h o r e s a n d / o r p r o t e i n e n v i r o n m e n t arises from the t e m p e r a t u r e d e p e n d e n c e of the stationary emission spectra (Fig. 2). T h e y u n dergo a p r o n o u n c e d n a r r o w i n g a n d c o n c o m i t a n t b a t h o c h r o m i c shift u p o n lowering the t e m p e r a t u r e . If these two effects were caused by a c o n f o r m a t i o n a l relaxation process, we would expect some slowing down

with decreasing t e m p e r a t u r e . This would lead to temp e r a t u r e d e p e n d e n c e opposite to that observed, i.e., a t e n d e n c y to increased emission from conformationally unrelaxed states at short emission wavelengths upon lowering the t e m p e r a t u r e . O t h e r effects may influence the stationary emission as well and the whole t e m p e r a tare d e p e n d e n c e of this spectrum is probably not easily explained by a single p h e n o m e n o n . For example, it is well known that the P * absorption b a n d u n d e r g o e s a strong b a t h o c h r o m i c shift at low t e m p e r a t u r e s [31]. In any case, a c o n f o r m a t i o n a l relaxation process can probably be excluded as the m a i n cause for the a p p a r e n t t i m e - d e p e n d e n t b a t h o e h r o m i c shift o f both the s p J n t a n e o u s as well as the s t i m u l a t e d emission spectra !10]. T h e t e m p e r a t u r e d e p e n d e n c e of the stationary emission seems to be best explained to a first approximation by a t e m p e r a t u r e - d e p e n d e n t mixture of at least two spectral c o m p o n e n t s with maxima at a r o u n d 8 7 0 88(I n m a d d 910-92(t nm. The spectral maxima of these two c o m p o n e n t s seem to agree reasonably well with the estimated emission spectra of the two states seen in the D A S (for an e s t i m a t e of the c o r r e s p o n d i n g species-associated spectra sec Ref. i). Using the Kenn a r d - S t e p a n o v relationship [32], o n e would expect the emission m a x i m u m of the u n r e l a x e d B*-state n e a r 820 nm, i.e., much shorter than the e x p e r i m e n t a l value. In view of this situation we c o n s i d e r an e n e r g y - t r a n s f e r process as the most likely cause for the 3 ps c o m p o n e n t . Hov, ever, the relatively large Stokes shift o f the d o n o r state n e e d s some explanation. Possibly the d o n o r state is not the initially excited BChI~_ state, but some BChI~ state that might have some charge transfer character mixed into it which could explain this larger t h a n expected Stokes shift, i n this case the K e n n a r d Stepanov relationship does not apply, it is also interesting in this c o n n e c t i o n that J e a n et ai. calculated for the BChl L ---, P e n e r g y transfer process a lifetime of about 3 ps for purple bacterial RCs [33]. However, according to the present i n t e r p r e t a t i o n of femtosecond transient a b s o r p t i o n experiments, this energy transfer step in p u r p l e bacterial RCs occurs on a time scale much shorter t h a n 1 ps, possibly even shorter if, an 100 fs [14,17].

The 7 ps and approx. 18 ps components T h e 7 ps and 18 ps c o m p o n e n t s can be best resolved in the decays m e a s u r e d with a ~ = 850 n m excitation, since u n d e r these c o n d i t i o n s the 3 ps c o m p o n e n t is absent (c.f. Fig. 7). At the fast sample p u m p rate both c o m p o n e n t s are present, b u t their a m p l i t u d e ratios vary d e p e n d i n g o n the laser intensities (Fig. 9). Decreasing laser intensity increases the a m p l i t u d e of the 7 ps c o m p o n e n t a n d in parallel decreases the a m p l i t u d e of the 18 ps c o m p o n e n t . Despite the substantial scatter in the data, which arises mainly from u n c e r t a i n t i e s in

the p u m p raite and also trom analysis errors, this tlep c n d e n c e R)llows from the results shown in Fig. 9. At y e w low sample p u m p rg'tes the a m p l i t u d e of ~hc 7 ps c o m p o n e n t disappear~.d a n d only the IN ps c o m p o n e n t r e m a i n e d . C o n c o m i t a n t l y the total fluorescence intensity increased reversibly 5- to 9-R~ld relative to that observed with fast pumping. Both c o m p o n e n t s have similar D A S both with respect to shape and spectral m a x i m u m . T h e s e findings suggest that the increase in a m p l i t u d e of the 18 ps c o m p o n e n t and in total fluorescence reflect the closing of RCs, i.e., reduction of Oa. W e thus interpret the approx. 18 ps lifetime as the charge s e p a r a t i o n time of closed RCs (QA reduced). Correspondingly the approx. 7 ps c o m p o n e n t should r e p r e s e n t the charge separation time for o p e n RCs (QA oxidized). Notably this lifetime is in a g r e e m e n t with the charge separation time for open RCs at room t e m p e r a t u r e m e a s u r e d by transient absorption by Bccker et al. [ll)]. In their data there was no significant c o n t r i b u t i o n from a 20-3(I ps c o m p o n e n t at room temp e r a t u r e . In contrast in o u r data (Fig. 9), even at the lowest laser intensities, the relative amplitude of the 18 ps c o m p o n e n t a m o u n t s to about 20% of the a m p l i t u d e of the 7 ps c o m p o n e n t . Given our laser repetition rate of 4 MHz, obvi,msly a still lower excitation intensity would be required in order to keep all RCs o p e n since the QA-State has a long lifetime as c o m p a r e d to the 0,25 # s spacing b e t w e e n pulses. W o r k i n g with 10 Hz r e p e t i t i o n rate this p r o b l e m did not arise in the transient a b s o r p t i o n m e a s u r e m e n t s of Becker et al. [22] at room t e m p e r a t u r e because the reoxidation of the OAstate should occur faster than the 100 ms interval b e t w e e n their excitation pulses. Thus. the kinetics observed in that experiment are likely to be those of completely o p e n RCs only, while we are observing a mixture of o p e n a n d closed RCs. It follows from these two lifetimes (7 and 18 ps) that the rate of the primary charge separation w h e n going from open to closed RCs is r e d u c e d by a factor of about 2.5. Interestingly, this value is similar to that reported for purple bacterial RCs [14,18,34,35], but still considerably smaller than the factor of about 6 m e a s u r e d for Photosystem I! of higher plants by Schatz et al. [36]. This reduction factor in the rate of primary charge separation could in principle also tell us something a b o u t the distance a n d electronic coupling b e t w e e n B P h e o a n d QA" O n the basis of this i n t e r p r e t a t i o n also the data with 812 n m excitation shown in Figs. 3a and 3b ( o p e n RCs) a n d in Fig. 5 (closed RCs) can now be b e t t e r u n d e r stood. It should be kept in m i n d that the approx. 3 ps c o m p o n e n t is resolved u n d e r all c o n d i t i o n s for 812 n m excitation. However, in that case the global analysis of a single time-resolved s p e c t r u m with free r u n n i n g par a m e t e r s for o p e n RCs (Fig. 3a) yielded a further lifetime of about 13 ps which was not found u n d e r o t h e r c o n d i t i o n s (closed RCs or A ~ = 850 nm). This

11} com|~ont.'l'll ,,plit i n t o I~,~,(+ ct+ntr}tWiclll~,, ht)~,~,t?~,t.,l-, t i n d e r the t~,~ an;dx,,is conditions dc,,cribt'd ilk the Rcstolt,, section, In the tirsl c4sc the IN p , , c o m p o n c n t Illclime v,a~ fixed ~,hilc 411 ,~thcr i~tramctcrn ".~.¢r~.' f r c c - l t m n i n g in the glob:d ~malysi,,. l h u s the lilctimcs ,,hoxvn in I:ig. 3b wcrc obtained. T h e lilt:times ;Hid the t ~ o I ) A % ol the ; I p p r o x , ,7 ps ;uld ;lppr~t\. I~ 13', COl111)onell|S ; l g i c c well ~,~ilii the C(~l-rcnpontling datlt ol~lained under the o t h e r nlc;~uring condilion~ ( e i th e r R ( ' s clo~cd or excitation at N511 llnl). SeCt)lit|. resolution of these two contr)onents a n d essentially the s41nc restllt, with respect to lifetimes and I ) A S ct~tlld bc o b t a i n e d with f r e e - r u n n i n g p a r a m e t e r s when all lhe t im e- r eso l v ed spectra taken u n d e r y a w i n g excitation intensities (Fig. 91 were ~malyzcd t o g e l h c l ill a global fashion. B o t h of these results ,'_l~gt'M th;H the 13 ps comp~}r~cnl ill Fig. 3a indeed rcsul|~ f r o m ~ln unrc,,t~lvcd mixture o f the applox. 7 I)~ and ,ippr(~x. IN 11~ C{mlpOllCnl.s. Th e tact that tllcsC tyro componcllt~ could not bc resolved directly in z~ single I ) A S is not ",urprising in ~,ic~ of the additional prc~,cncc of the 3 ps c o m p o n e n t . T h e most desirable e x p e r i m e n t clearly would bc to e l im i n at e the IN ps c o m p o n e n t by reducing the laser intensity. Howe v e r , this ~ccm~ to bc impossible ;tt present, given the low d e t e c t o r sensitivity. From the a m p l i t u d e in the D A S (Fig. 3b) v,c conclude thai ttp It~ 4()r; of the RCs were in the closed slate in that e x p e r i m e n t .

O t h e r lilt'time cootpol~cnts A 55 + 15 ps c o m p o n e n t is always observed indep e n d e n t of laser intensity, excitation wavelength or p u m p i n g speed. Its a m p l i t u d e is small, d e p e n d i n g on the sample used, typically 2 - 1 2 " ; of the sum of the a m p l i t u d e s of the 7 and 18 ps ¢onlponenls, but can not be ignored. This c o m p o n e n t has a I ) A S very similar to those of the 7 ps and 18 ps c o m p o n e n t s . T h e lifetime of this c o m p o n e n t drops to about 311 ps if the 7 ps and 18 ps c o m p o n e n t s are nol resolved (Fig. 3A). This occurs due to mixing of several lifetime c o m p o n e n t s . Th e real value of this c o m p o n c n l (about 55 ps) is, however, well s e p a r a t e d from the o t h e r short-lived c o m p o n en t s . With fast p u m p rates, w h e r e a significant proportion of the RCs are in the o p e n state, we observed a weak c o m p o n e n t in the 15()-300 ps range. Its D A S - i s very similar to that of the 7 ps and 18 ps c o m p o n e n t s . It is assigned to the c h a r g e - t r a n s f e r process in o p e n RCs r e p o r t in g on the e l e c t r o n transfer to QA in a g r e e m e n t with the rates r e p o r t e d by o t h e r authors [1(},37}. W h e n the p u m p rate is low and RCs arc closed, a 5.3 ns c o m p o n e n t of low but significant a m p l i t u d e appears, c o n c o m i t a n t with the increase in total fluorescence. W c assign this to a c h a r g e - r e c o m bination process in the state P * H Q , [ , r e p r e s e n t i n g the lifetime of the radical pair in the closed state. Since its lifetime is quite hmg. it can bc d e t e r m i n e d quite

~vcll in o u r c,q~erimcnts, despite its I,,~w' a m p l i t u d e (Fig. 5}

1)o~'~ rmc ~ f the' componctlls /~llh'ct B(7~ll reduc'ti~m? A sequential two-step e l e c t r o n - t r a n s f e r m e c h ; m i s m ha, been p r o p o s e d for RCs of purple b a c t e r i a ll8] as well as for (7'. a , r r m t i a c u . s R C s [1%20]. A full discussion of vhc various hypothetical kinetic m o d e l s for e l e c t r o n transl'¢r is beyond the scope of this paper. W c have cl.cckcd, however, w h e t h e r any of o u r resolved lifetime c o m p o n e n t s could reflcct an e l e c t r o n transfer process including BChl ~i as an i n t e r m e d i a t e . Clearly, t he strong 3 ps c o m p o n e n t can bc excluded as a c a n d i d a t e . First, its blue-shifted f l u o r e s c e n c e does not arise f r o m t he P*-st~ttc and s e c o n d wc would expect only positive a m p l i t u d e s for such a c o m p o n e n t . F u r t h e r m o r e , it is not obscr~'cd w h e n the P*-statc is excited directly. W e t h e r e f o r e do not a g r e e with the i n t e r p r e t a t i o n o f Shuvah}v c! al. [21] v, ho assigned an approx. 3 ps c o m p o nent ttp ihe seco n d ar y e l e c t r o n - t r a n s f e r step BChI~. ---} BPlaet h . O f course, wc could not entirely ex c l ude from our data such a seco n d ar y e l e c t r o n - t r a n s f e r step, BChl I ~ B P h e o v , if its rate w e r e to be significantly faster than the (7 ps) ~ rate for the primary e l e c t r o n lransfcr step, since in this case we would expect a small a m p l i t u d e only in the f l u o r e s c e n c e kinetics. H o w e v e r , no indication for BChlil f o r m a t i o n in R C s of C. a u r a n tiactts has b e e n found by B e c k e r el al. [10] b a s e d o n t h ei r transient a b s o r p t i o n data. W e thus would be left with one of the 7 ps and 18 ps c o m p o n e n t s as candid at es tor such a model. A strong a r g u m e n t against such an i n t e r p r e t a t i o n is the widely varying a m p l i t u d e ratio of these two c o m p o n e n t s upon varying the laser intensity. Such b c h a v i o u r supports t h e o p e n / c l o s e d R C i n t e r p r e t a t i o n p r o p o s e d above, but w o u l d be inconsistent with a s e q u e n t i a l e l e c t r o n - t r a n s f e r m o d e l involving both the 7 ps and the 18 ps c o m p o n e n t s , in o r d e r to p e r f o r m a q u a n t i t a t i v e check we have solved the c o r r e s p o n d i n g rate e q u a t i o n s for the coltsccutive two-step e l e c t r o n transfer and have fitted it to o u r data, as has b c c n d o n e by Bcckcr et ai. [10]. T h e r e are two possibilities, taking the 7 ps c o m p o n e n t e i t h e r in t h e first o r the second step. We have c a l c u l a t e d for both m o d e l s the BChI~I and B P h e o L c o n c e n t r a t i o n as a /unction o f time. A l t h o u g h formally such a fit is possible in o u r data in cases w h e r e the 18 ps c o m p o n e n t is p r e s e n t in addition to the 7 ps c o m p o n e n t , in all cases the p r e dicted rise of BChl/~ was m u c h t o o slow as c o m p a r e d to that m e a s u r e d e x p e r i m e n t a l l y [ 10]. F u r t h e r m o r e , t he rate constants for forward and back e l e c t r o n t r a n s f e r d e d u c e d from these formal fits did not s e e m to be r e a s o n a b l e . W e thus a g r e e with B e c k e r et al. [10] that such a possibility can be excluded. F o r the same reasons, each o f the still slower lifetimes can be e x c l u d e d as a possible c a n d i d a t e .

II is tlu'n' heterog,:teeity iu the rate o f primuo' cl~arge sepa rat ton ? A n intrinsic h e t e r o g e n e i t y in the rate
c o n s i d e r e d as a possible cause of the observed rate h e t e r o g e n e i t y . W e should like to propose closed RC~ as a possible cause of this reported ralc hcterogencit3. Th e b eh ax i o u r could bc easily u n d e r s t o o d if the fast c o m p o n e n t arises from open i C s and the slower one from closed RCs. For o p e n R C s the potential energy curves {if excited state an d radical pair state cross at or near the p o t e n t i a l m i n i m u m o f the P* state. C h a r g e s e p a r a t i o n is activationlcs~ and probably even speeds up at cryogenic t e m p e r a t u r e s , an al o g o u s to the situation with p u r p l e bacterial R ( s [10,24]. For closed RCs, however, the i n t e r a c t i o n b e t w e e n close-lying B P h e o and O,,, shifts the potential e n e r g y curve for the radical pair up in e n e r g y relative to the o n e for o p e n RCs. C h a r g e s e p a r a t i o n then would r eq u i r e an activation energy. At room t e m p e r a t u r e this leads to a 2.5-fold d e c r e a s e in the charge s e p a r a t i o n rate for closed centres as discussed above. C o n s e q u e n t l y , upon lowering the t e m p e r a t u r e the charge s e p a r a t i o n rate is e x p e c t e d to slow down further, as is o b s e r v e d experimentally. T h e a n a l o g o u s ch an g es in rates upon r ed u cti on o f OA have b e e n well studied in open and closed Photosystem !1 R C s [36] and wc suggest that bacterial R C s behave in a similar way. This m o d e l in fact would explain well the d i f f e r e n c e s in free energy, A G ( ' P * - P ' H ), that were held responsible for the rate h e t e r o g e n e i t y in the conclusions of the work by Feick ct al. [24]. W e stress the point that b ased on o u r data and the above discussion, we can clearly not generally exclude an intrinsic rate h e t e r o g e n e i t y for the primary charge separation. However. such a rate h e t e r o g e n e i t y remains still to be shown u n d e r p r o p e r conditions w he r e o p e n / c l o s e d R C h e t e r o g e n e i t y can definitely be excluded.

Acknowledgements We t h an k Mr. M. Reus, Mrs. K. Hehl and Mrs. D. Pagirnus for their help in the p r e p a r a t i o n of RCs. Financial su p p o r t from the Alerted Krupp yon Bt, hlen und H a l b a c h - S t i f t u n g (K.G.) an d f r o m the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t is gratefully acknowledged. We also t h an k Professor K. S ch af f n cr for his interest and su p p o r t in this work. We like to thank Professor W.W. Parson for providing results prior to publication.

References 1 Miiller. M.G.. Griebcnow. K. and tlolzv,'arth. A.R. (1990) in Structure and Function of Bacterial Reaction Centers (MichelBeyerle, M.E.. ed.L Springer Series in Bi~physics, Vol. 6. pp. 169-180, Springer. Berlin. 2 Ilolzwarth, A.R. (1989) Q. Rev. Biophys. 22. 239-326. 3 Parson. W.W. (1991) in The Chh~rophytls (Scheer. H., ed.L pp. 1153-118;t}. CRC Press. Be)ca Raton. 4 Michel. It. and Deisenholcr. J. (1988) Pure Appl. ('hem. 6t). 953-958.

12 5 Deisenhifer, J.. Epp. {}, Miki, K.. t t u b e r R and Michel, It. 119841J. Mol. Biol. IN{I. 385-3cJ8. f} ('hante!, C t l . . l'icdc, I ) . M . . ] : l n g . J.. Smith. I L . Nl~ri'i~. ,1. and Schilfer, M (1{}86) FF.BS ;,eli, 2{15. 112-f41~. 7 {kchinnik{~<~. Y . & . . Abdutat:'.. N.{i,. Shmtlklt'i. B , I Z{~ll~t;irc'v. A.S.. t#,art2~tl~l%', A . A . . Knlttz{iv, ~.!.,>, ~[elo/hinskaya. I N . and l,evina N.B. It},~} F t ' I t S I.eli. 232..t,34-.3f~. 11 ()vchinnit~ov. ~l'.A.. Abdulat'~. N.(i.. )~}hlllirex, A.S.. Shrllukhsr. B.Ir].. Zarg~tro~. A.A,. K u l u z o v . M , A . . l'clezhinskaya, I . N and l_<:vina. N.B. { It ~;~4i F E l l S Loll. 231, 237 _4_. ~ ~ ij ~h;o;za,.~.a, J.A.. l~t~llspcich. F.. {)cstcrh,:ll. I). itntl Ft'Jt'k. R. (19~91 Eur. J. Bit, :hem. 1140, 75-114. I11 ]¢el-er, M.. Nagai ijan. V.. M i d d e n d o r f , D. Parson, W.V¢.. ~i,|illtin, i . E . and Blanl, m.~hip. R . [ . (ltl~}ll Biochem. Biophys. A e l a Ili57. 299-312. 11 Nagar~+ian.e.. Prarson, ti~J.ltli'.. Gaul, D. and ]c|ltrnck, C. l lgt~{i} Prol2. Nail. Acad. Sci. L~gA 147, 7888-711iJ2. 12 Thornhe:'. J.P.. Cogdcll. R.J., Pierson, I}.K. a~d Softer. R . E . B . (19S31 J. ('ell. Biochcnl. 2~, 15~J-1130. I a Pierson. 13 K. arid T h o r n i e r . J.P. (IriS3} Ptoc. ~atl. Acad. ScJ. LISA gll, 111J-~4. 14 Breton, J., ~lalrlin. J.-L, Mr,tun, A.. Antoltetti. A. tnd Orszag. A. (10861 Prec. Natl. Acad. Sci. USA 83, 5121-5125. 15 Kirmaicr, {2 a,ld Ito|len. D. {It1901 Prec. Nail. Atad. Sci. USA 87. 3552 3556. 16 Bixon, M.. Jortr, er, J.. MicheI-Beyerle, M.E., {}gro~lnik, A. and Lersch, W. { tt~87, (`'hem. Phys. |+ell. 1411, 626-6311. 17 I]rct{~n. J., Marlin. J.-I... Flcrnint!. G.R, and I .arnbr3', J.-C. { 198111 Biochernisl~' 27, b1276-112~4. 111 Ih}lzapft'l, W., Fink,'le, U.. K~iist r, W., Oeslcrhelt, ")., Scht:er, I!., S:ilz, II.LJ. and Z~lilh, W. (19891 Chem. Phys. Letl. 1~0. 1-7. 19 Shuvalo~, V.A. and Kltvanik, A.V. ~19831 FEBS Letl. 1611,51-55. 211 Shu,,illov. V.A., Amcsz, ,I. and Duyrens, I,.N.M. (Iti116~ Biochirn. Bi~lphys, Acta 851, 327-331.1. 21 Shuvalo~', V.A., Vasrnel, tt., Amesz J. and Duysen.~. LN.M. {198fl) Biochirn. Biophys. Acta 85 I. 361-368. 22 Becker. M.. Middendorf, D._ Nagarajal~, V., Parson, W.W.. Martin, I.E. and Blankenship. R.F.. (lqt~lD in Current Re,~earch in Pholosynthesis (Baltseheffsk$, M.. ed3 Vol. I, pp. 121-124. Kluwcr, rh}rdrcchl.

23 Feick, R.. Martin. J.-L, Breton. 1. Volk. M., Scheidel, G., I_angenbachcr, T., t.rrb;ino, C., Ogrodnik. A. and MicheI-Beycrle. M.F-. (199~1i in Structure and Function of Baclerial Reaction Center, MicheI-Beycrle. M.E., ed.}. Splinger Series in Biophysics, Vol. 6. pp. t81-181-;. Springer, Berlin. 24 Marlin. J.-L. 1,:imbry. J.C., Ashokkultlar, M.. MicheI-Beyerle, M.F.. Feick, R. and Breton, J. (Itiqll) Ultrafasl Phenomena VII, Vol. 53, 524-528. Springer Series (?hern. Phys., Springer, Berlin. 25 Ciricl.enow, K. and Holzwarth, A R . (19891 Biochim. Biophys. Acta '173, 235-2411. 26 Grit~bt:now, K., MOiler, M.{i. and t t o l ~ ' a r t h . A.R. {1991) Bioellma. Biophys. Acta 11t59, 226-232. 27 Griebe~ow, K. and Itolzwarth. A.R. (199111 in Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria {Drews, G. and Dawes, E A . , eds.L pp. 375-381, Plenum, New York. 28 Griebenow, K., Itolzwarth, A.R. and Schaffner, K. (19911) Z. Naturfl}rsch. 45C, 823-828. 29 flolzwarth. A.R.. Wendler. J. and Surer, G.W.