M.-E.Michel-Beyerle (Editor)

Reaction Centers of Photosynthetic Bacteria Feldafing-II-Meeting

With 165 Figures

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Contents

P A R T I Native Reaction Centers: Structure and Spectroscopy

F. R E I S S - H U S S O N , B . A R N O U X , A . D U C R U I X , M . R O T H , M . P I C A U D and C . A S T I E R :

Reaction Center from Wi ld Type Y Rhodobacter sphaeroides: Primary Structure of the L and M Subunits; Carotenoid and Detergent Structures in the Three-Dimensional Crystals 3

Q. Z H O U , T . A . M A T T I O L I and B . R O B E R T Probing Reaction Center Asymmetry Us ing L o w Temperature Absorpt ion Spectroscopy of Rhodobacter sphaeroides Reaction Centers Containing Bacteriopheophytin Anions 11

A . V E R M E G L I O , D . G A R C I A and J . B R E T O N Cytochrome Arrangement in Reaction Centers of Different Species of Photosynthetic Bacteria 19

W. M Ä N T E L E , M . L E O N H A R D , M . B A U S C H E R , E . N A B E D R Y K , J . B R E T O N and D . A . M O S S

Infrared Difference Spectroscopy of Electrochemically Generated Redox States in Bacterial Reaction Centers 31

E J . L O U S , M . H U B E R , R . A . I S A A C S O N and G . F E H E R E P R and E N D O R Studies o f the Oxidized Primary Donor in Single Crystals of Reaction Centers of Rhodobacter sphaeroides R - 2 6 45

F . L E N D Z I A N , B . E N D E W A R D , M . P L A T O , D . B U M A N N , W. L U B I T Z and K . M Ö B I U S

E N D O R and T R I P L E Resonance Investigation of the Primary Donor Cat ion Radica l P g 6 5 in Single Crystals o f Rhodobacter sphaeroides R - 2 6 Reaction Centers 57

A . A N G E R H O F E R , J . G R E I S , V . A U S T , J . U . V O N S C H Ü T Z and H . C . W O L F Triple t State A D M R of Bacterial Reaction Centers at L o w Transit ion Frequencies 69

S. B U C H A N A N and H . M I C H E L Investigation of Quinone Reduction in RhodoPseudomonas viridis by F T I R Difference Spectroscopy and X - R a y Diffract ion Analysis 75

D . L . T H I B O D E A U , J . B R E T O N , C . B E R T H O M I E U , K . A . B A G L E Y , W. M Ä N T E L E and E . N A B E D R Y K

Steady-State and Time-Reso lved F T I R Spectroscopy of Quinones in Bacterial Reaction Centers 87

VIII

P A R T II Native Reaction Centers: Electron Transfer Dynamics

G . J . S M A L L , R . J A N K O W I A K , M . S E I B E R T , C . F . Y O C U M and D . T A N G Spectral Hole Burning Studies o f Photosystem II Reaction Centers: Exc i ted State Structure, Charge Separation and Energy Transfer Dynamics 101

C . K I R M A I E R and D . H O L T E N Evidence for an Inhomogeneous Distr ibut ion of Reaction Centers on the Timescale o f the Pr imary Electron Transfer Events 113

U . F I N K E L E , K . D R E S S L E R , C . L A U T E R W A S S E R and W. Z I N T H Analysis o f Transient Absorpt ion Data from Reaction Centers of Purple Bacteria 127

K . D R E S S L E R , U . F I N K E L E , C . L A U T E R W A S S E R , P. H A M M , W. H O L Z A P F E L , S. B U C H A N A N , W. K A I S E R , H . M I C H E L , D . O E S T E R H E L T , H . S C H E E R , H . U . S T I L Z and W. Z I N T H

Similarities o f the Pr imary Charge Separation Process in the Photosynthesis of Rhodobacter sphaeroides and RhodoPseudomonas viridis 135

G . H . A T K I N S O N , H . H A Y A S H I , M . T A S U M I and S. K O L A C Z K O W S K I Picosecond Resonance Raman Spectroscopy of Rhodobacter sphaeroides Reaction Centers 141

S.G. B O X E R , D J . L O C K H A R T , S. F R A N Z E N and S .H. H A M M E S Electr ic F ie ld Modula t ion of the Fluorescence Lineshape for Photosynthetic Reaction Centers: A N e w Probe of the Primary Electron Transfer Mechanism 147

A . O G R O D N I K , U . E B E R L , R . H E C K M A N N , M . K A P P L , R . F E I C K and M . E . M I C H E L - B E Y E R L E

One Step Electron Transfer to P + H " in Reaction Centers of Rhodobacter sphaeroides Derived from Dichroic Excitation Spectra of Electr ic F ie ld Modulated Fluorescence Y i e l d 157

M . G . M Ü L L E R , K . G R I E B E N O W and A . R . H O L Z W A R T H Energy Transfer and Charge Separation Kinet ics in the Reaction Center of Chloroflexus aurantiacus Studied by Picosecond Time-Resolved Fluorescence Spectroscopy 169

R. F E I C K , J . L . M A R T I N , J . B R E T O N , M . V O L K , G . S C H E I D E L , T. L A N G E N B A C H E R , C . U R B A N O , A . O G R O D N I K and M . E . M I C H E L - B E Y E R L E

Biexponential Charge Separation and Monoexponential Decay of P + H ~ in Reaction Centers of Chloroflexus aurantiacus 181

P. S E B B A N , P. P A R O T , L . B A C I O U , P. M A T H IS and A . V E R M E G L I O Analog Effects of L o w Temperature and L i p i d Rig id i ty on the Distr ibut ion o f T w o Conformational States o f the Reaction Centers 189

P A R T III Modified Reaction Centers: Effects of Mutagenic and Chemical Modifications

I. S I N N I N G , J . K O E P K E and H . M I C H E L Recent Advances in the Structure Analysis of RhodoPseudomonas viridis React ion Center Mutants 199

M . M . Y A N G , W.J . C O L E M A N and D . C . Y O U V A N Genet ic Coding Algor i thms for Engineering Membrane Proteins 209

M . H U B E R , E . J . L O U S , R . A . I S A A C S O N , G . F E H E R , D . G A U L and C . C . S C H E N C K

E P R and E N D O R Studies of the Oxid ized Donor in Reaction Centers of Rhodobacter sphaeroides Strain R - 2 6 and two Heterodimer Mutants i n which Hist idine M 2 0 2 or L173 was Replaced by Leucine 219

C . C . S C H E N C K , D . G A U L , M . S T E F F E N , S .G. B O X E R , L . M c D O W E L L , C . K I R M A I E R and D . H O L T E N

Si te-Directed Mutations Affec t ing Primary Photochemistry in React ion Centers: Effects o f Dissymmetry in the Special Pair 229

W.W. P A R S O N , V . N A G A R A J A N , D . G A U L , C . C . S C H E N C K , Z . - T . C H U and A . W A R S H E L

Electrostatic Effects on the Speed and Directionali ty of Electron Transfer in Bacterial Reaction Centers: The Special Role of Tyrosine M - 2 0 8 239

K . A . G R A Y , J.W. F A R C H A U S , J . W A C H T V E I T L , J . B R E T O N , U . F I N K E L E , C . L A U T E R W A S S E R , W. Z I N T H and D . O E S T E R H E L T

The Ro le of Tyrosine M210 in the Initial Charge Separation in the React ion Center o f Rhodobacter sphaeroides 251

H . U . S T I L Z , U . F I N K E L E , W. H O L Z A P F E L , C . L A U T E R W A S S E R , W. Z I N T H and D . O E S T E R H E L T

Si te-Directed Mutagenesis o f Threonine M222 and Tryptophan M 2 5 2 in the Photosynthetic Reaction Center of Rhodobacter sphaeroides 265

W.J . C O L E M A N , E . J . B Y L I N A , W. A U M E I E R , J . S I E G L , U . E B E R L , R . H E C K M A N N , A . O G R O D N I K , M . E . M I C H E L - B E Y E R L E and D . C . Y O U V A N

Influence of Mutagenic Replacement of Tryptophan M250 on Elect ron Transfer Rates Involving Primary Quinone in React ion Centers o f Rhodobacter capsulatus 273

S.J. R O B L E S , J . B R E T O N and D . C . Y O U V A N Transmembrane H e l i x Exchanges Between Quasi-Symmetric Subunits o f the Photosynthetic Reaction Center 283

J . B R E T O N , J . - L . M A R T I N , J . - C . L A M B R Y , S.J. R O B L E S and D . C . Y O U V A N G r o u n d State and Femtosecond Transient Absorption Spectroscopy of a Mutant o f Rhodobacter capsulatus which Lacks the Initial Electron Acceptor Bacteriopheophytin 293

X

N . W . W O O D B U R Y , A . K . T A G U C H I , J.W. S T O C K E R and S .G . B O X E R

Prel iminary Characterization of p A T - 3 , a Symmetry Enhanced Reaction Center Mutant o f Rhodobacter capsulatus 303

A . S T R U C K , D . B E E S E , E . C M I E L , M . F I S C H E R , A . M Ü L L E R , W. S C H Ä F E R and H . S C H E E R

M o d i f i e d Bacterial Reaction Centers: 3. Chemically M o d i f i e d Chromophores at Sites B A , B B and H A , H B 313

K . W A R N C K E and P . L . D U T T O N Effect o f Cofactor Structure on Control of Electron Transfer Rates at the Q A Site o f the Reaction Center Protein 327

P A R T I V Reaction Centers: Modelling of Structure/Function-Relationship

A . F R E I B E R G and T . P U L L E R I T S Energy Transfer and Trapping in Spectrally Disordered Photosynthetic Membranes 339

M . R . W A S I E L E W S K I , G . L . G A I N E S , III, M . P . O ' N E I L , W . A . S V E C , M . P . N I E M C Z Y K and D . M . T I E D E

Mul t i -S tep Electron Transfer in R i g i d Photosynthetic Models at L o w Temperature: Requirements for Charge Separation and Spin-Polar ized Radical Ion Pair Formation 349

P.O.J. S C H E R E R , W. T H A L L I N G E R and S.F. F I S C H E R Electronic Couplings for Light Induced Charge Transfer in Covalently Bonded Donor-Acceptor Systems 359

J . F A J E R , K . M . B A R K I G I A , K . M . S M I T H , E . Z H O N G , E . G U D O W S K A - N O W A K and M . D . N E W T O N

Mic ro -Env i ronmenta l Effects on Photosynthetic Chromophores 367

A . S C H E R Z , J . R . E . F I S H E R and P. B R A U N Simulation o f the Absorpt ion and Circular Dichroism Spectra for the Primary Electron Donor in Reaction Centers of Purple Bacteria and Photosystem II 377

M . B I X O N , J . J O R T N E R and M . E . M I C H E L - B E Y E R L E On the Primary Charge Separation in Bacterial Photosynthesis 389

P.O.J. S C H E R E R Mul t ip le Exci ted States o f Photosynthetic Reaction Centers 401

J. V R I E Z E and A . J . H O F F Exci ton Band M i x i n g in RhodoPseudomonas viridis 409

XI

A . L . M O R R I S , J .R . N O R R I S and M . C . T H U R N A U E R A n Extended M o d e l for Electron Spin Polarization in Photosynthetic Bacteria 423

E.W. K N A P P and L . N I L S S O N C a n Elect ron Transfer be Influenced by Protein Dynamics: The Transfer f rom Cytochrome C to the Special Pair in Photosynthetic Reaction Centers 437

Concluding Rcmarks

M . E . M I C H E L - B E Y E R L E and H . S C H E E R Beyond Nat ive Reaction Centers 453

Subject Index 464

Beyond Native Reaction Centers M . E . Miche l -Beye r l e 1 a n d H . Scheer 2

1 Institut für Physikalische und Theoretische Chemie,Technische Univers i tä t M ü n c h e n , W 8046 Garching, F R G

2 Botanisches Institut der Ludwig-Maximi l ians-Univers i tä t , Menzinger St raße 67, W 8 0 0 0 M ü n c h e n 19, F R G

The past five years after solving the crystal structure of the bacterial reaction center from

RhodoPseudomonas (Rp.) viridis [1] - and the first Feldafing meeting [2] - have witnessed a

remarkable concentration of interdisciplinary research on this pigment-protein complex. The

importance of the reaction center is related to its key function in transforming photons into

electrochemical energy and, in more general terms, to the fact, that it is the first membrane

protein of which the three-dimensional structure is known. Beyond these central biological

bearings, the reaction center has a stimulating effect on many developments in related fields.

Its complex pigment-protein and pigment-pigment interactions required refinement of existing

and development of novel spectroscopic techniques. Since the reaction center constitutes the

best-defined supramolecular electron donor/acceptor System in chemistry, it has the potential

of becoming a test case for electron transfer theories. This year's Feldafing II Conference

intended to provide a status-report on the prescnt understanding of reaction center function,

highlighting the new biochemical, spectroscopic and theoretical approaches. Still under the

impression of the Conference we w i l l try to give our personal views and some leading

references , preferentially to papers within this book, on the spectacular progress and

limitations related to biochemical and genetic modifications of the reaction center collected in

Part III.

U n t i l recently, there were only few ways to introduce specific modifications into the

reaction center structure which were suited for a parametric study of Charge Separation

processes. Sclective modifications were limited to the two quinones, Q A and Q B (Warncke &

Dutton), the presence and nature of the divalent metal ion located between Q A and Q B [3],

and the removal of the H-protein subunit [4]. The only possibility to interfere wi th one of the

four bacteriochlorophylls, the one at the Site B B in reaction centers of Rhodobacter (Rb.)

sphaeroides, consisted in the borohydride treatment [5] and the chemistry of this procedure is

still under debate [6].

454

In the meantime, this Situation has been fundamentally improved by the development o f

two new tools: Site-directed mutagenesis of the Polypeptides and, most recently, thermally

induced exchange of four o f the six pigments, i.e. the two monomeric bacteriochlorophyll

molecules (BChl) at the sites B A B and the two bacteriopheophytins (BPh) at the sites H A B by

chemically modified analogues (see Append ix for nomenclature).

M U T A G E N E S I S

Site-directed mutagenesis o f reaction centers has been pioneered wi th Rb. capsulatus [7], a

bacterium which is genetically more stable than Rb, sphaeroides. However, reaction centers

from this species have not yet been crystallized. A s shown in various reports in this volume

(Gray et al . , Huber et a l . , Schenck et al . , Stilz et al.), site-specific mutagenesis has also been

successful in reaction centers from Rb. sphaeroides for which the three-dimensional structure

is known (Reiss-Husson et al.) [8]. The techniques are presently established in three

laboratories and rapidly advancing. It should be pointed out, however, that the structures of

the "native" Rb. sphaeroides reaction center (from the wi ld type strain 2.4.1 and the

carotenoid-less mutant R26) are by c±Q. less resolved than the best defined structure o f the

Rp.viridis reaction center (2.2%). Unfortunately, directed mutagenesis in the latter species still

meets considerable Problems and remains a challenge. Some interesting, spontaneously formed

mutants are already now available (Sinning et al.). They show Single- or multiple replacements

of amino acid residues in the vicini ty of (or at) the quinone ( Q B ) binding site and for some of

them a differential crystal structure has been performed.

In summary, comparing reaction centers from these three purple bacteria there is at present

an inverse relationship between the capability to mutate selectively and the extent of structural

Information available, and a continuing effort is needed to balance the two opposing trends.

In this context, it is still rewarding to explore reaction centers from other species which may

be more amenable to both, crystallographic and genetic manipulations. The reaction center

from the green bacterium Chloroflexus (Cf.) aurantiacus may be such a candidate. It shows

some very interesting variations with respect to pigment composition and protein structure

(Feick et al.), and has been crystallized recently.

The mutations focused on were designed or selected to influence specific electron transfer

Steps and thus addressed the protein environment of the cofactors participating in the electron

transfer sequence. These are the primary donor, the B C h l dimer (P) with its components P A

and P B , the B C h l monomer and the B P h at the sites B A and H A , respect ively , and the two

quinones, Q A and Q B .

455

(1) The first class o f mutations are those in which amino acid residues close to the central

four N-a toms of any of the six tetrapyrroles are exchanged. In "native" reaction centers the

B C h l molecules carry histidines (His) ligated to the central M g - i o n , while the BPhs have

hydrophobic amino-acids l ike isoleucine (He) instead at the respective positions. Interchange of

any one o f these His-residues leads to a change of the pigment bound at the respective site,

e.g. a Hi s - I l e mutation results in a reaction center in which the respective B C h l is replaced by

B P h , and vice versa (Schenck et al . , Woodbury et al.). U p to now there is only one report on

an "intermediate" amino acid, the glutamate (Glu) , which at least partly leads to B C h l binding.

Remarkably, in the reaction center of the green bacterium Cf. aurantiacus which has He

instead of Hi s at the relevant position of the B B binding site, one B C h l is replaced by B P h ,

most l ike ly at the B B - s i t e (Feick et al.).

The pigment exchange experiments (see below) on reaction centers from Rb. sphaeroides

and Rp.viridis ful ly corroborate this selection rule o f the binding sites. Presently without any

exception, pigments are selectively introduced at the sites B A B and H A B according to the

presence or absence of a central M g - i o n , respectively. This selection principle has been

observed irrespective of the structural modifications at the pigment periphery, provided an

exchange was possible at all (Struck et al.).

(2) The second set o f site-directed mutations involved aromatic amino-acids located in (or

close to) the electron transfer path and preferentially those which are highly conserved in

type-II bacterial reaction centers and photosystem-II of plants.

In photosystem-II, two such amino acids, tyrosine (Tyr-160 on D 2 - subun i t and Tyr-161 on

D x - subun i t ) seem to actively participate in the electron transfer sequence between the water-

splitting site and the primary donor radical cation, P680 + [9]. The homologous T y r - M 1 6 2 in

reaction centers from Rb. sphaeroides is located between P and the putative binding site for

the cy tochrome-c 2 (acting as electron donor to P + ) ; its replacement results in a decreased rate

of electron transfer (Gray et al . , unpublished results).

O f special interest wi th respect to primary Charge Separation was the tyrosine T y r - M 2 0 8 in

reaction centers of Rb. capsulatus (Parson et al.) and analogous T y r - M 2 1 0 of Rb. sphaeroides

(Gray et al.). This amino acid residue is located between the P A component of the primary

donor and the B P h at H A and is not conserved in Cf. aurantiacus. Another aromatic amino

acid (conserved in all reaction centers sequenced up to now) is the tryptophane T r p - M 2 5 0 in

Rb. capsulatus (Coleman et al.) and the analogous T r p - M 2 5 2 in Rb. sphaeroides (Stilz et al.)

which is the only amino acid residue between the B P h at H A and Q A and in van der Waals-

distance to both cofactors. The so-far studied replacements of these aromatic amino acids at

456

both positions lead to a pronounced decrease of the respective electron transfer rates; they also

tend to reduce the binding affinity o f the primary quinone, Q A .

(3) A third set of mutations addressed the asymmetry of Charge Separation which seems to

proceed exclusively along the active A - b r a n c h , the ratio of relative primary rates being

k A / k B > 2 5 in Rb. sphaeroides [10]. Replacement of glutamate, G l u - L 1 0 4 , located close to the

1 3 1 - C O - g r o u p of H A in Rb. capsulatus was the first example [11] investigated; the latest

attempts towards "symmetrization" o f the two pigment branches involve the exchange of large

structural motifs between the two protein subunits, L and M (Robles et al. , Woodbury et al.).

So far, it has not been possible to remove or diminish the asymmetry of Charge Separation.

Not even after complete blocking o f the A - b r a n c h , electron transfer along the B-branch

became detectable (Robles et al.).

P I G M E N T E X C H A N G E

In a complementary approach to study structure-function relationships in the reaction center,

the tetrapyrrole pigments are directly modified. Site-directed mutagenesis is at present

restricted to B C h l - B P h exchange (see above), and potentially to introducing biosynthetic

precursors. A more general method suitable for exchanging the native B C h l and BPh by

chemically modified analogues, was developed very recently (Struck et al.).

Accessible sites are B A and B B , into which a variety o f pigments bearing a central metal-

ion have been introduced, and H A and H B , into which a corresponding set of free bases

(bacteriopheophytins) have been exchanged. So far, the majority of studies has been

performed wi th reaction centers from the carotenoidless Rb. sphaeroides R26 , but the method

is applicable to other purple bacteria. Aga in , B C h l a - or BPha-containing species are

favourable, while in Rp. viridis containing b-type pigments, only the BPh sites are accessible,

probably due to the stiffening by the bound cytochrome-c. By the proper choice of reaction

center, pigment and reaction conditions, there is even the possibility of non-symmetric

modifications. A s an example, only the B A site is accessible in reaction centers of the

carotenoid-containing wi ld- type strain Rb. sphaeroides 2.4.1. The primary donor has as yet

resisted any exchange, but it may be anticipated that the successful exchange of the other

pigments triggers research which w i l l eventually render the P-site accessible, too. Whenever a

pigment exchange was possible, the reaction center showed reversible bleaching, although

electron transfer was slowed down e.g. in the B C h l a - B A B - ( 3 - v i n y l ) - B C h l a modification (see

Append ix for the proposed nomenclature). Pigments like the aforementioned, which show

distinctly blue-shifted absorption maxima, were also useful to study electronic interactions,

and for the assignment of absorption bands in the crowded regions of the spectrum.

457

S T R U C T U R A L A N D F U N C T I O N A L C O N T R O L O F M O D I F I E D R E A C T I O N C E N T E R S

The f inal objective of modifications is certainly only achieved in control experiments which

relate the modifications to changes in structure, interactions and electron transfer dynamics.

Control by c ry s t a l structure analysis. It is self-evident that the control o f a modified

reaction center by crystal structure analysis would be highly desirable, although hard to

achieve. However , there are some arguments which make us think on this point in relative

terms. For many Problems related to electronic couplings in electron transfer and, in

particular, for a füll quantitative analysis, ultrahigh resolved structural data are needed.

Calculations on the basis o f the structure of Rp. viridis revealed much less charge-

transfer character o f the excited B C h l - d i m e r X P * than the ones based on the "intermediate"

refinement [12](Scherer et al.). However, even on the basis of this ultimate refinement

calculations of electronic overlaps turned out to be problematic. This is especially true when

these depend on the proximity o f two methyl groups, as it seems to be the case for the

electronic overlap between B C h l at B A and B P h at H A [12]. Moreover , it should be kept in

mind that even the best-resolved structure o f the ground State configuration at room

temperature might differ from the one encountered after protein relaxation, or at the low

temperatures to be used in many spectroscopic studies. A n d , last, but not least, non-periodic

water molecules might play a large and different role in native and modified reaction centers.

Thus, such limitations together with the requirement o f high resolution certainly lower the

expectations l inked to future differential crystal structures of modif ied reaction centers, in

particular of those from Rb. sphaeroides, as long as the native structure is only resolved to

Furthermore, the resolution o f crystals from modified reaction centers might suffer from

additional complications, such as an increase of structural or energetic inhomogeneity and

stability problems, as previously encountered in e.g. borohydride treated reaction centers from

Rb. sphaeroides (A l l en et al . , private communication).

S t r u c t u r a l and f u n c t i o n a l c o n t r o l by spec t roscopy . In order to study interactions and

dynamics in the native and modified reaction centers the entire arsenal o f spectroscopy has to

be employed. The interpretation of dynamic data profits from more general developments in

electron transfer theory [13] and new experimental results in the field of artificial

donor/acceptor Systems where specific features can be influenced in a parametric way.

(Scherer et al . , Wasielewski et al.).

The road to the identification of detailed changes in pigment-pigment and pigment-protein

interactions opens through steady State optical (i) and magnetic resonance (ii) spectroscopy.

458

Temperature-dependent absorption (Sebban et al . , Z h o u et al.) and fluorescence spectroscopy,

is complemented and supplemented by structure-based quantum calculations (Vrieze & Hoff)

and more complex techniques, as L D - (Breton et al.), C D - (Scherz et al.), F T I R - (Buchanan

& M i c h e l , Män te l e et al . , Thibodeau et al.) and Stark-spectroscopy [14], together with the

diversity o f magnetic Single- and multiple resonance methods (Angerhofer et al . , Lendzian et

al . , Lous et al . , Mor r i s et al.). A s , for instance, changes i n the absorption spectrum of the

pigments may occur, modified reaction centers could favour probing of selected states.

Pigment exchange, l ike the replacement o f the 3-acetyl- by a v inyl -group in B C h l has already

proven its potential towards disentanglement of congested spectral absorption features (Struck

et al.). Modif ica t ion induced spectral changes could also lead to the revelance of spectroscopic

features which in native reaction centers are buried below congested absorption bands. In this

context, a long-hunted State is the radical pair P + B C h l " at the site B A .

O n the basis of such spectroscopic data, electron transfer dynamics and energetics of

intermediate Charge-transfer states can be investigated. This is achieved by the appplication of

a large variety of methods working in the frequency- or time domain (hole-burning

experiments (Small et al.), femto- to millisecond-time-resolved pump-probe absorption

(Breton et al . , Dressler et al . , Finkele et al . , K i r m a i e r & Holten), R a m a n - (Atkinson et al.)

and fluorescence measurements (Mül le r et al.), including absorption experiments in static

magnetic [15] and microwave fields [16]. The congested absorption bands of the different

neutral (ground and excited State) and charged species in the reaction center and the necessity

to avoid multiple excitation, have initiated considerable improvements in the sensitivity of

detection ( A O D < 10" 3) (Finkele et al.), a condition which is especially diff icul t to meet in

experiments with high temporal resolution (femto- to nanoseconds).

Apar t from these well-established techniques, the knowledge of the three-dimensional

structure of the reaction center stimulated the development of an experimental approach [17]

which yields the orientation of the dipole moment of the radical pair formed in competition

to the prompt fluorescence from the excited primary donor State, 1 P * . The method is based on

the measurement of the anisotropy of electric field induced change of the pr imary Charge

Separation rate, as reflected in the stcady State fluorescence yield (Boxer et al. , Ogrodnik et

al.). Especially when applied to time-resolved fluorescence measurements, the technique

carries high potential for identifying the mechanism of primary Charge Separation; i.e. to infer

on the relative contributions o f a two-step, sequential vs. an unistep, superexchange

mechanism (Bixon et al.). The electric field induced energy shift of radical pairs carrying a

large dipole moment is also reflected in field dependent absorption transients i n the

picosecond- [18] and nanosecond time domain [19] and might be very useful in mapping out

energetics in reaction centers.

459

Data on electron transfer dynamics are interpreted wi th in the conventional nonadiabatic

approach [20,21] which provides the conceptual framework for the description o f electron

transfer processes i n the reaction center [22](Bixon et al.). The analysis o f the rates i n terms of

the electronic coupling matrix dement and a F ranck-Condon factor requires the independent

determination of energy Parameters, such as the free energy gap between equi l ibr ium nuclear

configurations o f ini t ia l and final states. With the assumption of sufficiently fast protein

relaxation times, it is then possible to extract from the rate and its temperature dependence

information on the effects a given modification has on the structure, thereby affecting the

electronic couplings and the energetics, changing free energy differences and protein

reorganization energies, and thus the F ranck-Condon factor. Such a treatment cannot,

however, account for possible thermal contraction (which might be even different i n modified

reaction centers) as wel l as for energetic and structural inhomogeneities.

Changes of electronic couplings can be induced by a variety of effects such as (i) differences

in the arrangement of the cofactors, either following from changes in the binding site or in

the nature o f the cofactors, or both; (ii) differences in the Charge distribution at the cofactors

in the ground, excited or charged states due to specific interactions wi th the environment, e.g.

with polar amino acids, and (iii) differences in superexchange mediated electronic interactions

between cofactors which rely on details of the structure (and energetics) of cofactors and

protein. In this context, the mutagenic replacements T r p - M 2 5 0 - P h e , L e u in Rb. capsulatus

(Coleman et al.) and T r p - M 2 5 2 - P h e , T y r in Rb. sphaeroides (Stilz et al.) were investigated.

Changes in the nuclear Franck-Condon factor may be encountered when modifications

influence energetics via a change in the free energy ( A G ) or the protein reorganization energy

(A) which is related to relaxation of the amino acid environment upon electron transfer. Such

changes can be either induced by changes in the nature o f the protein or of the cofactors. It

is one of the characteristic features o f native reaction centers that the membrane-spanning,

Charge Separation rates are temperature-independent, i.e. they proceed under conditions where

A G roughly equals the protein reorganization energy A. The investigation o f reaction centers

with modif ied energetics is very attractive, since these may constitute "new species" where this

"optimization principle" is violated.

Moreover , the still controversially debated question of the mechanism of primary Charge

Separation (Dressler et al . , Finkele et al . , K i r m a i e r & Holten) is directly related to the energy

of the radical pair P+BChl " at the site B A , i n that it determines the relative contributions of

sequential and superexchange mechanisms (Bixon et al.). In this context, the first explicit

studies addressed the role of T y r - M 2 0 8 in reaction centers of Rb. capsulatus (Parson et al.)

and the respective T y r - M 2 1 0 in Rb. sphaeroides (Gray et al.) in femtosecond time-resolved

460

experiments and electrostatic calculations (Parson et al.). The phenomenology of the primary

electron transfer rate and its temperature dependence can be modeled wi th in the frame of the

nonadiabatic theory and is consistent wi th the notion of a smaller free energy of the State

P+BChl" at the site B A (Bixon et al.). A controlled free energy shift of the State P + B C h l " at

the site B A is also the goal o f the more recent pigment exchange experiments (Struck et al.).

Apar t from its role in determining both, the free energy differences within the electron

transfer cascade and the reorganization energy related to the structural relaxation of the amino

acid residues upon electron transfer, the protein might even undergo larger structural changes

upon electron transfer and do so in a complex time-dependent pattern (Knapp & Nilsson).

This might be influenced also by modifications, leading ultimately to changes in both, the

Franck-Condon factor and electronic couplings.

In view of this diversity o f complex effects possibly exerted by the cofactors and the

protein environment on electron transfer dynamics, the spectroscopic characterization of

modif ied, de facto new reaction centers can only aim at a crude differentiation between

effects on structure and energetics. Such a global characterization should enable us, however,

to screen those modifications for which special efforts towards crystallization and X - r a y

structure analysis appear rewarding.

O U T L O O K

O n the basis of the strategies outlined above, a cri t ical evaluation of the goals, of unwanted

side-effects, and of the design of experiments on modified reaction centers is useful. Some

immediate goals, already addressed before and especially emphasized in Parts 1,11 and IV of

this volume are related to basic questions o f the electron transfer mechanism in reaction

centers, e.g.: (1) Al though the most recent, highly sensitive femtosecond data (Dressler et al. ,

Finkele et al.) can be readily explained by a sequential electron transfer mechanism, the extent

of a parallel "superexchange" unistep contribution to the primary Charge Separation is not yet

clear (Bixon et al.). (2) Al though the structural symmetry in terms of cofactor arrangement

and thereby electronic couplings favours electron transfer along the active branch, the

contribution of asymmetry in energetics to unidirectionality is not understood (Parson et al.).

(3) While the present X - r a y analysis gives only a static structure, structural or energetic

changes due to protein relaxation (Knapp & Nilsson) related to electron transfer may be

functionally important.

Addressing these questions requires criteria for the selection of suitable modifications, and

an open mind for possible interferences. In a naive expectation, all modifications should show

461

the largest influenae in their immediate environment. However, more far-reaching effects

have to be envisaged, even upon seemingly "minor" modifications, and are expected to become

more pronounced wi th "major" ones. For example, a change of the polar tyrosine versus

Phenylalanin ( T y r - L 2 2 2 - P h e in Rp.viridis) at roughly equal molar volume exerts "a dramatic

effect on the structure" of this reaction center (Sinning et al.), and the Ty r -M208(M210) -Phe

mutation close to P leads to a loss of Q A wh ich is located more than 20% away "on the other

side" o f the reaction center (Parson et al . , Gray et al.). T o define and eventually forecast just

what may constitute a "minor" or "major" modification and its possible side effects w i l l be one

of the most challenging problems of the future. The design of changes introduced into the

reaction center by replacing amino-acids or pigments has to account for steric factors, such as

e.g. molar volume and specific geometry (Fajer et al . , Yang et al.), and interactions as e.g.

electrostatic, dipolar and hydrophobic ones, and polarizabilities. It w i l l require all possible

techniques including optical and magnetic spectroscopies, X - r a y crystallography and ultimately

spectroscopic structure-determining techniques (applicable now only to much smaller proteins),

which allow to identify the implanted changes on a microscopic scale. Moreover, the design of

modified reaction centers as well as the exprimental efforts thereupon have to be accompanied

by quantum-chemical and improved molecular dynamics calculations, specifically parametrized

for reaction centers (Scherer; Parson et al . , K n a p p & Nilsson).

While these questions address mainly the reaction center as a System by itself, its relations

to other photosynthetic functional units is becoming more and more important. These include

the interaction with the antennas (Freiberg & PuIIerits), in particular the core ones, and the

interactions wi th secondary electron donors, the cytochromes (Vermeglio et al.) and possibly

also the acceptors (cyt b /c complexes). As a transducer between excitation energy and

membrane potential, the function of the reaction center depends on its co-action with the

antennas on one side, and electron donors and acceptors on the other. Since the Short t ime-

scale of excitation energy transfer prohibits any diffusional collision process to be involved,

the antenna probably has to be tightly associated with the reaction center. There are several

lines of biochemical evidence to this, but the site(s) and nature of the interactions can

currently only be speculated on. It is also unknown, i f certain pigment(s) present in the

reaction center, act(s) as the preferred acceptor(s) of excitation energy, and i f there are

similarly preferred energy donors in the antennae. In view of the rapid equilibration of

excitation energy wi th in the antenna, the transfer to the reaction center is a cri t ical Step

(Mül ler et al.). The same reasoning applies to electron transfer to and from the reaction

center. Al though it occurs on a much slower time-scale, its distance dependence is much more

cri t ical . A n y modificat ion, pigment or Polypeptide, should therefore not only be scrutinized

regarding its effect wi th in the isolated reaction center. In view of this, it appears mandatory

(i) to characterize modif ied reaction centers as extensively as possible, and (ii) to go beyond

462

isolated reaction centers and include their natural environment and functional partners.

Last, but not least, there is an abundance o f questions which are not related to the function

of the reaction center, but rather to its being the first member o f a large and important class

of membrane proteins. Such questions address the folding o f the individual protein subunits,

the incorporation o f the pigments and other cofactors, the assembly of the subunits with each

other and with neighbouring complexes and the mechanisms regulating these processes. There

is f inally the challenging problem, whether significant molecular properties relate D N A

sequences to the respective translation products (Yang et al.). The reaction center is a

fortunate case in this respect, because it has a variety of intrinsic probes which can be used in

following Organization and turn-over without distorting the System.

B y the same token, however, these processes are also complicated in reaction centers

because they are heavily loaded wi th cofactors, which are l ikely to exert a considerable

influence on the reaction center structure. The effects o f the tetrapyrrolic cofactors w i l l be

even more pronounced i n antenna complexes, where the pigments constitute an even larger

Proportion. A satisfactory modeling of the Organisation principle of the reaction center is

therefore expected to give valuable insight, too, into the construction principles of antennae.

R E F E R E N C E S References addressing articles in the book are referred by the names only, references to

other work are cited by numbers.

1. (a) J . Deisenhofer, O. E p p , K . M i k i , R . Huber, and H . M i c h e l , J . M o l . B io l . 180, 385-398 (1984); (b) J . Deisenhofer, H . M i c h e l , E M B O J . 8, 2149-2170 (1989)

2. "Antennas and Reaction Centers o f Photosynthetic Bacteria" ( M . E . Miche l -Beyer le , ed.) Springer 1985

3. R . J . Debus, G . Feher, and M . Y . Okamura, Biochemistry 24, 2488-2500 (1985) 4. R . J . Debus, G . Feher, and M . Y . Okamura, Biochemistry 25, 2276-2287 (1986) 5. P. Maro t i , C . K i r m a i e r , C . Wraight, D . Holten, and R . M . Pearlstein, Biocheim.

Biophys. Ac t a 810, 132-139 (1985) 6. (a) Shuvalov, V . A . and L . N . M . Duysens, Proc. Nat l . A c a d . Sei. U S A 83, 1690-1694

(1986); (b) A . Struck et al . , to be published 7. E . J . Byl ina and D . C . Youvan , Proc.Natl .Acad.Sci . U S A 85, 7226-7230 (1988) 8. (a) H . K o m i y a . T . O . Yeates, D . C . Rees, J.P. A l l e n and G . Feher, Proc. Nat l . A c a d . Sei.

U S A 85, 9012-9016 (1988); (b) D . E . B u d i l , P. Gast, C . H . Chang, M . Schiffer and J .R. Norr is , Biochemistry 24, 7516-7521 (1985)

9. (a) S. Gerken , K . Brettel, E . Schlodder, an H . T . Witt, F E B S Lett. 237, 69-75 (1988); (b) R . J . Debus, B . A . Barry, G . T . Babcock, and L . Mcln tosh , Proc. Nat l . Accad . Sei. U S A 85, 427-430 (1988)

10. W. Aumeier , U . Ebe r l , A . Ogrodnik, M . V o l k , G . Scheidel, R . Feick and M . E . Miche l -Beyer le in "Progress in Photosynthetic Research" ( M . Baltscheffsky, ed.), Martinus N i j h o f f Publ . (1989)

11. E . J . By l ina , C . K i r m a i e r , L . M c D o w e l l , D . Holten, and D . C . Youvan , Nature 336, 182-184 (1988)

463

12. M . Plato, K . M ö b i u s , M . E . Miche l -Beyer le , M . B ixon and J . Jortner, J . A m . Chem. Soc. 110, 7279-9285 (1988)

13. "Perspectives in Photosynthesis" (J. Jortner and B . Pulman, eds.), K l u w e r Press, Dordrecht 1990

14. (a) A . Gopher , M . Schönfe ld , M . Y . Okamura, and G . Feher, Biophys. J . 48, 311 (1984); (b) D J . Lockhar t , R . F . Goldstein, and S .G. Boxer, J . Chem. Phys. 88, 1408-1415 (1988); M . Lösche , G . Feher, and M . Y . Okamura, Proc. Nat l . A c a d . Sei. U S A 85, 107-111 (1987)

15. A . Ogrodnik , M . V o l k , R . Letterer, R . Feick and M . E . Miche l -Beyer le , , B ioch im. Biophys. A c t a 936, 361-371 (1988)

16. W. Lersch and M . E . Miche l -Beyer le in "Advanced E P R in Biology and Biochemistry (Hof f , A . J . , ed.), pp.685-705 Elsevier Amsterdam 1989

17. D . J . Lockhar t , R . F . Goldstein, and S .G. Boxer, J . Chem. Phys. 89, 1408 (1988) 18. D J . Lockhar t , C K i r m a i e r , D . Hol ten, S .G. Boxer, J . Phys. Chem. 94, 6987-6995

(1990) 19. M . V o l k , Thesis, i n preparation 20. R . A . Marcus , J . Chem. Phys. 24, 966-978 and 979-989 21. J . Jortner, J . Chem. Phys. 64, 4860-4867 (1976) 22. R . A . Marcus , Isr. J . Chem. 28, 205-213 (1988)

A P P E N D I X

The many modifications now possible pose some nomenclature Problems which are currently

unsettled. For bacterial reaction centers we suggest to use the fol lowing self-consistent

approach.

N a t i v e r eac t ion centers. Prolein subunits are labeled L , M and H . Amino acid residues are

named either by three or one letter code and labeled by the respective subunit and their

Position wi th respect to the N-terminus . The two pigment branches are labeled by A (active)

and B (inactive), as suggested by George Feher. The co/actor binding sites are labelled by

P A B for the primary donor, B A B for the neighbouring monomeric pigments, H A B for the

subsequent ones, Q A B for the two quinones, and M B S for the metal binding site.

A s long as the P - and B - sites contain only bacteriochlorophyll a or b, and H contains

bacteriopheophytins a and b, the binding sites are equivalent to the pigments, B C h l a , B C h l b

and BPha , BPhb in the nomenclature. If this is not the case, as e.g. in the reaction centers of

Cf. aurantiacus the pigments have to be used together with the binding site. Hence, B B (=

B C h l a - B B ) i n the purple bacterium Rb. sphaeroides becomes B P h a - B B i n Cf. aurantiacus .

M o d i f i e d reac t ion centers: Amino acid exchanges are characterized in the conventional way.

Native amino acid residue, followed by protein subunit and Position, followed by the new

amino acid residue. A s an example, E - M 1 5 8 - Q = G l u - M 1 5 8 - G l n denotes mutation of

glutamate at position 158 on the M-subun i t to glutamin. Pigment exchanges are treated in a

similar way. As an example, B C h l a - B A - 1 3 2 O H - B C h l a denotes an exchange of B C h l a at the

site B A against 1 3 2 - O H - B C h i a .