Bertoluzza, A.

Fagnano, C.

Finkele, Ulrich

Holzapfel, Wolfgang

Monti, P.

Zinth, Wolfgang

English

Universität München: Elektronischen Publikationen

2 9 1 
SEQUENTIAL ELECTRON TRANSFER IN THE PRIMARY PHOTOSYNTHETIC 
REACTION OF RHODOBACTER SPHAEROIDES. 
W. Zinth, W. Holzapfel and U. Finkele 
Physik Department E l l der Technischen Universität München, 
Ar c i s s t r a s s e 21, D-8000 Manchen 2, FRG 
The primary photochemistry in the photosynthesis of 
bacteriochlorophyll containing organisms i s a light-induced charge 
separation within a protein complex ca l l e d reaction center (RC). 
Recently, the c r y s t a l structures of the reaction centers could be 
determined for the f i r s t time /1,2/. For Rhodopseudomonas (Rps.) 
v i r i d i s and Rhodobacter (Hb*) sphaeroides the structure analysis 
gave very s i m i l a r arrangements of the prosthetic groups /3/. They 
are disposed as two branches (A, B ) in an approximate C-2 symmetry* 
Two bacteriochlorophyll molecules from the so-called special pair 
(P) located on the symmetry axis. Starting at the s p e c i a l pair each 
branch consists of an accessory bacteriochlorophyll {Β Λ , B B ) , a . 
bacteriopheophytin (H A , H B ) , and a quinone ( O A , Q B ) . 
A number of femtosecond spectroscopic experiments have 
been undertaken in order to resolve the primary electron transfer 
reaction. Most recent data for Rb. sphaeroides (as well as for Rps. 
v i r i d i s ) give the following picture /4-6/: Light absorption excites 
the primary donor, the s p e c i a l p a i r P, to the e l e c t r o n i c a l l y 
excited s t a t e P*. With a time constant of 2.8 ps an electron i s 
transferred along the Α-branch d i r e c t l y to the bacteriopheophytin 
HA forming the s t a t e P*H". I t was shown in the l i t e r a t u r e that 
a f t e r the decay of P* and p r i o r to the formation of s t a t e P*H~ 
there i s no intermediate electron carrying state or, i f such a 
st a t e e x i s t s , i t s li f e t i m e must be shorter than 100 f s /5,6/. 
However, i n the context of e a r l i e r experiments - not performed 
under optimum conditions - evidence for such an intermediate was 
discussed, based on the finding of rapid transient absorption 
changes with approximate time constants of 1 ps /7-9/» In sight 
of t h i s controverse discussion we decided to reinvestigate the 
k i n e t i c s of the primary processes of Rb. sphaeroides under 
optimized experimental conditions. 
Reaction centers from Rb. sphaeroides R26.1 and ATCC 17023 
were prepared according to the- procedure in Ref./10/. In the time-
resolved absorption measurements we used femtosecond excit i n g 
pulses (tp * 60 f s , repetition rate 10 Hz) at 860 run in the lowest 
energy absorption band of the s p e c i a l pair (see inset in F i g . l ) . 
Probing of the light-induced absorption changes at various wave­
lengths Xpr was performed with synchronized pulses as a function 
of time delay tD. The excitation energy density was kept low, only 
13% of the reaction centers were excited by each excitation pulse. 
2 9 2 
O l 1 0 100 1000 
Deity Τ I m (ps] 
F i g . l . Time-resolved absorption data ( f u l l c i r c l e s ) 
at a probing wavelength of 545 run for 
reaction centers of Rb. sphaeroides R26.1 
( e x c i t a t i o n wavelength 860 nn)« The curves 
are calculated according to two models given 
i n the text. Inset: absorption spectrum of 
the sample. 
The time resolved absorption measurements are shown i n 
F i g . l and 2 /10/. The experimental data points are indicated as f u l l 
c i r c l e s . The curves are calculated according to two k i n e t i c models. 
The f i r s t model ( s o l i d curve) i s d i r e c t l y deduced from the present­
l y accepted electron t r a n s f e r scheme;-i.e. electron tra n s f e r occurs 
from P * d i r e c t l y to s t a t e P * H ~ , Consequently, the complete reaction 
k i n e t i c s u n t i l the formation of s t a t e P+Q~ involves three i n t e r ­
mediates connected by two time constants. The second model (dashed 
curve, schematic shown i n Fig.3) considers an additional i n t e r ­
mediate s t a t e with the l i f e t i m e of 0.9 ps* The experimental r e s u l t s 
of F i g . l and 2 were taken at Apr = 545 nm (HA absorption band), 785 
nm ( H , B absorption), and 665 nm ( H ~ , B ~ absorption range). A l l three 
measurements indicate that the model of Fig*3 (dashed curve, four 
intermediates, three time constants) i s required to explain the 
experimental data. E s p e c i a l l y at λ = 665 nm and 785 nm the 0.9 ps 
k i n e t i c i s outstanding, while the 3,5 ps and the 200 ps k i n e t i c s 
a l s o appear. 
The amplitudes of the absorption changes connected with 
s t a t e s I i to I 4 can be used to r e l a t e the various intermediate 
states to molecular configurations. The molecular interpretation i n 
Fig.3 i s based on the following arguments: The f i r s t s t a t e I i i s 
the excited e l e c t r o n i c s t a t e . I t s monoexponential decay with a time 
constant of 3.5 ps i s observed around 920 nm. This time constant i s 
the f i r s t one (τι) i n the sequence of our model. The subsequent 
s t a t e s are intermediates of the electron t r a n s f e r process. The next 
st a t e i s I 2 decaying r a p i d l y with a time constant of 0.9 ps. The 
most obvious assignment of I 2 i s the configuration P * B * containing 
0.02 
0.01 
c • 
δ 
ο c 
0.006 
0.004 
0.002 -
ι ι ι ι I 
- 7 Θ -
1 i ι 
- 785 η· 
I 
ι ι 
- // 
Γ 
. 1 
^ ^ ^ ^ ^ ^ ^ ^ 
I 
- 663 na 
\ ®_ 
• · ι i 1 1 
10 100 
Delay TiM [ps] 
1000 
Fig.2. Tine-resolved absorption data ( f u l l c i r c l e s ) 
for probing wavelengths of (a) 785 nm and 
(b) 665 nm for reaction centers of wild-type 
Rb. sphearoides ATCC 17023 (excitation 860 nm) 
I 1 
hV 
35 ps 
I 3 
0.9 ps 
200ps 
Ρ Β HQ •P*B"HQ- p*BH"a *Ρ*ΒΗ0Γ 
Fig.3· Scheme of the sequence of states involved 
in the primary electron transfer in reaction 
centers. 
f 
294 
a bacteriochlorophyll anion r a d i c a l . This interpretation i s 
supported by the following observation /10/. ( i ) Iz shows strongly 
reduced absorption at the wavelength of 797 nm, where B~ ( i n v i t r o ) 
has strongly reduced absorption, ( i i ) During the rapid decay of I 2 
t h i s absorption recovers, and ( i i i ) I 2 does not show absorption 
decrease i n the wavelength range where bacteriopheophytin absorbs. 
State I 3 represents the next intermediate where the electron has 
reached the bacteriopheophytin Η forming the configuration to P*BH~Q. 
The r e l a t e d decrease of* the Η absorption at 545 nm i s c l e a r l y seen 
a f t e r the decay of s t a t e Iz and p r i o r to the formation of I 4 . 
F i n a l l y , with a time constant of 200 ps, the electron a r r i v e s in 
st a t e I4 at the quinone OK ( s t a t e P+BBQ-). 
I n conclusion: We have performed an experimental study of 
the primary charge transfer process i n the reaction centers of 
Hb. sphaeroides. The ana l y s i s of the k i n e t i c data ( i n a l i n e a r 
model) strongly suggests that the primary charge t r a n s f e r to the 
bacteriopheophytin proceeds v i a the accessory bacteriochlorophyll 
as a true intermediate. The following reaction scheme r e s u l t s : 
After e x c i t a t i o n of the s p e c i a l p a i r Ρ an electron i s transferred 
with a time constants of 3*5 ps to the accessory bacteriochloro­
phy l l * Here the electron resides f o r the short time of 0.9 ps 
before i t reduces the bacteriopheophytin. . 
Acknowledgement 
The authors acknowledge valuable collaboration with and supply of 
the Hb. sphaeroides samples by D. Oesterhelt, H. Scheer, and 
H.U. S t i l z . 
The work was supported by the Deutsche Forschungsgemeinschaft, SFB143. 
REFERENCES 
1. J . Delsenhofer, 0. Ερρ, Κ. Miki, R. Huber, Η. Michel, 
J . Mol. B i o l . 180, 385 (1984) 
2. H. Michel, 0· Epp, J . Delsenhof er, EMBO J . 5, 2445 (1986) 
3. J.P. Allen, G. Feher, T.O. Yeates, H. Komiya, D.C. Rees, 
Proc. Natl. Acad. S e i . USA 84, 5730 (1987) 
4· J#-L. Martin, J . Breton, A.J. Hoff, A. Migus, A. Antonetti, 
Proc. Natl. Acad. S e i . USA 83, 957 (1986) 
5· G.R. Fleming, J.~L. Martin, J * Breton, Nature 333, 190 (1988) 
6. J . Breton, J.-L. Martin, A. Migus, A. Antonetti, A. Orszag, 
Proc. Natl. Acad. S e i . USA 83, 5121 (1986) 
7. D. Holten, C. Hoganson, M.W. Windsor, C.C. Schenck, W.W. Parson, 
Α. Migus, R.L* Fork, C.V. Shank, ΒJochim. Biophys. Acta 592, 461 
(1980) 
8. W. Zinth, M.C. Nuss, M.A. Franz, W. Kaiser, H. Michel, i n 
"Antenna and Reaction Centers of Photosynthetic Bacteria*» 
Springer Se r i e s in Chemical Physics, Vol. 42, Ed. M.E. Michel-
Beyerle, Springer, Heidelberg 1985, p. 286 
9. V.A. Shuvalov, A.V. Klevanik, FEBS Lett . 160, 51 (1983) 
V.A. Shuvalov, J . Amesz, L.N.M. Duysens, ΒJochim. Biophvs. Acta 
851, 327 (1986) 
10. W. Holzapfel, ü. Finkele, W. Kaiser, D. Oesterhelt, H. Scheer, 
' H.U. S t i l z , W. Zinth, Chem. Phvs. L e t t . , to be published (1989) 


Sequential electron transfer in the primary photosynthetic reaction of Rhodobacter sphaeroides

Open Access LMU

2 9 1 SEQUENTIAL ELECTRON TRANSFER IN THE PRIMARY PHOTOSYNTHETIC REACTION OF RHODOBACTER SPHAEROIDES. W. Zinth, W. Holzapfel and U. Finkele Physik Department E l l der Technischen Universität München, Ar c i s s t r a s s e 21, D-8000 Manchen 2, FRG The primary photochemistry in the photosynthesis of bacteriochlorophyll containing organisms i s a light-induced charge separation within a protein complex ca l l e d reaction center (RC). Recently, the c r y s t a l structures of the reaction centers could be determined for the f i r s t time /1,2/. For Rhodopseudomonas (Rps.) v i r i d i s and Rhodobacter (Hb*) sphaeroides the structure analysis gave very s i m i l a r arrangements of the prosthetic groups /3/. They are disposed as two branches (A, B ) in an approximate C-2 symmetry* Two bacteriochlorophyll molecules from the so-called special pair (P) located on the symmetry axis. Starting at the s p e c i a l pair each branch consists of an accessory bacteriochlorophyll {Β Λ , B B ) , a . bacteriopheophytin (H A , H B ) , and a quinone ( O A , Q B ) . A number of femtosecond spectroscopic experiments have been undertaken in order to resolve the primary electron transfer reaction. Most recent data for Rb. sphaeroides (as well as for Rps. v i r i d i s ) give the following picture /4-6/: Light absorption excites the primary donor, the s p e c i a l p a i r P, to the e l e c t r o n i c a l l y excited s t a t e P*. With a time constant of 2.8 ps an electron i s transferred along the Α-branch d i r e c t l y to the bacteriopheophytin HA forming the s t a t e P*H". I t was shown in the l i t e r a t u r e that a f t e r the decay of P* and p r i o r to the formation of s t a t e P*H~ there i s no intermediate electron carrying state or, i f such a st a t e e x i s t s , i t s li f e t i m e must be shorter than 100 f s /5,6/. However, i n the context of e a r l i e r experiments - not performed under optimum conditions - evidence for such an intermediate was discussed, based on the finding of rapid transient absorption changes with approximate time constants of 1 ps /7-9/» In sight of t h i s controverse discussion we decided to reinvestigate the k i n e t i c s of the primary processes of Rb. sphaeroides under optimized experimental conditions. Reaction centers from Rb. sphaeroides R26.1 and ATCC 17023 were prepared according to the- procedure in Ref./10/. In the time-resolved absorption measurements we used femtosecond excit i n g pulses (tp * 60 f s , repetition rate 10 Hz) at 860 run in the lowest energy absorption band of the s p e c i a l pair (see inset in F i g . l ) . Probing of the light-induced absorption changes at various wave­lengths Xpr was performed with synchronized pulses as a function of time delay tD. The excitation energy density was kept low, only 13% of the reaction centers were excited by each excitation pulse. 2 9 2 O l 1 0 100 1000 Deity Τ I m (ps] F i g . l . Time-resolved absorption data ( f u l l c i r c l e s ) at a probing wavelength of 545 run for reaction centers of Rb. sphaeroides R26.1 ( e x c i t a t i o n wavelength 860 nn)« The curves are calculated according to two models given i n the text. Inset: absorption spectrum of the sample. The time resolved absorption measurements are shown i n F i g . l and 2 /10/. The experimental data points are indicated as f u l l c i r c l e s . The curves are calculated according to two k i n e t i c models. The f i r s t model ( s o l i d curve) i s d i r e c t l y deduced from the present­l y accepted electron t r a n s f e r scheme;-i.e. electron tra n s f e r occurs from P * d i r e c t l y to s t a t e P * H ~ , Consequently, the complete reaction k i n e t i c s u n t i l the formation of s t a t e P+Q~ involves three i n t e r ­mediates connected by two time constants. The second model (dashed curve, schematic shown i n Fig.3) considers an additional i n t e r ­mediate s t a t e with the l i f e t i m e of 0.9 ps* The experimental r e s u l t s of F i g . l and 2 were taken at Apr = 545 nm (HA absorption band), 785 nm ( H , B absorption), and 665 nm ( H ~ , B ~ absorption range). A l l three measurements indicate that the model of Fig*3 (dashed curve, four intermediates, three time constants) i s required to explain the experimental data. E s p e c i a l l y at λ = 665 nm and 785 nm the 0.9 ps k i n e t i c i s outstanding, while the 3,5 ps and the 200 ps k i n e t i c s a l s o appear. The amplitudes of the absorption changes connected with s t a t e s I i to I 4 can be used to r e l a t e the various intermediate states to molecular configurations. The molecular interpretation i n Fig.3 i s based on the following arguments: The f i r s t s t a t e I i i s the excited e l e c t r o n i c s t a t e . I t s monoexponential decay with a time constant of 3.5 ps i s observed around 920 nm. This time constant i s the f i r s t one (τι) i n the sequence of our model. The subsequent s t a t e s are intermediates of the electron t r a n s f e r process. The next st a t e i s I 2 decaying r a p i d l y with a time constant of 0.9 ps. The most obvious assignment of I 2 i s the configuration P * B * containing 0.02 0.01 c • δ ο c 0.006 0.004 0.002 -ι ι ι ι I - 7 Θ -1 i ι - 785 η· I ι ι - // Γ . 1 ^ ^ ^ ^ ^ ^ ^ ^ I - 663 na \ ®_ • · ι i 1 1 10 100 Delay TiM [ps] 1000 Fig.2. Tine-resolved absorption data ( f u l l c i r c l e s ) for probing wavelengths of (a) 785 nm and (b) 665 nm for reaction centers of wild-type Rb. sphearoides ATCC 17023 (excitation 860 nm) I 1 hV 35 ps I 3 0.9 ps 200ps Ρ Β HQ •P*B"HQ- p*BH"a *Ρ*ΒΗ0Γ Fig.3· Scheme of the sequence of states involved in the primary electron transfer in reaction centers. f 294 a bacteriochlorophyll anion r a d i c a l . This interpretation i s supported by the following observation /10/. ( i ) Iz shows strongly reduced absorption at the wavelength of 797 nm, where B~ ( i n v i t r o ) has strongly reduced absorption, ( i i ) During the rapid decay of I 2 t h i s absorption recovers, and ( i i i ) I 2 does not show absorption decrease i n the wavelength range where bacteriopheophytin absorbs. State I 3 represents the next intermediate where the electron has reached the bacteriopheophytin Η forming the configuration to P*BH~Q. The r e l a t e d decrease of* the Η absorption at 545 nm i s c l e a r l y seen a f t e r the decay of s t a t e Iz and p r i o r to the formation of I 4 . F i n a l l y , with a time constant of 200 ps, the electron a r r i v e s in st a t e I4 at the quinone OK ( s t a t e P+BBQ-). I n conclusion: We have performed an experimental study of the primary charge transfer process i n the reaction centers of Hb. sphaeroides. The ana l y s i s of the k i n e t i c data ( i n a l i n e a r model) strongly suggests that the primary charge t r a n s f e r to the bacteriopheophytin proceeds v i a the accessory bacteriochlorophyll as a true intermediate. The following reaction scheme r e s u l t s : After e x c i t a t i o n of the s p e c i a l p a i r Ρ an electron i s transferred with a time constants of 3*5 ps to the accessory bacteriochloro­phy l l * Here the electron resides f o r the short time of 0.9 ps before i t reduces the bacteriopheophytin. . Acknowledgement The authors acknowledge valuable collaboration with and supply of the Hb. sphaeroides samples by D. Oesterhelt, H. Scheer, and H.U. S t i l z . The work was supported by the Deutsche Forschungsgemeinschaft, SFB143. REFERENCES 1. J . Delsenhofer, 0. Ερρ, Κ. Miki, R. Huber, Η. Michel, J . Mol. B i o l . 180, 385 (1984) 2. H. Michel, 0· Epp, J . Delsenhof er, EMBO J . 5, 2445 (1986) 3. J.P. Allen, G. Feher, T.O. Yeates, H. Komiya, D.C. Rees, Proc. Natl. Acad. S e i . USA 84, 5730 (1987) 4· J#-L. Martin, J . Breton, A.J. Hoff, A. Migus, A. Antonetti, Proc. Natl. Acad. S e i . USA 83, 957 (1986) 5· G.R. Fleming, J.~L. Martin, J * Breton, Nature 333, 190 (1988) 6. J . Breton, J.-L. Martin, A. Migus, A. Antonetti, A. Orszag, Proc. Natl. Acad. S e i . USA 83, 5121 (1986) 7. D. Holten, C. Hoganson, M.W. Windsor, C.C. Schenck, W.W. Parson, Α. Migus, R.L* Fork, C.V. Shank, ΒJochim. Biophys. Acta 592, 461 (1980) 8. W. Zinth, M.C. Nuss, M.A. Franz, W. Kaiser, H. Michel, i n "Antenna and Reaction Centers of Photosynthetic Bacteria*» Springer Se r i e s in Chemical Physics, Vol. 42, Ed. M.E. Michel-Beyerle, Springer, Heidelberg 1985, p. 286 9. V.A. Shuvalov, A.V. Klevanik, FEBS Lett . 160, 51 (1983) V.A. Shuvalov, J . Amesz, L.N.M. Duysens, ΒJochim. Biophvs. Acta 851, 327 (1986) 10. W. Holzapfel, ü. Finkele, W. Kaiser, D. Oesterhelt, H. Scheer, ' H.U. S t i l z , W. Zinth, Chem. Phvs. L e t t . , to be published (1989) 

Delsenhof er,

in &quot;Antenna and Reaction Centers of Photosynthetic Bacteria*»

https://epub.ub.uni-muenchen.de/3575/1/3575.pdf

Sequential electron transfer in the primary photosynthetic reaction of Rhodobacter sphaeroides

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