Crystallized reaction centers from Rhodopseudomonas viridis (i) are photochemically active with electron transfer from the special pair to the quinones, (ii) show dichroism giving valuable information on the orientation of the different chromophores and (iii) allow chemical treatment in the crystalline phase

Clayton

Den Blanken

H. Michel

Holten

Michel

Paillotin

Philipson

Prince

Shuvalov

Thornber

Trosper

Vermeglio

W. Kaiser

W. Zinth

English

Zinth, Wolfgang

Kaiser, Wolfgang

Michel, H.

Universität München: Elektronischen Publikationen

128 Btochlmtca et Btophyswa Acta, 723 (1983) 128-131 
Elsevier Blomedtcal Press 
BBA Report 
BBA 40019 
E F F I C I E N T  P H O T O C H E M I C A L  ACTIVITY AND S T R O N G  D I C H R O I S M  OF S I N G L E  CRYSTALS 
OF REACTION CENTERS F R O M  R H O D O P S E U D O M O N A S  V I R I D I S  
w ZINTHa, W KAISERaandH MICHELb 
a Physlk Departement der Techmschen Umversltat Munchen, D.8000 Munchen, and b Max-Planck-lnsntut fur Btochemw, D-8033 
Martmsrwd (F R G ) 
(Received December 6th, 1982) 
Key words Reactton center crystal, Bactertal photosynthests, Chromophore orwntatton, (Rps wrtdts) 
Crystallized reaction centers from Rhodopseudomonas viridis (i) are photochemicaily active with electron 
transfer from the special pair to the quinones, (ii) show dichroism giving valuable.information on the 
orientation of the different chromophores and (iii) allow chemical treatment in the crystalline phase. 
In the past, reaction centers from photosyn- 
thetic bacterm have been investigated m intact 
cells, in photosynthetic membranes or isolated m 
detergent solutions. Comparison of the optical 
spectra of intact cells with those of isolated reac- 
tion centers suggested that careful ~solatlon does 
not seriously affect the properties of the reaction 
centers [1-6]. The spectral properues and the pig- 
ment  composmon of isolated reaction centers from 
Rhodopseudomonas  vtrtdts have been studied ex- 
tenswely [1-6] Recently, the photosynthetic reac- 
tion centers from thas photosynthetic bacterium 
were crystallized [7]. In this paper  we report on the 
properties of these crystals 
Reaction centers isolated w~th the detergent 
N,N-dlmethyldodecylarmne N-oxide contain four 
BChl b and two BPh b molecules [1] The pr imary 
electron donor, called P-960 due to its maxamum 
of absorbance, is made up of two of the four BChl 
b molecules, whale the 'p r imary '  electron acceptor 
is a qulnone-lron complex [1]. There are one 
menaqumone and one to two ub~qumones per 
P-960 [8]. Isolated reacUon centers from Rps  vtrt- 
A bbrevlatlons BChl, bactenochlorophyll, BPh, 
bactenopheophytm 
dts also contain four heme groups (two 'cyto- 
chrome c-558' and two 'cytochrome c-553'), wluch 
are probably bound to the same protein subumt 
[6], They feed electrons to P-960 +. 
The reaction centers were ~solated as described 
recently with the following modification [7]. For 
the molecular sieve chromatography step we used 
as column material Fractogel TSK HW 55 S 
(Merck, Darmstadt) ,  thus step was repeated. In 
contrast to earher preparations [7] using different 
column material, we now observe bleaching of 
P-960 upon lllumanatlon Thus finding indicates 
that the qumones are still present in the new 
preparations. The crystals were grown as described 
previously [7]. The space group of these crystals IS 
P4~(3)2~2 with a tetragonal unit cell dlmens~on of 
223 × 223 x 114 ,~ containing eight asymmetric 
umts per crystallographac umt cell. The volume of 
the asymmetric umt and the molecular wetght of 
the reactton centers make it more hkely that each 
asymmetric umt ~s occupied by two reaction center 
molecules than by one or three With these figures 
were calculate 2.8- l0 )8 reaction center molecules 
per cm 3, corresponding to 4 . 7 - l 0  -3 mol/1. In 
space group P41(3)212, a 4-fold screw axas is paral- 
lel to the z-direction whereas nomntersectlng 2-fold 
screw axes (perpen&cular to the 4-fold axis) are 
0005-2728/83/0000-0000/$03 00 © 1983 Elsevier Science Pubhshers 
parallel to the x- and y-dlrecUon. In most cases the 
crystals grow in the form of tetragonal columns 
with the preferential growth dtrectlon parallel to 
the z-axis. Fortunately, the crystals can be ob- 
tained in the form of tlun platelets of several 
hundred micrometers in length and width and 
with a uniform thickness of a few micrometers 
whilst the surfaces of the crystals are stall parallel 
to the xy, xz, or yz planes In these platelets the 
orientauon of the crystaUographlc axes can be 
determined upon visual inspection 
Spectral transmission measurements and ob- 
servations of the photochemical activity were made 
with the help of two spectrometers, a microscope, 
and various photodetectors Care was taken to 
keep the measuring light at the lowest possible 
level to avoid absorption changes due to photo- 
chemical activity during the spectral transmission 
measurements. Photoreactions were studied by s~de 
illumination of the crystal with actinic light of 
)~ > 850 nm (incandescent lamp plus cutoff filter). 
The photodetector was protected against scattered 
hght by one of the spectrometers. During our 
investigations the crystals were kept m a closed 
cell, which contained a salt solution of 2 7 M 
ammonium sulfate, 20 mM sodium phosphate, 
0 1% N, N-dImethyldodecylarmne N-oxide, 1% tri- 
e thylammonlum phosphate, and 1% heptane- 
1,2,3-trlol, pH 6 5. 
The crystals are uniform in their absorption 
properties Scanning the cross-section of the 
crystals or determining the extihctlon coefficient 
for different crystal tlucknesses suggests a h i g h  
degree of crystal homogeneity. It should be em- 
phasized at tlus point that the same absorption 
spectrum was obtained from different crystals pre- 
pared over a period of several months. 
The optical spectrum of the reaction centers in 
the starting solution is shown m F~g. la; it ~s 
essentmlly identical to absorption data found in 
the literature for Rps. wrtdts m solution [1-5]. 
The optical absorption spectrum of reaction 
center crystals from Rps vmdls is presented in 
Fig la  (broken line) and the change m absorption 
versus wavelength for irradlauon with infrared 
hght at ~ >_ 850 nm is shown in Fig. lb. All spectra 
are recorded with unpolarized hght. The difference 
spectrum gives positive and negauve contmbutions 
to the absorption spectrum taken in the 'dark '  
129 
LO0 600 800 1000 
"- - 'T "1 T 1 1 "1 
® 
"~® 2 Solut,0n - - ~  
u 
r, ~ ^ 
.D I I ~ "~ 1 / 
/ /  
® 
.< o 
,el 
-01 
400 600 800 1000 
Wavelength ~. l" nm'l 
Fig I (a) Unpolanzed absorption spectra of reaction centers 
from Rps wrldts of a solution (sohd curve) and of a smg|¢ 
crystal (broken hnc) The crystal was oriented with its z-axis 
pcrperdlcular to the mcasunng hghI beam The reaction center 
concentration of the solution was adjusted to have equal ab- 
sorbance at ~. = 530 nm (b) Absorbanc¢ change of the crystal 
upon fl]urmnauon with actinic hghI with ~ > 850 nm 
Quahtatlvely, the difference spectrum is smular to 
that in pubhshed data on reaction centers in solu- 
tion, but the dlchrolsm of the crystals (see below) 
gives rise to quantitative changes (e.g., at 960 and 
around 830 nm) [3,9,13]. 
Comparing the absorption spectrum of the re- 
action centers in solution and in the crystal (Fig. 
l a) we note drastic differences of the strong ab- 
sorption around 830 nm and of the absorption 
band at 960 nm. It  is generally accepted [1] that 
the strong band at 830 nm is mainly due to two 
molecules of BChl b and that the maxamum at 
P-960 is indicative of the exclton-bound dlmer 
consisting of the other two BChl b molecules. The 
smaller absorption of unpolarlzed hght at 830 nm 
in the crystal is due to the strong dlchrolsm which 
will be discussed below The reduced absorption at 
960 nm in the crystals has two causes First, it 
results from the dlchrolsm of our samples and, 
second, it is caused - to some extent - by the 
oxidation of special pairs. In fact, we find absorp- 
tion at 1 3/~m (without excess illumination) in the 
130 
crystals suggesting the exastence of P-960 ÷ states 
frozen in at room temperature. Furthermore, we 
made the interesting observation that chemical 
treatment of the small crystals is possible. The 
addmon  of the reducing agent ascorbate to the 
hquld surrounding of the crystals leads to an m- 
crease m the absorbance at 960 nm by approx 
20%, quite likely, P-960 ÷ radicals are reduced At 
the same time we find an enhanced absorption at 
558 nm which points to a reduction of cytochrome 
c present in the reaction center. This chemical 
treatment does not disorder the crystals as checked 
by X-ray diffraction. 
Of special interest is the finding that the P-960 
band in our crystals bleaches completely and re- 
versibly upon dlummation with radiation of 2~ > 
850 nm The photoeff ioency appears to be high, 
we estimate a value between 0.1 and 1. Our data 
show that photoactlvlty, 1 e., the charge separation 
in the special pair and the charge transfer to an 
acceptor (probably the qumones), is preserved 
upon crystalhzaUon. In fact, the difference ab- 
sorbance spectrum of Fig. lb is sirmlar to pub- 
hshed spectra reported to be characteristic of the 
P+Q- states [3,9] 
Fig. 1 a shows a shoulder around 850 nm and m 
Fig. lb we found a distinct bleaching at the same 
1 I I I I [ 
E£ 
,../~.,..~...~ -,\,, \ 
) I ) I " t ' - - -  ~ 4,,,- _ 
?.00 600 800 I000 
Wavelength ~l'nm2 
Fig  2 Polarized absorp t ion  spect ra  of  a single crystal  of  reac- 
t ton center  p repara t ions  f rom Rps wnd~s T h e  electric vector of  
the  measu r ing  light was  chosen  pe rpen&cu la r  ( sohd  curve) and  
paral lel  (broken  hne)  to the z-axis 
wavelength. This observation agrees with the inter- 
pretation that the absorption at 850 nm represents 
the second dlmer band of the special parr [1,9,10]. 
C D  investigations [11], studies of oriented cells 
[12], and our measurements on crystals with 
polarized light (see below) lead to the same conclu- 
sions 
The small absorption band at 680 nm is an 
oxidation product of BChl b [2]. Special care m 
avoiding oxygen during the preparation reduces 
this minor absorption band. 
In Fig 2 we present transmission studies of the 
crystals, where the light was polarized with the 
electric vector either parallel or perpendicular to 
the 4-fold screw axis (z-axis) with the beam per- 
pendicular to the surface of the platelets. The two 
absorption spectra of Fig 2 show drastic dif- 
ferences over the whole spectral range. Most strik- 
ing is the disappearance of the dimer peak P-960 
for the Ell polarlsatlon and the very strong absorp- 
tion of the BChl b molecules at 830 nm for E2 
The data of Fig. 2, while not sufficient for a final 
assignment of the orientation of the different chro- 
mophores,  give a series of new reformation on 
pigment orientation. (1) The strong absorption of 
P-960 for E± suggests that the major dimer axis is 
located perpendicular to the z-axis of the crystals, 
while the absorption band at 850 nm (assumed to 
be the second &met  absorption) has components 
parallel to the x-axis as seen from the shoulder in 
the E± spectrum. (11) The two BChl b molecules, 
which are thought to absorb at 830 nm at room 
temperature, are also strongly oriented m the 
crystals. They have their major Qv absorption 
perpendicular to the z-axJs. The Qx transntlon of 
the same molecules give rise to the enhanced ab- 
sorption m the Eli spectrum at 600 nm (in) The 
two BPh b molecules absorb at 790 and 810 nm, 
according to the literature [9] Our absorpuon data 
of the Ell spectrum show a maximum at 805 nm 
and a shoulder around 780 nm. Furthermore, m 
the E± spectrum a shoulder appears around 800 
nm These data suggest that the BPh b molecules 
have an orientation substantially different from 
that of the BChl b molecules. The Qv band of the 
two BPh b molecules absorbs around 800 nm in 
the Eli and E± spectrum and the enhanced absorp- 
tion at 530 nm in the E± spectrum is due to the 
Q ~ transitions 
More  de taded  studies on the d lchrolsm and the 
pho tochemica l  activity of  the crystals are in pro- 
gress 
The  authors  hke to thank Professor  D. Oester-  
helt  for suppor t  and interest  m this work and M 
Sander  for exper imenta l  assistance. 
No te  added in proof (Received March 24th, 1983) 
Protein de te rnunat ions  using crystals of  known 
vo lume  suggest that  the crystals conta in  only one 
react ion center  molecule  per asymmetr ic  unit  
References 
1 Thornber, J P, Dutton, P L, Faler, J ,  Forman, A Holten, 
D, Olson, J M, Parson, W W, Prince, R C, Tlede, D M 
and Wmdson, M W (1978) m Proceedings of the 4th Inter- 
national Congress on Photosynthesis, 1977 (Hall, D O, 
Coombs, J and Goodwm, T W, eds), pp 55-70, The 
B~ochemlcal Society, London 
131 
2 Trosper, T L, Benson, D L and Thornber, J P (1977) 
Bloclum Biophys Acta 460, 318-330 
3 Holten, D, Windsor, M W, Parson, W W and Thornber, 
J P (1978) Blochlm Blophys Acta 501, 112-126 
4 Clayton, RK and Clayton, BJ (1978) Blochlm Blophys 
Acta 501,478-487 
5 Prince, R C, T,ede, D M, Thornber, J P and Dutton, L 
(1977) Btoclum Blophys Acta 462, 467-490 
6 Thornber, J P, CogdeU, R J,  Seftor, R E B and Webster, 
G D (1980) Btochlm Blophys Aca 593, 60-75 
7 Michel, H (1982)J Mol Blol 158, 567-572 
8 Trosper, T L (1982) Abstr PS-I-20, 10th Annual Meetmg 
of American Society for Photoblology, Vol 10, p 88, 
Umverslty of Brmsh Columbia 
9 Vermegho, A and Pafllotm, G (1982) Blochlm Blophys 
Acta 681, 32-40 
10 Den Blanken, H J and Hoff, A J (1982) Biochlm Blophys 
Acta 681,365-374 
11 Phlhpson, KD and Sauer, K (1973) Biochemistry 12, 
535-539 
12 Padlotm, G, Vermegho, A and Breton, J (1979) Btochlm 
Blophys Acta 545, 249-263 
13 Shuvalov, V A, Krakhmaleva, 1 N and Khmov, V V (1976) 
Biochlm Blophys Acta 449, 597-601 


Efficient photochemical activity and strong dichroism of single crystals of reaction centers from Rhodopseudomonas viridis

Crossref

Zinth, W.

Kaiser, W.

Michel, H. 

MPG.PuRe

Open Access LMU ( Ludwig-Maximilians-Univ. München)

128 Btochlmtca et Btophyswa Acta, 723 (1983) 128-131 Elsevier Blomedtcal Press BBA Report BBA 40019 EFFICIENT PHOTOCHEMICAL  ACTIVITY AND STRONG DICHROISM OF S INGLE CRYSTALS OF REACTION CENTERS FROM RHODOPSEUDOMONAS V IR ID IS  w ZINTHa, W KAISERaandH MICHELb a Physlk Departement der Techmschen Umversltat Munchen, D.8000 Munchen, and b Max-Planck-lnsntut fur Btochemw, D-8033 Martmsrwd (F R G ) (Received December 6th, 1982) Key words Reactton center crystal, Bactertal photosynthests, Chromophore orwntatton, (Rps wrtdts) Crystallized reaction centers from Rhodopseudomonas viridis (i) are photochemicaily active with electron transfer from the special pair to the quinones, (ii) show dichroism giving valuable.information on the orientation of the different chromophores and (iii) allow chemical treatment in the crystalline phase. In the past, reaction centers from photosyn- thetic bacterm have been investigated m intact cells, in photosynthetic membranes or isolated m detergent solutions. Comparison of the optical spectra of intact cells with those of isolated reac- tion centers suggested that careful ~solatlon does not seriously affect the properties of the reaction centers [1-6]. The spectral properues and the pig- ment composmon of isolated reaction centers from Rhodopseudomonas vtrtdts have been studied ex- tenswely [1-6] Recently, the photosynthetic reac- tion centers from thas photosynthetic bacterium were crystallized [7]. In this paper we report on the properties of these crystals Reaction centers isolated w~th the detergent N,N-dlmethyldodecylarmne N-oxide contain four BChl b and two BPh b molecules [1] The primary electron donor, called P-960 due to its maxamum of absorbance, is made up of two of the four BChl b molecules, whale the 'pr imary'  electron acceptor is a qulnone-lron complex [1]. There are one menaqumone and one to two ub~qumones per P-960 [8]. Isolated reacUon centers from Rps vtrt- A bbrevlatlons BChl, bactenochlorophyll, BPh, bactenopheophytm dts also contain four heme groups (two 'cyto- chrome c-558' and two 'cytochrome c-553'), wluch are probably bound to the same protein subumt [6], They feed electrons to P-960 +. The reaction centers were ~solated as described recently with the following modification [7]. For the molecular sieve chromatography step we used as column material Fractogel TSK HW 55 S (Merck, Darmstadt), thus step was repeated. In contrast o earher preparations [7] using different column material, we now observe bleaching of P-960 upon lllumanatlon Thus finding indicates that the qumones are still present in the new preparations. The crystals were grown as described previously [7]. The space group of these crystals IS P4~(3)2~2 with a tetragonal unit cell dlmens~on of 223 × 223 x 114 ,~ containing eight asymmetric umts per crystallographac umt cell. The volume of the asymmetric umt and the molecular wetght of the reactton centers make it more hkely that each asymmetric umt ~s occupied by two reaction center molecules than by one or three With these figures were calculate 2.8- l0 )8 reaction center molecules per cm 3, corresponding to 4 .7- l0  -3 mol/1. In space group P41(3)212, a 4-fold screw axas is paral- lel to the z-direction whereas nomntersectlng 2-fold screw axes (perpen&cular to the 4-fold axis) are 0005-2728/83/0000-0000/$03 00 © 1983 Elsevier Science Pubhshers parallel to the x- and y-dlrecUon. In most cases the crystals grow in the form of tetragonal columns with the preferential growth dtrectlon parallel to the z-axis. Fortunately, the crystals can be ob- tained in the form of tlun platelets of several hundred micrometers in length and width and with a uniform thickness of a few micrometers whilst the surfaces of the crystals are stall parallel to the xy, xz, or yz planes In these platelets the orientauon of the crystaUographlc axes can be determined upon visual inspection Spectral transmission measurements and ob- servations of the photochemical ctivity were made with the help of two spectrometers, a microscope, and various photodetectors Care was taken to keep the measuring light at the lowest possible level to avoid absorption changes due to photo- chemical activity during the spectral transmission measurements. Photoreactions were studied by s~de illumination of the crystal with actinic light of )~ > 850 nm (incandescent lamp plus cutoff filter). The photodetector was protected against scattered hght by one of the spectrometers. During our investigations the crystals were kept m a closed cell, which contained a salt solution of 2 7 M ammonium sulfate, 20 mM sodium phosphate, 0 1% N, N-dImethyldodecylarmne N-oxide, 1% tri- ethylammonlum phosphate, and 1% heptane- 1,2,3-trlol, pH 6 5. The crystals are uniform in their absorption properties Scanning the cross-section of the crystals or determining the extihctlon coefficient for different crystal tlucknesses uggests a h igh  degree of crystal homogeneity. It should be em- phasized at tlus point that the same absorption spectrum was obtained from different crystals pre- pared over a period of several months. The optical spectrum of the reaction centers in the starting solution is shown m F~g. la; it ~s essentmlly identical to absorption data found in the literature for Rps. wrtdts m solution [1-5]. The optical absorption spectrum of reaction center crystals from Rps vmdls is presented in Fig la (broken line) and the change m absorption versus wavelength for irradlauon with infrared hght at ~ >_ 850 nm is shown in Fig. lb. All spectra are recorded with unpolarized hght. The difference spectrum gives positive and negauve contmbutions to the absorption spectrum taken in the 'dark' 129 LO0 600 800 1000 "-- 'T "1 T 1 1 "1 ® "~® 2 Solut,0n - -~  u r, ~ ^ .D I I ~ "~ 1 / / /  ® .< o ,el -01 400 600 800 1000 Wavelength ~. l" nm'l Fig I (a) Unpolanzed absorption spectra of reaction centers from Rps wrldts of a solution (sohd curve) and of a smg|¢ crystal (broken hnc) The crystal was oriented with its z-axis pcrperdlcular to the mcasunng hghI beam The reaction center concentration of the solution was adjusted to have equal ab- sorbance at ~. = 530 nm (b) Absorbanc¢ change of the crystal upon fl]urmnauon with actinic hghI with ~ > 850 nm Quahtatlvely, the difference spectrum is smular to that in pubhshed ata on reaction centers in solu- tion, but the dlchrolsm of the crystals (see below) gives rise to quantitative changes (e.g., at 960 and around 830 nm) [3,9,13]. Comparing the absorption spectrum of the re- action centers in solution and in the crystal (Fig. l a) we note drastic differences of the strong ab- sorption around 830 nm and of the absorption band at 960 nm. It is generally accepted [1] that the strong band at 830 nm is mainly due to two molecules of BChl b and that the maxamum at P-960 is indicative of the exclton-bound lmer consisting of the other two BChl b molecules. The smaller absorption of unpolarlzed hght at 830 nm in the crystal is due to the strong dlchrolsm which will be discussed below The reduced absorption at 960 nm in the crystals has two causes First, it results from the dlchrolsm of our samples and, second, it is caused - to some extent - by the oxidation of special pairs. In fact, we find absorp- tion at 1 3/~m (without excess illumination) in the 130 crystals suggesting the exastence of P-960 ÷ states frozen in at room temperature. Furthermore, we made the interesting observation that chemical treatment of the small crystals is possible. The addmon of the reducing agent ascorbate to the hquld surrounding of the crystals leads to an m- crease m the absorbance at 960 nm by approx 20%, quite likely, P-960 ÷ radicals are reduced At the same time we find an enhanced absorption at 558 nm which points to a reduction of cytochrome c present in the reaction center. This chemical treatment does not disorder the crystals as checked by X-ray diffraction. Of special interest is the finding that the P-960 band in our crystals bleaches completely and re- versibly upon dlummation with radiation of 2~ > 850 nm The photoeffioency appears to be high, we estimate a value between 0.1 and 1. Our data show that photoactlvlty, 1 e., the charge separation in the special pair and the charge transfer to an acceptor (probably the qumones), is preserved upon crystalhzaUon. In fact, the difference ab- sorbance spectrum of Fig. lb is sirmlar to pub- hshed spectra reported to be characteristic of the P+Q- states [3,9] Fig. 1 a shows a shoulder around 850 nm and m Fig. lb we found a distinct bleaching at the same 1 I I I I [ E£ ,../~.,..~...~ -,\,, \ ) I ) I " t ' - - -  ~ 4,,,- _ ?.00 600 800 I000 Wavelength ~l'nm2 Fig 2 Polarized absorption spectra of a single crystal of reac- tton center preparat ions from Rps wnd~s The electric vector of the measur ing light was chosen perpen&cular  (sohd curve) and parallel (broken hne) to the z-axis wavelength. This observation agrees with the inter- pretation that the absorption at 850 nm represents the second dlmer band of the special parr [1,9,10]. CD investigations [11], studies of oriented cells [12], and our measurements on crystals with polarized light (see below) lead to the same conclu- sions The small absorption band at 680 nm is an oxidation product of BChl b [2]. Special care m avoiding oxygen during the preparation reduces this minor absorption band. In Fig 2 we present ransmission studies of the crystals, where the light was polarized with the electric vector either parallel or perpendicular to the 4-fold screw axis (z-axis) with the beam per- pendicular to the surface of the platelets. The two absorption spectra of Fig 2 show drastic dif- ferences over the whole spectral range. Most strik- ing is the disappearance of the dimer peak P-960 for the Ell polarlsatlon and the very strong absorp- tion of the BChl b molecules at 830 nm for E2 The data of Fig. 2, while not sufficient for a final assignment of the orientation of the different chro- mophores, give a series of new reformation on pigment orientation. (1) The strong absorption of P-960 for E± suggests that the major dimer axis is located perpendicular to the z-axis of the crystals, while the absorption band at 850 nm (assumed to be the second &met absorption) has components parallel to the x-axis as seen from the shoulder in the E± spectrum. (11) The two BChl b molecules, which are thought to absorb at 830 nm at room temperature, are also strongly oriented m the crystals. They have their major Qv absorption perpendicular to the z-axJs. The Qx transntlon of the same molecules give rise to the enhanced ab- sorption m the Eli spectrum at 600 nm (in) The two BPh b molecules absorb at 790 and 810 nm, according to the literature [9] Our absorpuon data of the Ell spectrum show a maximum at 805 nm and a shoulder around 780 nm. Furthermore, m the E± spectrum a shoulder appears around 800 nm These data suggest hat the BPh b molecules have an orientation substantially different from that of the BChl b molecules. The Qv band of the two BPh b molecules absorbs around 800 nm in the Eli and E± spectrum and the enhanced absorp- tion at 530 nm in the E± spectrum is due to the Q ~ transitions More detaded studies on the dlchrolsm and the photochemical  ctivity of the crystals are in pro- gress The authors hke to thank Professor D. Oester- helt for support and interest m this work and M Sander for experimental ssistance. Note added in proof (Received March 24th, 1983) Protein deternunations using crystals of known volume suggest hat the crystals contain only one reaction center molecule per asymmetric unit References 1 Thornber, J P, Dutton, P L, Faler, J, Forman, A Holten, D, Olson, J M, Parson, W W, Prince, R C, Tlede, D M and Wmdson, M W (1978) m Proceedings of the 4th Inter- national Congress on Photosynthesis, 1977 (Hall, D O, Coombs, J and Goodwm, T W, eds), pp 55-70, The B~ochemlcal Society, London 131 2 Trosper, T L, Benson, D L and Thornber, J P (1977) Bloclum Biophys Acta 460, 318-330 3 Holten, D, Windsor, M W, Parson, W W and Thornber, J P (1978) Blochlm Blophys Acta 501, 112-126 4 Clayton, RK and Clayton, BJ (1978) Blochlm Blophys Acta 501,478-487 5 Prince, R C, T,ede, D M, Thornber, J P and Dutton, L (1977) Btoclum Blophys Acta 462, 467-490 6 Thornber, J P, CogdeU, R J, Seftor, R E B and Webster, G D (1980) Btochlm Blophys Aca 593, 60-75 7 Michel, H (1982)J Mol Blol 158, 567-572 8 Trosper, T L (1982) Abstr PS-I-20, 10th Annual Meetmg of American Society for Photoblology, Vol 10, p 88, Umverslty of Brmsh Columbia 9 Vermegho, A and Pafllotm, G (1982) Blochlm Blophys Acta 681, 32-40 10 Den Blanken, H J and Hoff, A J (1982) Biochlm Blophys Acta 681,365-374 11 Phlhpson, KD and Sauer, K (1973) Biochemistry 12, 535-539 12 Padlotm, G, Vermegho, A and Breton, J (1979) Btochlm Blophys Acta 545, 249-263 13 Shuvalov, V A, Krakhmaleva, 1 N and Khmov, V V (1976) Biochlm Blophys Acta 449, 597-601 

Abstr PS-I-20, 10th Annual Meetmg of American Society for Photoblology, Vol 10, p 88, Umverslty of Brmsh Columbia

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Efficient photochemical activity and strong dichroism of single crystals of reaction centers from Rhodopseudomonas viridis

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