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2

Recent EPR Studies on the Bacterial


Photosynthetic Reaction Centre
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

~ ~ _ _ _ _ _ _ _ _ _ _ ~ ~

BY STEFAN WEBER
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1 Introduction

The primary processes of bacterial photosynthesis, the conversion of electro-


magnetic energy (light) into chemical energy, are mediated by an integral
membrane protein complex called the bacterial photosynthetic reaction centre
(RC) in which a sequence of photoinduced electron-transfer and proton-
transfer reactions takes place. Our understanding of these processes was
greatly enhanced through the determination of the three-dimensional struc-
tures of the RCs from two purple photosynthetic bacteria: Rhodopseudomonas
viridis (Rps. viridis)1-3 and Rhodobacter sphaeroides (Rb. ~phaeroides).~-~
Recently, a major breakthrough has also been achieved with the determination
of the crystal structure of one of the light-harvesting complexes found in
bacteria, LH2, from Rhodopseudomonas acidophila (Rps. acidophila).lo-
Knowing what both assemblies, the photosynthetic RC and the light-har-
vesting complexes, look like at the atomic level, we are now close to a complete
picture of the bacterial photosynthesic unit (PSU).12-14 Figure 1 depicts
schematically the intracytoplasmic membrane of purple bacteria with its
primary photosynthetic apparatus.
In most purple bacteria, the photosynthetic membranes contain two types of
light-harvesting complexes, LH 1 and LH2. LH 1 immediately surrounds the
RC,15-17 whereas LH2 is not in direct contact with the RC but transfers
energy to the RC through LH 1.I8 A 1: 1 stoichiometry exists between the RC
and LHl l 9 whereas the number of LH2 complexes varies according to growth
conditions. In contrast to the bacterial RC, bacterial light-harvesting com-
plexes are paradigms of symmetry and simplicity: most contain either bacterio-
chlorophyll a (BChl a) or bacteriochlorophyll b (BChl b) as light-absorbing
chromophores, while carotenoids serve as accessory light-harvesting pigments

Abbreviations: BChl ulblg, bacteriochlorophyll ulblg; BPhe ulb, bacteriopheophytin ulb; Chl u,
chlorophyll u; CRPM, correlated radical pair mechanism; CW, continuous-wave; ENDOR,
electron-nuclear double resonance; EPR, electron paramagnetic resonance; ESE, electron spin
echo; ESEEM, electron spin echo envelope modulation; ESP, electron spin polarization; hf,
hyperfine; hfc, hyperfine coupling constant; HOMO, highest occupied molecular orbital; LUMO,
lowest unoccupied molecular orbital; MO, molecular orbital; RC, reaction centre; RF, radio
frequency; TRIPLE, electron-nuclear-nuclear triple resonance.

Electron Paramagnetic Resonance, Volume 17


0The Royal Society of Chemistry, 2000
43
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44 Electron Paramagnetic Resonance

/
Light
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LH2

Figure 1 The photosynthetic unit of bacteria consisting of the RC surrounded by the


antenna complexes LHI and LH2. The diagram shows a view from above the
membrane. The BChl pigments are represented as black lines. Incident light is
absorbed by LH2 and the excitation energy is transferred via the LHI to the
specialpair in the RC (modiJiedfromi4)

and provide photoprotection. In LH2, the chromophores are arranged in a


ring-like structure of high symmetry: see Figure 2.
When a photon is absorbed by one of the BChl molecules, the absorbed
energy spreads extremely rapidly (in less than 1 ps!) to the others in the ring,
on account of their favourable spacing and orientation. Where the rings touch
in the close-packed membrane, the energy can jump the distance to an adjacent
complex. LH1 absorbs at longer wavelengths and, hence, lower energy than
LH2. It serves as an energy funnel for the RC in which the special pair21
having the most red-shifted absorption, acts as an energy sink. Unfortunately,
for the EPR spectroscopist, the light-harvesting complexes are energy-transfer
systems and do not operate via electron transfer. This is perhaps the main
reason why there are only very few EPR studies on light-harvesting complexes
found in the l i t e r a t ~ r e . ~Nevertheless,
~-*~ if the EPR community were to focus
a similar level of imagination and ingenuity to the study of antenna systems as
they have done in the past with the exploration of the bacterial RC then a
bright future for these systems is ahead.
Exciting research has not come to an end with the photosynthetic RCs
either: (1) The high-resolution X-ray structure of the plant photosystem I at
A
2.5 resolution is just around the corner; (2) The forthcoming publication of
the three-dimensional structure of the plant photosystem I1 at a resolution
better than 4 A will be another milestone in photosynthesis research; (3) In
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 45


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Figure 2 Schematic representation of the LH2 nonamer complex'ofrom Rps. acidophila


viewed from the periplasmatic side of the membrane as obtained from the X-ray
structure analysis (Protein Data Bank of the Research Collaboratory for
Structural Bioinformatics (RCSB): accession number I KZU). The top (A) and
bottom (B, C) images show LH2 with and without the a and polypeptides. The
arrangement of the two rings of BChl a molecules within the walls formed by the
protein subunits are shown: the ring of eighteen B850 BChl a moleculesforms an
overlapping array perpendicular to the membrane plane (B) and the second ring
of nine B800 BChl a molecules approximately parallel to the membrane (C). The
carotenoid molecules (drawn in grey, B ) span the membrane coming into close
contact with the two BChl a rings. Figure A was produced using the package
Molscript *O

bacterial photosynthesis, a comparison of the X-ray structures of the Rb.


sphaeroides photosynthetic RC collected in the dark and under illumination at
2.2 and 2.6 A resolution, respectively, has been presented recently25unravelling
light-induced structural changes in the RC: see Figure 3.
This is just to mention a few of the highlights coming out at the dawn of the
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46 Electron Paramagnetic Resonance


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Figure3 Arrangement of the cofactors in the RC of Rb. sphaeroides R-26 as obtained


from the X-ray structure analysis (Protein Data Bank of the Research Colla-
boratory for Structural Bioinformatics (RCSB): accession number I AIJ). The
Figure shows the light structure of the RC.25 The electron transfer along the A
branch is indicated by arrows and the electron transfer times are given

new millennium. As will be shown in this overview, EPR did and does
significantly contribute with its unique features: (1) Its high specificity for
reactants, intermediates or products that carry unpaired electron spins makes
it favorably suited to detect the working states in primary photosynthetic
charge separation; (2) Decisive evidence for the identity of photoproducts as
well as information on the molecular structure and relative spatial arrangement
of intermediates is obtained by EPR due to its high spectral and temporal
resolution; (3) From electron spin polarization (ESP) phenomena important
information is obtained on mechanistic aspects of the primary photosynthetic
charge separation. Many excellent review articles on various subjects relevant
to EPR in photosynthesis have been published in the last couple of year^.^^-^^
In this monograph series, the article EPR Studies of Photosynthesis by Klaus
Mobius was published back in 1994. At the beginning of the third millennium
it is now time to present an update covering selected EPR studies on bacterial
photosynthesis published in the last five to six years. In order to keep this
article and the references list reasonably short, work published before then is
only sparingly mentioned and the reader is referred to earlier reviews for a
glimpse deeper into the past. Also, where possible, preference was given to
include full research articles rather than conference proceedings because the
latter are typically hard to obtain for non-attendees of the respective meetings
and sometimes contain preliminary work that is later often revised.
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 47


2 The Primary Electron Donor, P

2.1 The Electronic Structure of P.+,Part I: Hyperfine Couplings. - In order to


understand the light-induced electron-transfer process in the RC from first
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

principles, the spatial structure of the system alone is not sufficient, but
additional knowledge of the electronic structure of all the pigments, including
their surrounding, in the initial and charge separated states is required.
Electronic wavefunctions - and their respective orbital energies - can, in
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principle, be calculated by modern semi-empirical or ab initio molecular orbital


(MO) theoretical methods.37 Experimentally, EPR, ENDOR and TRIPLE
resonance techniques have been used quite extensively to identify and char-
acterize the radical cation of the primary donor in bacterial photosynthetic
RCs. Feher has given a chronological account, up to early 1992, of the EPR/
ENDOR characterization of P.' in bacterial photosynthesis, both in its native
and heterodimer mutant forms.26 An update focusing on studies of the
electronic structure of P.+published in the early 1990s up to 1996 was given by
Huber in which also ENDOR on the photoexcited triplet state, 3P, was
covered38(see below).
ENDOR and TRIPLE resonance spectroscopies are excellent tools for the
determination of asymmetries in the electronic structures of the primary donor
cation radicals in photo~ynthesis.~~ However, the distinction, for example,
between a disturbed monomer and a strongly asymmetric dimer is not easy to
make. Clearly, for a final interpretation of the hyperfine (hf) data obtained for
the in vivo systems it is necessary to have a good model system in vitro.
Consequently, the monomeric BChl a cation and the chlorophyll a
(Chl a) cation radical4*were studied in great detail in organic solvents. The
influence of different substituents on the electronic structure of the BChl a
cation radicals has also been meticulously inve~tigated~~ and together with
MO calculations of the spin density distributions was a great aid to the
understanding of the factors determining structure and function of BChl
pigments in the RC.
Nitrogen hyperfine couplings (hfc) of the primary donor cation radical P&
from frozen solutions of l5N-labelled RCs of Rb. sphaeroides have been
obtained from three-pulse ESEEM and HYSCORE (four-pulse ESEEM)
experiments by KaD and co-workers.44In P& up to eight different *'N hfc
tensors can be expected in the case of an asymmetric BChl a dimer. In the past
a maximum number of only four different nitrogen hfc tensors was ob-
tained.45-48All these prior investigations failed in detecting further couplings
with smaller hfc tensor components. By means of 2D HYSCORE a complete
set of principal values including the smaller couplings has now been obtained
and assigned with the help of semiempirical MO calculations at the RHF-
INDO/SP The results were interpreted in terms of an asymmetric spin
density distribution over the halves of the 'special pair' in the reaction centre
with a ratio of 5.1:l in favor of the BChl a bound to the protein subunit L.
This contrasts with results obtained from ENDOR near room temperature,
which gave a ratio of only 2:l. A possible explanation could lie in a change of
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48 Electron Paramagnetic Resonance

the spatial andlor electronic structure of the dimer and its surrounding upon
freezing the RC so that there exist two distinct conformational states of P&
(see below).
Very different electron spin density distributions for the primary donors of
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

RCs from four different species of purple photosynthetic bacteria, Rb. sphaer-
oides, Rb. capsulatus, Rhodospirillum rubrum (Rs.rubrum) and Rs. centenum,
were obtained from an analysis of proton hfcs measured in EPR and ENDOR/
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TRIPLE experiments by Rautter and c o - w o r k e r ~All . ~ ~four RCs contain a


BChl a dimer as the primary donor. For Rb. sphaeroides and Rs. rubrum the
donor Q, optical absorption band is at 865 nm, compared to 850 nm for Rb.
capsulatus and Rs. centenum. Both spectral forms can be interconverted by the
addition of charged detergents, such as deoxycholate, which shows that the
electronic structure of the primary donor is directly influenced by interactions
with the detergent. The EPR/ENDOR studies of the donor cation radicals of
the two forms revealed that both EPR line width and spin density distribution
varied dramatically between the two groups. The spin density distribution of
P& was slightly asymmetric, favoring the L-half of the dimer by a ratio of 2:l
and 1.6:1 for Rb. sphaeroides and Rs. rubrum, respectively. In contrast, the
spectra of P&, in reaction centres from Rb. capsulatus and Rs. centenum show
an almost complete localization of the unpaired electron on the L-half of the
dimer with a ratio of d : I over the M-half. On the other hand, the spin density
distribution for all four cases is identical when the primary donor cation
radical is investigated in RCs contained in their original membrane environ-
ment, i. e. x2:1, reflecting the natural state.
A second, distinct conformation of the primary electron donor has also been
found in RCs from Rb. sphaeruidesS0 and was characterized using the
ENDOWSpecial TRIPLE technique. It has been designated according to
the wavelength of the optical Q,-band in the neutral state. An equilibrium
between P& and exists that can be shifted by changing the detergent/RC
ratio and the temperature. The exact experimental conditions for the occur-
rence of the second blue-shifted state, however, differs from species to
species.49 In Rb. sphaeroides, P& is found in chromatophores and in RCs
solubilized with nonionic detergents and bile salts, whereas is induced by
zwitterionic and ionic detergents with aliphatic hydrophobic chain^.^'
The electronic structure of the cation radical of the primary electron donors
in the BChl-BPhe heterodimer mutants HL (M202) and HL (L173) of Rb.
sphaeroides have been characterized by Huber and co-workers using EPR and
ENDOR spectro~copy.~~ As a result of the mutations, BChl a at PM (HL
(M202)) and BChl a at PL (HL (L173)) are each replaced by a bacteriopheo-
phytin (BPhe). In both RC mutants, the unpaired electron is localized on the
BChl a half of the heterodimer, i.e. on the L-side (PL) in the HL (M202)and
on the M-side (PM)in the HL (L173) mutant. This is consistent with the fact
that BChl a is easier to oxidize than BPhe. Despite the very different electron
spin density distributions of the electron donors in the two heterodimer
mutants, electron transfer still proceeds via the A-branch only, and no transfer
along the B-branch in the HL (L173) mutant could be observed?
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 49

The electronic structure of the primary donor cation radical can also be
dramatically influenced by altering the amino acid environment in order to
change the hydrogen-bond situation of P'+.This has been shown in a series of
ENDOWTRIPLE studies on single and double mutants of RCs from Rb.
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

sphaeroides presented by Lubitz and co-workers.54-57 Site-directed mutations


were designed to add or remove hydrogen bonds between the conjugated
carbonyl groups of the primary donor and histidine residues of the protein and
were introduced at the symmetry-related sites L168, and M197, near the
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2-acetyl groups of the dimer and at sites M160, and L13 1, in the vicinity of the
9-keto carbonyls of the dimer. The changes in the hydrogen bond situation of
the primary donor were accompanied by changes in the dimer oxidation
midpoint potentials. The Special TRIPLE resonance spectra showed that the
asymmetric spin density distribution in the wild type is mainly caused by the
hydrogen bond between His L168 and the acetyl oxygen of PL. Depending on
the different combinations of hydrogen bonds to PL and PM,the spin density
in Irg's5 can be shifted almost completely from PL to PM. The observed shifts
have been explained by an asymmetric dimer model, relating the spin density
ratio p ~ / to p ~the difference of the energies of the valence orbitals of the
monomers PL and PM which are affected by the hydrogen bonds and the
interaction energy between PL and PM .58
A simple relationship between the oxidation potential and the electron spin
density of the primary electron donor in RCs from Rb. sphaeroides was found
by Artz and co-workers in a study of a series of eight RC mutants with
different amino acids replacing leucine at the M 160 position.59The P865/P&
midpoint potentials, Em,were measured by electrochemical titrations and the
fraction of spin density on PL, p ~ was, calculated from the l-methyl and 5-
methyl group hfc's of PL and PM as obtained from ENDORBpecial TRIPLE
experiments. A plot of AE, (the change of the midpoint potential when going
from the wild type to the mutant, i.e. E,(wildtype) - E,(mutant)) as a
function of A ~ (theL change in spin density on PL when going from the wild
type to the mutant, i.e. pL(wi1dtype) - pl(mutant)) yields a linear relationship,
AE, = VAPL, (1)
where V is the potential required to move an electron from PL to PM.For the
M160 mutants, V = 280 f 40 mV, a value that is consistent with an approx-
imate value that can be estimated based on the RC structure. These results
demonstrate, that the midpoint potential can be fine-tuned by electrostatic
interactions with amino acids near the dimer and show that the properties of
the electronic structure of a donor or acceptor in a protein complex can be
directly related to functional properties, such as the oxidation-reduction
midpoint potential.
In contrast to the oxidized primary electron donors P& from Rb. sphaer-
oides and P& from Rps. viridis the primary donor radical cation P& from the
green sulfur bacterium Chlorobium limicola J: sp. thiosulfatophilum consisting
of two BChl a molecules shows only very small deviations from a 1:l electron
spin density distribution over the dimer as has been revealed by ENDOR and
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50 Electron Paramagnetic Resonance

Special TRIPLE spectroscopies.60Since the electron spin density distribution


of P i is thought to be determined by the protein framework, this is consistent
with the proposal that the photosynthetic RC of C,ZimicoZa is a homodimer
providing a symmetrical environment for Observation of the small hfcs
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to the ring nitrogens by 14N ESEEM6*gives results that are in agreement with
those obtained by ENDOR measurements of large hfcs to the methyl group
protons.60
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2.2 The Electronic Structure of Pa+, Part 11: g-Tensor. - Since the breakage of
C2 symmetry in the electronic structure of the primary donor cation radical P
might represent an important functional factor in controlling the vectorial
properties of photoinduced electron transfer in bacterial RCs, such as the high
quantum yield and the unidirectionality along the L protein branch, other
independent probes for the electronic structure of P have lately found
increasing attention. Such a probe is the electronic g-tensor contained in the
leading Zeeman interaction term of the spin Hamiltonian describing an EPR
experiment. In contrast to hfc constants, which probe the electronic wavefunc-
tion locally and directly reflect the unpaired electron spin density at the
magnetic nuclei, the g-tensor reflects the more global properties of the
wavefunction, and thus, can also serve as a sensitive probe for the symmetry
characteristics of the electronic structure of P . Furthermore, the principal
+

components and the orientation of the principal axes of the g-tensor play a
crucial role in the interpretation and correct simulation of the spin-polarized
EPR spectra of the light-induced radical pairs that occur upon the primary
photosynthetic charge separation (see below). Since the g-anisotropy of F +is
on the order of only it is necessary to perform an EPR experiment at
high magnetic field in order to fully resolve the g-tensor whose spectral features
are often obscured by a Gaussian envelope dominated by unresolved hf
splittings. Here, high field means that

is fulfilled, i. e. the anisotropic Zeeman interaction must exceed the inhomoge-


neous line broadening. In Eqn. (2), Ag are the differences between the
principal elements of the g-tensor, gjso is the isotropic g-factor, is the
hyperfine-broadened EPR line width, and BOis the externally applied magnetic
field. For large organic molecules, such as the primary electron donor in
photosynthetic RCs, this usually requires magnetic fields above 11 T and
correspondingly microwave (mw) frequencies above 300 GHz, or, for fields
between 3 and 11 T, full sample deuteration and/or single-crystal work.
The principal values of the g-tensor of the primary donor cation radical Pi&
of deuterated and protonated RCs of Rb. sphaeroides have been reported by
three independent groups using W-band (95 GHd3.5 T) and D-band (135
GHd5 T) continuous-wave (CW) EPR.62-66Using illuminated single crystal
RCs of Rb. sphaeroides the orientation of the principal axes of the g-tensor of
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 51

Table 1 Principal values of the g-tensors of P+in various organisms and the
monomeric BChl a*+
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Rb. sphaer. R-26 2.00323 2.00241 2.00197 82 44 2.00254 67


2.00326 2.00240 2.0019 1 86 49 2.00252 69
2.00324 2.00246 2.00221 88 25 2.00267 65
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2.00329 2.00239 2.00203 90 36 2.00257 63"


2.00329 2.00239 2.00208 90 31 2.00259 63"nh
2.00330 2.00250 2.00210 80 40 2.00263 64
2.00333 2.00246 2.00204 87 42 2.00261 66
2.00402 2.00224 2.00159 178 65 2.00262 62
Rb. sphaer. wild type 2.00333 2.00250 2.00201 83 49 2.00261 69
2.00333 2.00246 2.00204 87 42 2.00261 696
Rb. sphaer. HL(M202) 2.00319 2.00246 2.00215 73 31 2.00260 66
2.00325 2.00247 2.00219 78 28 2.00264 66"
Rps. viridis 2.00309 2,00251 2.00205 58 46 2.00255 69

BChl a' ' 2.00330 2.00260 2.00220 70 40 2.00270 64


" Single crystal work, fully deuterated RCs.

P& relative to the crystal axes of the unit cell could be determined.63 Even the
magnetically inequivalent sites in the unit cell of the RC crystal could be
resolved. With the recent availability of EPR at yet higher magnetic fields and
mw frequencies the g-tensor of P& has been revisited by Bratt and co-
w o r k e r ~ . ~An
~ ~EPR
~ * experiment 'beyond the limits of superconducting
magnets' at 670 GHz/23.9 T has been performed on light-induced and
chemically oxidized P&. A Bitter magnet providing up to 25 T with high
magnetic field homogeneity was used in these experiments. As expected, the
spectra show excellent resolution, where the gYY and gzzprincipal components
of the g-tensor are completely resolved. The g-values reported from this study,
however, are only marginally different from the ones reported pre-
viously62-66y69and closely resemble those of monomeric BChl a*ksee Table 1.
This is evidence for the very asymmetric 5:l spin density distribution over the
dimer.44
High-field EPR (95 GHz) on RC single crystals was also used to characterize
the g-tensor of P& in the heterodimer mutant HL(M202).66In this RC the
histidine at position M202 is replaced by a leucine, resulting in a primary
electron donor in which the BChl is on the L side and the BPhe at the M side.
Since in this mutant the unpaired electron of P& is localized on the BCh1,52
the g-tensor reflects the monomer properties. The directions of the principal
axes of the g-tensor are similar for the mutant and the wild type RC.
No spectral differences have been observed between the different prepara-
tions - light induced versus chemically oxidized - of the donor cation radical.
A decrease of the g-anisotropy when going from cryogenic temperatures to
room temperature that has been reported for the primary donor P;b, of plant
photosystem 170 could not be observed in P&.67 This apparent insensitivity of
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52 Electron Paramagnetic Resonance

the g-values to temperature changes in RCs of Rb. sphaeroides is surprising


given that other spectroscopic parameters, such as optical absorption bands,
the vibrational spectra of P865 or even the zero-field splitting parameters of the
primary donor triplet state, react quite strongly to temperature and have been
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interpreted as due to temperature-induced changes in geometric and electronic


structure. Since this is not observed with the g-tensor of PAs, this raises the
question whether g-factor data alone are able to distinguish between different
electronic structures on the special pair. One of the most striking findings of
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the high-field experiments is that there is negligible g-strain in the primary


donor cation radical. This signals a remarkable homogeneity of the primary
donor in their samples quite unlike the spectra of oxidized monomeric Chl
species in frozen organic solvents where g-strain dominates the line width
above 300 GHz.
With the availability of precise g-tensor data, there is an increasing demand
for a reliable quantum-mechanical analysis of g-matrices of biomolecules.
Semiempirical MO calculations of the electronic g-tensor have been performed
by Plato and MObius7l on the primary donor cation radical of Rb. sphaer-
oides. Their calculations were based on Stones for free radical
doublet states and RHF-INDO wavefunctions and orbital energies. The result
for the primary donor cation radical P& is in qualitative agreement with the
experimental values obtained from W-band EPR studies on RC single
crystals. Remaining quantitative discrepancies concern mainly the orientation
of the calculated principal axes with respect to the experimentally determined
ones.71Hsiao and Zerner have calculated the principal values of the P& g-
tensor using the semiempirical intermediate neglect of differential overlap
model parameterized for spectroscopy (INDO/S).74 They obtain
g,, = 2.00319, gyy = 2.00266, and gzz= 2.00220 which agrees reasonably well
with the experimental values see Table 1. The reader should stay tuned to the
various attempts to calculate g-matrices using density functional theory
(DFT), which is also often used to calculate hyperfine coupling constants,75or
modern ab initio implementations that comprise a complete treatment of all
relevant terms at the Hartree-Fock level of theory (see e.g.76v77and references
therein).

2.3 The Primary Electron Donor Triplet State. - The state of the primary
donor most thoroughly investigated is the cation radical P t in which the
unpaired electron resides in the HOMO of P. In most primary donor cation
radicals an asymmetry of the electron spin density distribution over the two
dimer halves was found. For the forward electron-transfer reactions knowledge
of the spin density distribution of the LUMO is important, however. The
distributions of both electrons in the HOMO and the LUMO determine the
electronic properties, e.g. the charge transfer character of the excited state, *P.
Unfortunately the excited singlet state is diamagnetic and short-lived and not
accessible by EPR techniques. However, information about the orbital coeffi-
cients of the HOMO and the LUMO can be obtained from the triplet state, 3P,
which is formed by recombination of the primary radical pair P t@i in pre-
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 53


reduced RCs in which the electron transfer to the quinone acceptors is
blocked. Singlet-triplet mixing in the singlet-born primary radical pair, P.+ax,
leads to a high yield of 3P which exhibits a characteristic ESP pattern.78 In
EPR spectra of 3P, the dominant interaction is the magnetic interaction of the
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two electrons, which results in the zero-field splitting tensor, D. Detailed data
were obtained from the determination of the zero-field splitting tensor
principal axes of 3P86S by EPR on RC single crystals of Rb. sphaeroides. The
triplet z-axis of 3P865 was found to agree within 5 degrees with the normal
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vector on the average plane of PL and PM and contributions of triplet


excitation delocalization, 55% 3PLPMr32% P L ~ P M and
, of charge transfer 13%
P i PL , have been c o n ~ l u d e d . ~ ~ * ~ ~
The electronic structure of the photoinduced triplet state of the primary
electron donor, 3P865 in frozen solution and RC single crystals from Rb.
sphaeroides was investigated by Huber and co-workers using CW ENDOR.81
The hf splittings found are consistent with an asymmetric spin density
distribution with a mirror image symmetry, in which the 2:l asymmetry of
PL:PMin the HOMO is matched by a 1:2 asymmetry in the LUMO. However,
since an experimental assignment of the hfcs is lacking, consequently there is
no quantitative proof for such a spin density distribution.81Based on the signs
of the hfcs and on a comparison with the cation and anion radicals of BChl a
an assignment of the experimental hfcs to nuclei in the different dimer halves
was proposed by Lendzian and co-workers in a subsequent study using pulsed
ENDOR spectroscopy.82However, the limited data available does not allow
to decide between two possible assignments that lead to different contributions
of charge transfer character (PLPL or vice versa) in 3P865.82Further experi-
ments on 3P865 in RC single crystals will be necessary to determine the
magnitude and the orientations of the hf tensors of 3P86s. This would allow
one to experimentally distinguish between hf tensors that belong to PL and PM
and thereby yield reliable assignments.
The origin of frequently observed negative (opposite phase) ENDOR lines
in the low-frequency region of triplet state ENDOR spectra was examined by
Dubinskii and c o - w o r k e r ~It. ~was
~ shown that longitudinal radio frequency
(RF) field components, which actually exist in most ENDOR experiments due
to slight misalignments of the transversal ENDOR field, can induce artifact
lines by means of an R F modulation satellite mechanism. On the other hand,
these R F modulation satellites provide information concerning lines distant
from the spectral position in the EPR spectrum chosen for ENDOR observa-
tion. This allows one to record the pattern of side holes burnt by mw
saturation through forbidden transitions that carries information about
ENDOR frequencies comparable to what can be extracted from electron spin
echo envelope modulation (ESEEM) experiments. Such comparability is
demonstrated for nitrogen ENDOR of 3P865 in frozen solutions of Rb.
sphaeroides.
The first time-resolved magnetophotoselection study of the primary donor
triplet states in Rb. sphaeroides and Rps. viridis RCs has been very recently
reported by Borovykh and c o - w o r k e r ~ .Using
~ ~ direct-detection EPR the
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54 Electron Paramagnetic Resonance

spherical coordinates of the optical transition moments of the primary donors


with respect to the principal triplet axes were determined with an accuracy of
f5".In QA-depleted RCs and Zn-substituted RCs of Rb. sphaeroides the
orientation of the optical transition moment is independent of temperature
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

within the range 10K 5 T 5 SOK. In non-treated RCs of Rb. sphaeroides and
even more pronounced in Rps. viridis RCs, however, a temperature-depen-
dence was observed which was ascribed to the magnetic interaction between
the primary radical pair, P'+@i, and the [QiFe2+]complex, which causes a
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deviation from a pure TOpopulation of 3P.


The temperature dependence of the ESP of the triplet state of the primary
donor 3P96()in RCs of Rps. viridis was examined by van den Brink and co-
w o r k e r ~In. ~contrast
~ ~ ~ ~to previous reports by van Wijk and co-workersS7it
was shown using direct-detection EPR, that at 100 K the initial ESP in frozen
RC solutions is not inverted for the canonical Y-direction of 3P960. In single
crystals the initial electron spin polarization is almost zero for the complete
YZ-plane of 3P960.The disappearance of the spectral features for the canonical
Y - and 2-orientations is explained with a fast anisotropic spin-lattice relaxa-
tion in 3[Pbko@i](k, > 109s-' for T > 25K). The relaxation in 3[PGko@i]is
enhanced by relaxation in [ Q i Fe2+]. The anisotropy of the temperature
dependence of the initial electron spin polarization indicates that vibrations of
the crystal field of the high-spin Fe2+-ion impose an anistropic phonon
distribution on the RC p r ~ t e i n . ~ ~ ? ~ ~
Magnetic interactions between the triplet state of the primary donor, 3P865,
and the reduced primary electron acceptor quinone, Q I , in modified RCs of
Rb. sphaeroides have been examined by Bosch and co-workers using time-
resolved CW EPR.88 Some time ago, de Groot and c o - ~ o r k e r sfound ~~
additional structure in the emissively polarized EPR spectrum of QA. This
structure consists mainly of a trough in the high-field wing of the EPR signal
and is only present at relatively early detection times (< lms) after an exciting
laser flash. From the time and temperature dependence of the trough in the
spin-polarized EPR spectrum of QX Bosch and co-workers have unambigu-
ously demonstrated that this spectral feature is a manifestation of a magnetic
interaction between QA and 3P8(55.The experimental spectrum at early times
after the laser pulse could be simulated Yery well using a dynamic model of
anisotropic ESP by Hore and c o - w o r k e r ~extended
,~~ with a dipolar interaction
between 3P865 and QA . The simulation is consistent with a dipolar coupling of
-0.125mT, calculated using the point-dipole approximation with the distance
of P865 and QA taken from the X-ray structure.
The triplet properties of the primary electron donor and antenna chromo-
phores in membranes of Heliobacterium chlorum have been studied by Vrieze
and co-workers using absorbance-detected magnetic resonance (ADMR).9*In
H. chlorum, probably two BChl g molecules form the (homodimeric) primary
electron donor. A variety of triplet states was detected, which were all localized
on single BChl g chromophores as concluded from a comparison with the
triplet state of monomeric BChl g in organic solvents.92The zero-field splitting
parameters of the primary donor could be unequivocally determined, being
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 55

(DJ= 727.5MHz and IEI = 254.5MHz. The small value of /El suggests that the
BChls of P are monoligated. Several triplet states of the antenna have also
been observed which are located on BChl g molecules absorbing at longer
wavelength than the primary donor.
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

3 The Intermediate Electron Acceptor, @A


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The intermediate electron acceptor in photosynthetic bacteria is bacteriopheo-


phytin a (BPhe a), abbreviated @ A . @A receives an electron from the primary
donor in approximately 3-4 ps via the 'accessory' BChl a, BA, as a real
intermediate in a predominantly sequential m e c h a n i ~ m . ~The
~ - ~electron
~ is
subsequently passed onto QA in about 200 ps. At low temperatures, the optical
Qx transitions of the two BPhe's in the L and M branch, @A and @*, can be
distinguished with absorption maxima at 546 and 534 nm, respectively. The
trapping of WA upon photoreduction of pre-reduced RCs in the presence of
cytochrome c2 occurs in three steps9? First, after chemical reduction of the
quinone acceptors, illumination in the presence of cytochrome yields the state
@IQA, In a second dark reaction, the diamagnetic state @ A Q ~ - is formed. A
final light reaction then yields @iQi-. Alternatively, the state @i can be
obtained directly in quinone-depleted RCs. The halftime of @i under inert gas
atmosphere is only 15-20 minutes at room temperature, but the radical anion
is stable at temperatures below 200 K. The electron spin density distribution of
WA in RCs from Rb. sphaeroides has been examined some time ago using
ENDOR spectroscopy (see e.g. and references therein).
56997-99

The paramagnetic species @I[QiFe2+]obtained by photoaccumulation of


RCs from Rps. viridis has been examined by van den Brink and co-workers
using Q- and X-band EPR.lm The EPR signal associated with @I[Qi Fe2+]
shows a characteristic splitting of 14.0mT which has been attributed to an
exchange interaction between and the complex [Qi Fe2+],consisting of the
reduced secondary quinone acceptor, QX , coupled to the divalent, high-spin
( S = 2) Fe2+-ion. The simulation of the EPR spectra was successful using an
isotropic exchange interaction of -7.5 mT between @I and QX together with
an anisotropic, purely dipolar interaction of - 1.75 mT between @i and the
Fe2+-ion. The parameter for the magnetic interaction between @i and Fe2+
obtained from the simulations agrees well with the value of -1.8 mT obtained
using the point-dipole approximation and the edge-to-edge distance between
Fe2+and @A of approximately 1.6 nm taken from the crystal structure.
In an ENDOR and Special TRIPLE study, Muh and co-workers have
shown that depending on the trapping conditions and the temperature at least
two distinct @i states designated 1; and I; , can be freeze-trapped in RCs
from Rb. sphaeroidesI0' One of the two states, I; , is metastable and decays
irreversibly between 135-160 K to the other state, 1; . The spectrum of the
pure I; -state can be obtained by subtracting the spectrum measured at 165 K
from that at 135 K. It was proposed that the two radical anions differ in the
orientation of the 3-acetyl group with respect to the macrocycle. Each of the
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56 Electron Paramagnetic Resonance

two conformations, 1; and I; , probably exists in different subconformers, for


example, conformations with a twist of the saturated rings or torsional isomers
of other sulptituents such as the 132-methylester. The hydrogen bond between
Trp LlOO and the 132-methyl ester is possibly weakened or absent in the
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

metastable @i. In a subsequent study of quinone-depleted RC mutants of Rb.


sphaeroides, Muh and co-workers have presented direct experimental evidence
that the 3-acetyl group plays a crucial role in the structural relaxation from I;
to I; Io2 The electronic structure of photochemically reduced @A has been
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investigated by ENDOR and Special TRIPLE resonance spectroscopies of RC


mutants in which Tyr at the M210 position has been replaced by either Phe,
A
Leu, His or Trp. The Tyr M210 residue is located within 5 of P, BA and @ A .
It has been shown that mutations at the M210 site only marginally affect the
electronic structure of the primary donor, Pt ,1039104 despite their marked
influence on the P / P t midpoint p ~ t e n t i a l . ~His
~ -at~ ~M210,
~ however, has
also a strong influence on the electronic structure of @ .; Muh and co-
workersIo2 demonstrated that the ability to undergo relaxation from 1;- to 1;-
depends on the type of residue at the M210 site. His and Leu at M210 suppress
the formation of two conformations of @x. Therefore, the interaction between
His M210 and @A must be such that only one orientation of the 3-acetyl group
is stable. To explain the effect of the Leu M210 a steric hindrance of the 3-
acetyl group was postulated. A Tyr, Phe or Trp residue at M210 essentially
leaves the 3-acetyl group free to rotate. Semi-empirical calculations at the
INDO/S level10*indicate that the contact between BA and @A is mainly via the
two 2 -methyl groups and hence, the electronic coupling is mainly determined
by the electron densities at ring A of both cofactors. The ENDOR/TRIPLE
results of Muh and co-workers demonstrate that a reorientation of the 3-acetyl
group predominantly affects the 7r-electron density in ring A. Therefore, the
coupling between BA and @A might be changed by the M210 mutations, in
particular by Tyr --+His (M210).
The importance of the 3-acetyl group orientation for an efficient electron
transfer from the primary electron donor to the intermediate electron acceptor
was also emphasized by OMalley who has recently presented a theoretical
study on the electronic structure of the BPhe radical anion of Rb. sphaer-
oides.09 Isotropic and anisotropic hfcs have been calculated using the DFT
method at an B3LYP/EPR-II level. The sibpicosecond electron transfer from
the preceding electron carrier, BA, to @ A is facilitated by the close orbital
contact caused by the extension of the LUMO onto the 3-acetyl group of
@A.O When a vinyl group replaces this acetyl group, a decreased LUMO
delocalization toward the preceding electron donor is expected, hence slowing
down the electron transfer between the two electron carriers. This retardation
of the @i-formation was shown previously by Huber and co-workers using
femtosecond optical spectroscopy on RCs of Rb. sphaeroides containing
different types of electron acceptors at the @A-site, 3-vinyl BPhe a among
them. 1 1 1 ,112
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 57


4 Quinone Acceptors

Quinones are found as electron acceptors in the primary photosynthetic charge


separation process in most RCs. In the photosynthetic bacterium Rb. sphaer-
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

oides, for example, the primary and secondary quinones, QA and QB, act as
one- and two-electron gates, respectively. QX just passes its extra electron to
QB which, in a second photo-induced electron-transfer step, becomes double
reduced, takes up protons from the environment and leaves the RC to serve as
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an electron donor in the subsequent dark reactions. Since in Rb. sphaeroides


QA and QB are the same ubiquinone- 10 molecules, their different properties in
the electron-transfer chain must result from specific interactions with the
protein environment. These interactions are believed to fine-tune the electronic
structures of QA and QB for optimum function in the RC. Details of the
electronic structure of the quinones in their paramagnetic form can be obtained
from the electronic g-tensor and from their hfc tensors.
An in-depth summary of the EPR work on the primary and secondary
electron acceptors in bacterial photosynthesis has been given in a series of
three review articles by Feher, Okamura and L ~ b i t z . ~A ~chronological
-~~
account of the work that led to the identification of the quinone acceptors in
photosynthetic RCs of Rb. sphaeroides was presented in the first part of the
series.33 In part 2, EPR studies and other experimental results on the
ferroquinone complexes [Qi Fe2+] and [Qb Fe2+] were reviewed.34The latest
part of this review series covers the present knowledge of the electronic
structure of Q and qB
i as deduced from EPR, ENDOR and ESE experiments
published up to 1999.35
In most studies of the g-matrices of qA and Q ; , RC preparations were used
in which the high-spin Fe2+, which is magnetically coupled to the q ~ i n o n e s , ~ ~
was replaced with a diamagnetic divalent metal ion, e.g. Zn2+, either by
chemical replacement, by biosynthetic replacement or via mutagenesis. In the
latter method the histidine amino acid at position M266 is replaced by cysteine
which facilitates the uptake of metals like Zn2+. The quinone anion radicals
QBor QA in the RC can be specifically created by one or three saturating laser
flashes in the presence of an excess of reduced cytochrome c to reduce the
oxidized primary donor P+back to P according to the following reaction
scheme3?
1st laser flash: PQAQB -% P'+QAQ'~ * PQAQ.B
2nd laser flash: PQAQ.~-% P'QAQ; PQAQ~-
3rd laser flash: PQ*Q;- -% P"QAQ;- * PQAQ~-.
The g-tensor anisotropies of quinone anion radicals in bacterial photo-
synthesis are much larger than those of the primary donor cation radicals.
Therefore, good spectral resolution is already obtained at relatively moderate
mw frequencies and corresponding magnetic fields (e.g. Q-Band: 35 GHdl.25
T or W-band: 95 GHd3.5 T). It is widely accepted that the principal axes of
the g-matrices of quinone radical anions are colinear with their molecular axes.
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58 Electron Paramagnetic Resonance

Table2 Principal values of the g-tensors of semiquinone anion radicals in Zn-


substituted RCs and of respective model systems in vitro
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

Rb. sphaeroides Qx 2.00649 2.00532 2.00210 2.00464 113"


2.0066 2.0054 2.0022 2.0047 64
Rb. sphaeroides (& 2.00626 2.00527 2.0021 3 2.00455 113"
Rps. viridis 2.00597 2.00492 2.00216 2.00435 114
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Ubiquinone-lo' 2.00646 2.00542 2.00222 2.00470 64


2.00632 2.00533 2.0021 5 2.00460 113
Ubiquinone-3' 2.00622 2.00526 2.00210 2.00453 115
Menaquinone-4' 2.00579 2.00498 2.00218 2.00432 114
" Single crystal work.

The axis with the smallest principal value g,= is oriented perpendicular to the
quinone plane (z-axis), the axis of the largest value gXx is along the line
connecting the two carbonyl groups (x-axis), and the axis of the third principal
value of g is perpendicular to the carbonykarbonyl axis lying in the quinone
plane (y-axis) (see e.g. 64-113), The g-tensor values for the semiquinone anion
radicals QA and QB of Zn-substituted RCs of Rb. sphaeroides in frozen
solution and single crystals have been determined some time a g 0 . ~ 7 " Re- ~
cently, Zn-substituted RCs have also been obtained from Rps. viridis in which
QA is a menaquinone-9 and Q B is a ubiquinone-9l l 4 see Table 2.
A comparison of the g-matrices of QX in frozen RC solution with that of a
menaquinone-4 anion radical in 2-propanol shows an increase of the g,,-value
of QX: this indicates a similar bonding for QA in Rb. sphaeroides and Rps.
viridis. g-tensor data for Qi- in RCs of Rps. viridis are not yet available.
Reliable calculations of the rather small shifts of the principal values of the
g-tensor of organic radicals are difficult, mainly due to the problem of
accurately accounting for the excited states that play a role in calculating the g-
tensor. Recently, Torring and co-workers have proposed new expressions for
the calculation of g-matrices from RHF MO calculations using a Rayleigh-
Schrodinger perturbation theory approach. Their expressions differ signifi-
cantly from those found previously in the literature, mostly due to the explicit
inclusion of a class of excitations where the total spin is reversed in the
excitation. Model calculations were presented for the para-benzosemiquinone
anion radical. The calculated principal values of its g-tensor showed very good
agreement with experimental data.64
All major hfc constants for the protons, carbonyl oxygens and carbons have
been determined from EPR and ENDOR experiments, see Table 3. Again, in
vitro model systems l 2 *3 as well as numerous model calculations 23- 33 on
22y1

quinone anion radicals served as an indispensable source for an unambiguous


assignment of hfc's to atom positions within the quinone and to nuclei from
the protein environment.
In early ENDOR experiments resonances from nitrogen nuclei were detected
for QX in Zn-substituted RCs of Rb. sphaeroides'20 and the suggestion was
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 59


Table 3 ' H , 13C and I7O hypei$ne tensor values Qj
and QBin Zn-substituted
RCs of Rb. sphaeroides
AJMHz AJMHz AJMHz Ai,,/MHz Ref.
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

Q A C(5W3 +6.9 +3.8 +3.2 +4.6 117


(+)6.8 (+)3.6 (+)3.2 (+)4.5 118
OCH3 2.7 2.0 1.8 118
CH2 (+)8.6 (+)5.6 (+)5.0 (+)6.4 118
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13c(1) (+)22.7 119


]3C(4) (+)35.0 119
17c( 1) (-194 120
'7C(4) (-175 120

QiB C(5W3 +7.8 +4.4 +3.9 +5.4 117


13c(1) (+)27.7 119
'3C(4) (+)32.2 119
17c(1) (-188 120
'7C(4) (-182 120
Signs in brackets indicate that the sign of the hfc has not been determined in the referenced
experiments.

made that these resonances originate from a coupling through a hydrogen


bond with the nearby histidine at M219. Bosch and c o - ~ o r k e r s 'as
~ ~well as
Lendzian and c o - ~ o r k e r applied
s ~ ~ ESEEM spectroscopy to QA in ZnbRCs.
ESEEM is particularly sensitive to nuclei with small magnetic moments and
small hfc's which are usually difficult to detect by ENDOR. Considering the
geometry of the QA binding site and based on a comparison of the nuclear
quadrupole resonance (NQR) parameters for the nitrogen resonances ex-
tracted from the ESEEM spectra of QA with those obtained for histidine,
tryptophane and other peptide nitrogens, the ESEEM data established unequi-
vocally that the N'('1-H group of His M219 participates in the formation of a
hydrogen bond to the C(4) carbonyl group of QA in RCs of Rb. sphaer-
aides.134,579135
ESEEM spectroscopy has also been used to probe the surrounding of QA in
ZnbRCs of Rps. viridis with respect to nitrogens in the vicinity of the
menaquinone."4 From the NQR parameters of the nitrogen resonances it was
concluded that in this RC one of the carbonyl oxygens is coupled to His M2 17.
In agreement with structural data from the recent X-ray crystallographic study
of Rps. viridis3 a peptide nitrogen was assigned to alanine at M258 which most
likely takes part in a second hydrogen bond to the other carbonyl oxygen of
Qi *

Hyperfine data including information on the direction of hydrogen bonds


from the protein environment to the Qi, acceptor in RCs of Rb. sphaeroides
was also obtained from Davies-type pulsed ENDOR experiments performed at
a magnetic field of 3.4 T and a mw frequency of 95 GHz (W-band).l'* By
taking advantage of the increased electron Zeeman interaction at high
magnetic fields the g anisotropy of QA in vivo could be resolved even in non-
oriented samples. Hence, the W-band ENDOR spectra could be taken at the
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60
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3.424 3.426 3.428 3.430 3.432 3.434


W T
Figure 4 Field-swept two-pulse echo decay spectrum of in frozen-solution Zn-
substituted RCs of the HC(M266) mutant of Rb. sphaeroides recorded at 95
GHz ( W-band).137Extrapolated 7 = 0 spectra of the ESE-detected EPR
spectra are shown at T = 115 K (solid line) and T = 175 K (dashed line), as
extracted from the echo decay function S(27, Bo) = So exp (-27/T2 (Ba)). Field-
dependent T2 relaxation times are depicted as filled circles (T = 115 K) and
open circles (T = 175 K)

well-separated canonical peaks of the rhombic powder pattern, thereby


becoming highly orientation selective with respect to the relative orientation of
QA to the external magnetic field. In addition to the magnitudes of the hfc
tensors, relative orientations of the tensor axes with respect to the principal
axes of the g-tensor were obtained from an analysis of the W-band powder
ENDOR spectra. One of the three large hfc tensors used for the simulations
has been assigned to a strong coupling of a hydrogen-bonded proton. This hf
tensor deviates from axial symmetry and yields an isotropic contribution of
approximately -0.4MHz. The largest component is oriented out of the
quinone plane by 12" and its projection onto the plane deviates 15" from the
C-0 bond direction (molecular x-axis). In contrast to the ENDOR data taken
at X-band, however, no evidence for the existence of two strong hydrogen
bonds has been obtained from the high-field pulsed ENDOR studies.
The direction of the symmetry axis of the dominating hydrogen bond close
to the molecular x-axis of Q i from Rb. sphaeroides seems also to be in
agreement with recent studies of the anistropic transverse relaxation behaviour
of QA using pulsed high-field ESE,136see also Figure 4. At low temperatures
(7'= 115 K) a motional anisotropy of Q i in its protein binding pocket has
been deduced with a preference for librations about the C-0 symmetry axis. It
remains to be seen if the functional differences between Q A and QBcontrolled
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 61

by the structural differences of their respective protein binding pockets will be


reflected in a different relaxation behaviour of QX and Q.i. High-field
relaxation studies of the QBcofactor in Rb. sphaeroides motional dynamics are
currently in progress.
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

It is widely accepted that besides motional dynamics, protein conforma-


tional changes play an important role in biological electron transfer. Recently,
Utschig and co-workers have detected a local protein environment, a Zn site,
that apparently regulates the electron transfer QxQB --+ QAQi from a remote
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position. 13* Isolated RCs from Rb. sphaeroides that bind Zn2+ stoichiometri-
cally and in a site distinct from the non-heme high-spin Fe2+ site have been
investigated using Q-band EPR and optical absorption spectroscopy. When
Zn2+ is bound to this surface metal site, electron transfer QAQB QAQRis --+

slowed and the room-temperature kinetics become distributed across the


microsecond to millisecond time domain with its slowest component
((3.2 f 0.7) ms) being markedly longer than the slowest component
((233 f 50) ps) observed in native, non-Zn2+containing RCs. From an inspec-
tion of the RC crystal structure a cluster of histidine ligands located beneath
the QB binding pocket has been suggested as a potential Zn2+ binding site.
Paddock and co-workers have confirmed these findings and extended the work
to an investigation of the Zn2+ and Cd2+ influenced proton-uptake of qB
A 100-fold reduction in the rate of proton transfer to Q; upon stoichiometric
binding of Zn2+ or Cd2+ implies that there is one dominant proton entry point
into the RC. From a preliminary X-ray structure analysis a Zn2+ and Cd2+
binding site near His H126, His H128 and Asp H124 was proposed where
Zn2+ or Cd2+ binding possibly disrupts proton donors or blocks proton
transfer pathways to QB.
Smirnova and co-workers have investigated light-induced structural changes
in RCs from Rb. sphaeroides using Cu2+as a paramagnetic structural probe.Iw
The EPR spectrum of Cu2+incorporated into metal-depleted RCs was affected
by 1,lO-phenanthroline, an electron-transfer inhibitor substituting QB, which
suggests a localization of Cu2+ in the vicinity of the QB site. Because the EPR
spectrum was not influenced by low temperature illumination of the sample, it
was suggested that the copper ion position is somewhat different from that of
the non-heme Fe2+ ion. Utschig and co-workers have picked up on the idea of
probing this surface metal site using Cu2+ treated native Fe2+-containing and
biochemically non-heme Fe2+-removedRCs from Rb. sphaeroides.14' ESEEM
spectroscopy has been applied to characterize the magnetic interactions
between Cu2+and weakly coupled magnetic nuclei in the protein environment.
Comparison of the ESEEM spectra obtained confirmed that the Cu2+ surface
site has a different geometry and is spatially distinct from Cu2+ bound to the
non-heme Fe2+ site: Cu2+coordinates at or near the same site as the Zn2+ and
Cd2+.The results are consistent with the proposed location of Cu2+ beneath
the QB binding pocket with histidines at H68, H126, H128 and L211 as
potential ligands. 39
Electron flow from QA to QB may also be disrupted by inhibitors binding to
the QB-site of the L-subunit and displacing the native ubiquinone molecule.
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62 Electron Paramagnetic Resonance

From X-ray crystallography, the position of two inhibitors, s-triazine terbu-


tryn and o-phenanthroline, within the QB binding pocket is well e~tab1ished.l~~
Recently, Sopp and co-workers showed that one single amino acid mutation in
the RC M-subunit of Rs. rubrum with Glu at M234 replaced by Lys causes
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

resistance against NH thiazoles, which are known to act as efficient inhibitors


in plant photosystem I1 and bacterial R C S . ' ~Compared
~ to the wild type, the
mutant exhibited strongly altered [QB Fe2+] and [Qi Fe2+] EPR signals. The
differences in the EPR spectra and the indication of impaired electron transfer
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between QA and QB presumably reflect structural changes around the quinone


sites which are responsible for the herbicide resistance and which perturb the
magnetic interactions between the semi-quinones and the iron.
Time-resolved EPR and optical studies on QA and QB site mutants of Rb.
capsulatus constructed to partially balance the differences in charge distribu-
tions between the two quinone binding sites have shown that sequential
forward electron transfer to QA is slowed in those strains containing altered
QA sites.144The charge recombination rates were also altered in these mutants.

5 Radical Pairs

Absorption of light by photosynthetic bacterial RCs starts out a series of well


known electron-transfer reactions which can be written in a simplified form as
follows:
a'
PBA@AQAFe2+QB P*BA@AQAFe2+QB-+Pt B i @AQAFe2+QB+
p i B A @QAFe2+QB
~ +Pt BA@A[(& F e 2 + ] Q ~ - + BA@AQA
P" [Fe2+QB1.
The first relatively stable intermediate of the electron-transfer reaction scheme
is the so-called primary radical pair P+@i, where the unpaired electrons are
localized on the primary donor, P, and the BPhe acceptor, @,A, respectively. In
native RCs Pt@ i of about 200 ps, which is too short for a direct
has a lifetime
EPR detection or even appreciable spin dynamics to occur (see below). The
lifetime of the primary radical pair is increased in modified RCs where forward
electron transfer beyond @i is blocked, and a transient intermediate may be
observed using EPR with a high enough time resolution. EPR work on P"@p',
will be summarized in Section 5.1. The secondary and the subsequent radical
pairs are generated in non-treated RCs where electron transfer may proceed
past @ A . P ' Q l was first detected with EPR in RCs in which the non-heme
Fe2+ was magnetically uncoupled from the q ~ i n o n e and' ~ ~later also in RCs in
which the Fe2+-Qp', coupling was intact (Section 5.2.).146- 149

5.1 The Primary Radical Pair, P t@i. - The first successful EPR observation
of the primary radical pair P"@i was reported by Proskuryakov and co-
workers.150RCs from Rb. sphaeroides and Rps. viridis have been examined
where the forward electron transfer beyond @A was blocked by quinone
depletion and photoreduction of the primary quinone acceptor, respectively. A
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 63

new transient signal in the g = 2.0 region was found with AE polarization
(absorptive at low magnetic field, emissive at high magnetic field) and a peak-
to-peak separation of ~ 3 . 8 5mT in Rb. sphaeroides and 4 x 8 mT in Rps.
viridis. The disappearance of these new signals paralleled the formation of the
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

triplet states, 3P, with time constants at 70 K of ~ 3 ns 8 for QA-reduced Rb.


sphaeroides, =58 ns for QA-depleted Rb. sphaeroides, and =lo ns for QA-
reduced Rps. viridis. From its kinetic and spectral properties the signals were
ascribed to the primary radical pair P.+@z. Spectral simulations using the
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correlated radical pair mechanism (CRPM) required a negative exchange


interaction between P.+ and @ I , Jpa = -0.9 mT for Rb. sphaeroides and
Jpa = -1.7 mT for Rps. viridis, to account for the AE spectral polarization
pattern. A dipolar interaction of Dpa = -3.0 mT was used in the simulations
for both species. The negative sign of J is surprising, given that the electron
spin polarization of the semi-quinone QA in prereduced chromatophores of
Rs. rubrum in which the magnetic interaction between QX and the Fe2+ was
uncoupled by detergent treatment rather indicates a positive sign of J88(see
] Rb, sphaeroides with different
also151).In addition, the ESP of P + [ Q i F e 2 +in
quinones as QA also indicates J > 0.152In a subsequent publication, the
CRPM mode1153,154 to describe the EPR spectra was extended to include the
time dependence of the EPR intensities, different lifetime broadenings, and an
averaging over the inhomogeneous distributions of hf interactions in the two
radicals. lS5 The electron spin-spin interactions have been adjusted to
Jpa = -0.7 mT in both Rb. sphaeroides and Rps. viridis together with a zero
dipolar coupling in Rps. viridis. In fact, it was stated that the spectra contained
very little information on the dipolar coupling in P.+@I Decay rates ks and
kT for the decay of the singlet radical pair '[P.'@x] to the ground state, P@A,
and the decay of the triplet radical pair 3[P.+@;;] to the triplet state, 3P@A,have
been extracted from the simulations and were discussed in the light of the
energetic heterogeneity of the radical pair energies. 56

5.2 The Secondary Radical Pair, P.+QA* . - The secondary radical pairs in
bacterial photosynthesis have been predominantly examined using time-
resolved CW EPR and ESE-detected EPR after laser flash excitation. Here,
similar to the EPR studies of the primary donor cation radical and the quinone
anion radicals, higher mw frequencies and correspondingly higher magnetic
fields are increasingly used to enhance resolution and to separate the spectral
contributions of Pt and Qfi in the radical pair P''QA EPR spectra.

5.2.I Radical Pair EPR Spectra. Time-resolved W-band (95 GHz) EPR
spectroscopy has been applied to examine the secondary radical pair state in
Zn-substituted protonated and fully deuterated RCs of Rb. sphaeroides, 57 The
ambiguity in the orientation of the g-tensor principal axes of the primary
donor P8k, with respect to its molecular axes system, that was left from single
crystal CW EPR studies at W-band,63 could be removed due to the high
spectral resolution at W-band. In a subsequent multi-frequency EPR study
including spectra taken at X-, K-, and W-band frequencies, clear differences
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64 Electron Paramagnetic Resonance

between the time-resolved EPR signals of the secondary radical pairs in


bacterial RCs and plant photosystem I have only been observed at W-band,
while the spectral AEA(E) polarization pattern recorded in K- and X-band
EPR have a very similar shape for both organism^.^^^^^^^ Tang has taken the
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complete set of P&Qx transient EPR spectra of fully deuterated and


protonated RCs from Rb. sphaeroides recorded at X-, K-, and W-band,160*157
respectively, and has performed a simultaneous fitting of g-tensor orientations
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using the geometries from different X-ray structures.'61 Contrary to the


suggestion of van den Brink and co-workers'62 no exchange coupling JPQ
between and Qfi-was required to obtain a satisfactory agreement between
the experimental and calculated P&Q;\ transient EPR spectra. 157*161
The secondary radical pair state P.&,oQ.A from Rps. viridis has been examined
by Gardiner and co-workers using time-resolved CW X- and Q-band EPR
s p e c t r o ~ c o p yThe
. ~ ~ ~analysis of the transient EPR spectra revealed a very
similar relative orientation of the secondary radical pairs in Rps. viridis and
Rb. sphaeroides. Using out-of-phase ESEEM to determine the distance
between P& and Q i (see also below) a dipolar coupling of
D = -(O. 116 f 0.004) mT corresponding to a cofactor distance of
(28.8f0.3)A was obtained together with an upper limit for the isotropic
exchange interaction between the two radical pair halves of JPQ 5 1 pT. The
P&Qi geometry and the cofactor distance agree well with the available X-ray
crystallographic structure for the ground ~ t a t e . ~ -This * ~ -indicates
~ that no
major cofactor reorientation takes place during charge separation.
Proskuryakov and co-workers pointed out that the shape of transient spin-
polarized P.+Q EPR spectra might be affected by selective optical excitation
of the RCs when plane-polarized laser light is used to initiate charge
separation. 163 Examples were given for the secondary radical pair state P&Qr\
in Zn-substituted RCs of Rb. sphaeroides using different excitation wave-
lengths and directions of the polarization plane of the laser light with respect
to the magnetic field. It has been suggested that the excitation wavelength
should be chosen with care and to use a depolarizing lightguide in front of the
EPR cavity to avoid potential photoselection effects.
The ESP of the transient EPR spectrum of P"Q;i is also strongly influenced
by the lifetime of the precursor radical pair state P t @ which i in modified
reaction centres may be prolonged, resulting in a more pronounced singlet-
triplet interconversion under the influence of the dipolar, exchange, hf and
Zeeman interactions of P t and @i. The initial state of the subsequently
formed secondary radical pair P ' Q i then will no longer be a pure singlet
state, with quite dramatic efl'ects on the EPR line shape. For a complete
characterization of the magnetic properties in P ' Q i one needs therefore to
consider the singlet and triplet decay rates of P"@p',(ks and k ~as) well as the
singlet-triplet mixing frequencies together with the dipolar and exchange
interactions for both the primary and the secondary radical pair. Several
models that extend the existing CRPM mode1153,154 with a contribution of an
intermediate radical pair have been described in the literature. 164,1529 165-167
Singlet triplet mixing becomes even more efficient at high magnetic fields due
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 65

to the stronger anisotropic Zeeman interaction and one may have to consider a
sequential electron-transfer model even for the case of a relatively short-lived
precursor radical pair. 165 This has been demonstrated experimentally by
Utschig and co-workers and Tang and co-workers in a comparative EPR
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study of two different preparations of Fe-removed/Zn-substituted bacterial


RCs from Rb. sphaeroides with native (200 ps) and increased (x4ns) lifetime
of the precursor radical pair state, P&@i, respectively. Minor differ-
1689169

ences between the spin-polarized transient EPR spectra of the samples with
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slow and fast electron transfer are observed at X-band frequency, become
more pronounced at Q-band and W-band. In contrast to studies by Till and
co-workers 155 and Hulsebosch and co-workers 5 1 a positive value for the
exchange interaction, Jpa = 0.7 mT, between Ft and Pi in the precursor
radical pair state was used for the spectral simulations, together with a dipolar
coupling of Dpa = -0.45 mT.169
The necessity to utilize a sequential electron-transfer model has also been
demonstrated by Hulsebosch and co-workers in a multi-frequency EPR study
of Zn-substituted RCs from Rb. sphaeroides where the lifetime of the primary
radical pair Pi;, @i was manipulated by replacing the native ubiquinone- 10
with duroquinone or 2-ethyl anthraquinone. 51 Relatively long lifetimes for
P8k5@i of 33 ns and 3.3 ns were obtained with 2-ethyl anthraquinone and
duroquinone as secondary electron acceptors, respectively. From spectral
simulations of the X-, Q- and D-band (130 GHz) EPR spectra information on
the magnetic interactions and dynamics of the intermediate primary radical
pair P&@i was obtained. When the lifetime of P&@x is longer than a few
nanoseconds, the influence of the magnitude and sign of the exchange interac-
tion J p on the shape of the observed EPR spectrum is significant. A value of
J p = - (0.9f 0.1) mT for both duroquinone and 2-ethyl anthraquinone-recon-
stituted RCs was obtained, which is not significantly different from that
extracted from a simulation of the EPR spectrum of the primary radical pair
P&@A in pre-reduced RCs of Rb. sphaeroides (Jpa = -(0.7 f 0.1) mT).155A
small but non-zero exchange interaction, JPQ = -(0.2 f 0.1) pT and
-(0.5 f 0.2) p T between and the radical anions of duroquinone and 2-
ethylanthraquinone, respectively, was obtained from the simulations. 5 1 Tran-
sient K-band EPR spectra of the secondary radical pair in Zn-substituted RCs
from Rb. sphaeroides with duroquinone d12 or naphthoquinone db replacing
the native ubiquinone-10 have been reported previously by van der Est and co-
worker~.'~'It was concluded from spectral simulations that substitution of QA
does not have a measurable effect on the quinone orientation in the QA-site,
although the agreement between the experimental and calculated spectra was
only mediocre and would allow for a slight quinone reorientation. However, in
their calculations no sequential electron transfer model was used, and conse-
quently the observed secondary radical pair was considered to be initially in a
pure singlet state without any admixture of triplet character from the precursor
radical pair.
Quinone-substituted non-heme iron-containing RCs from Rb. sphaeroides
have been examined by Morris and co-workers in a time-resolved X- and Q-
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66 Electron Paramagnetic Resonance

band EPR study. 152 Replacing the native ubiquinone-10 with various anthra-
quinones and naphthoquinones alters both the free energy and the rate of
electron trpsfer from @A to [QFe2']. A characteristic AEA polarization
pattern was observed for Fe2+-containing RCs which has been attributed to
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

the P& part of the spin-correlated radical pair Fe2+]because the EPR
signal of the polarized [aA Fe2+]radical is much too broad to be observed. The
spin-polarized EPR spectrum was described by the sequential electron-transfer
polarization model in which the chemically induced dynamic electron polariza-
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tion developed in the primary radical pair was projected onto the correlated
radical pair polarization developed in P& [a. Fe2+]. The contribution gave
rise to criticism, 171 however, mainly because the strong g-anisotropy of
[QiFe2+]was neglected and an unrealistically large value for the exchange
interaction between P& and [QXFe2'] had to be assumed ( J ~ ~ Q 2 . pT).
5 Van
den Brink and co-workers have demonstrated that an extended CRPM model
to account for the g-anisotropies of P& and [QXFe2' yields accurate
[Qx
simulations of the spin-polarized EPR signals of Pt FJ'] for both Rps.
viridis and Rb. sphaeroides, without taking recourse to a large value for
Furthermore, the orientation of the principal axes of the g-tensor of the
[QAFe2+]complex with respect to the crystal axes had been assessed. It was
concluded that the z-axis of the g-tensor is parallel to the axis between Fe2+
and N E of ~ His at M266 in Rb. sphaeroides (M264 in Rps. viridis) and the y -
axis almost parallel to the axis between Fe2+ and 0 1 of Glu at M234 in Rb.
sphaeroides (M232 in Rps. viridis). The rapid decay of the spin-polarized EPR
signal was discussed in terms of the relaxation of the quinone-iron complex
and it was shown that it probes the cross-relaxation of Q i and Fe2+.
Low-temperature spin-polarized X- and K-band transient EPR spectra of
Mn-substituted RCs of Rb. sphaeroides Y have been examined by van der Est
and c o - w o r k e r ~ . A
' ~ ~strong component with a spectral width of -0.4 mT
appears near g = 2 and a weak component with a width of ~ 5 7 0mT is
observed in the spectral range 1 5 g 5 6. The narrow spectrum is primarily
due to P& and the broad component was assigned to [QIMn2']. Both signals
are polarized as a result of the correlation of the unpaired spins on P& and
[QAMn2']. The effect of the magnetic properties of the metal on the spin
polarization and the expected differences between the spectra of P& [aA Mn2+]
and Pi165[qA Fe2'] were discussed.

5.2.2 Coherence Phenomena. Information on the magnetic interactions within


the secondary radical pair state may also be obtained from the time course of
transient EPR signals. Quantum beat oscillations have been observed to
modulate the time traces of the spin-polarized EPR signals of the secondary
radical pairs in native non-heme Fe*+-containing and Fe2+-removed RCs of
Rb. sphaeroides. 149 These oscillations arise because the initial configuration of
the radical pair (singlet or triplet) is not an eigenstate of the corresponding spin
Hamiltonian, and hence, the radical pair starts out in a coherent superposition
of two of the four spin states (zero-quantum coherence) which manifests itself
as quantum beats in an EPR experiment with adequate time resolution.
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 67


Quantum beat oscillations have been predicted for photosynthetic R C S ' ~74~ ~ '
and observed for the first time for the secondary radical pair P&Ai in plant
photosystem I. 1753176 Observation of this coherence phenomenon in bacterial
RCs confirms previous interpretations of transient EPR spectra employing the
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CRPM c 0 n c e p t . ' ~ ~ The


9 ' ~ ~quantum beat frequency depends on the magnetic
interactions between the two radical pair halves, and thus, information on the
relative orientation of FA,and QK may be obtained from a thorough analysis of
the modulation pattern. In a subsequent paper, quantum beats have also been
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presented for the secondary radical pair of Zn-substituted RCs from Rb.
sphaeroides using time-resolved EPR at X- and K-band (24G H z ) . ' Additional
~~
oscillations in the frequency ranges 1.5-2.0 MHz and 2.5-3.0MHz have also
been observed which have been attributed to the interaction between the nuclear
spins and the unpaired electron spins. This attribution was later confirmed by
Weber and co-workers in a comparative study of 14N and I5N enriched
photosystem I RCs where significant differences in the modulation patterns of
both samples were observed' 78 (see also'79). A theoretical treatment of light-
initiated nuclear modulations in the time course of transient EPR signal was
presented by Weber and co-workers180and Jeschkelsl (see also ref. 182).
Quantum beat oscillations may also be observed as a change of the Hahn
echo shape in a two-pulse electron spin echo experiment following the
photogenerating laser flash (flash - t - pulse 1 - 7 - pulse 2). This has been
demonstrated experimentally by Dzuba and co-workers who have examined
protonated Zn-substituted RCs from Rb. ~phaeroides'~~ In their experiment,
the transient echo signal shape after the laser flash and mw pulse application
was measured as a function of the delay time t . Significant changes in the shape
and intensity of the first echo appearing at T = r were observed for t < 40 ns.
In addition, a second echo signal was found when t was chosen to be strictly
identical to the delay time T between the two mw pulses. This echo signal
appears at time 27 after the second mw pulse and is ascribed to a double-
quantum coherence appearing after the first mw pulse. The existence of
multiple-quantum coherence in a spin-correlated radical pair was noted by
Tang and Norris. 184
For a two-pulse echo experiment following a laser pulse to generate a spin-
polarized radical pair, Salikhov and co-workers have predicted an unusual
out-of-phase ESE showing a deep envelope modulation (ESEEM) as a
function of the delay time between the two mw pulses.185The modulation
frequency of the echo amplitude is dominated by the spin-spin coupling
between the two halves of the radical pair. This coupling can be divided into
two parts: the anisotropic dipolar coupling D which reflects the magnetic
dipole interaction between the two involved electron spins, and the isotropic
coupling J describing the overlap of the two electronic wavefunctions. Since
the dipolar coupling D is directly related to the distance between the two
electron spins,
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68 Electron Paramagnetic Resonance

it is possible to deduce the distance between the two radical pair halves from a
pulsed EPR experiment on the spin-polarized signal of the light-induced
radical pairs of photosynthesis. In weakly coupled radical pairs, D is typically
much smaller than the inhomogeneous linewidth dominated by unresolved hf
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

interactions, and hence, difficult to detect from other experiments. Following


the prediction by Salikhov and co-workers, several authors have subsequently
provided analytical calculations of echo decay signals after sequences of one,
two, or three mw pulses.186-189 Refined models to simulate out-of-phase
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ESEEM spectra have been introduced to account for sequential electron


transferIgOand for anisotropic hf interactions. For an instructional pre-
sentation of the various coherence phenomena associated with spin-correlated
radical pairs, including zero-quantum coherences, double-quantum coherences
and out-of-phase ESEEM, the reader is referred to a publication by Hoff and
co-workers.19*
The possibility to extract the dipolar and isotropic couplings separately from
a pulsed EPR experiment was first demonstrated by Dzuba and co-workers
using Zn-substituted RCs of Rb. sphaeroides.193 The dipolar couplings ob-
tained for P&QA were about 30% larger than the one derived from the
distance (see Eqn. (3)) obtained from the X-ray structure of Rb. sphaer~ides.~
Additionally, a rather large exchange coupling J = (1 1 f 2) pT was found
which in this magnitude is inconsistent with transient EPR spectra recorded in
different frequency bands.IS7Zech and co-workers have re-examined the out-
of-phase echo envelope modulations of P&Q* from different Zn-substituted
RC preparations of Rb. sphaeroides and obtained D = -(0.121 f 0.004) mT
and J = (1.0 f 0.5) pT, which, using Eqn. (3), leads to a distance of
r = (28.4f 0.3) between P& and QX.194,195 This value is in excellent
agreement with the distance between the midpoint of the two Mg atoms of P865
and the centre of the QA ubiquinone ring derived from the X-ray ~ t r u c t u r eIn
.~
an extended study of the temperature dependence of the out-of-phase echo
envelope modulations of P&QA, Dzuba and co-workers finally obtained
D = -(0.115 f 0.005) mT and J = (0.7 f 1.5) pT for temperatures above 100
K.193Abrupt changes of the linewidth of the Fourier transformed ESEEM
spectra were observed near 25,40 and 80 K. The lineshapes could be simulated
assuming that the distance between the two radicals is distributed within a
range of about 4 A and that the distribution depends stepwise on the
temperature, however, becoming quite narrow at higher temperatures.
Light-induced structural changes in bacterial RCs have been studied using
out-of-phase ESEEM together with transient EPR spectroscopy on the spin-
correlated radical pair state P&QX .1967197 A light-induced structural change of
the RC was first suggested by Kleinfeld and c o - w o r k e r ~ 'to~ ~explain the
observed difference in the rate of forward electron transport from QA to QB
and in the recombination kinetics of Pi&(& at cryogenic temperatures after
freezing QB-depletedRCs in the dark or under continuous illumination (ie. in
the charge-separated state). The charge recombination is slowed down con-
siderably and shows a nonexponential behaviour in light-frozen RCs. A shift
A
of the quinone by more than 1 resulting in a larger distance between P&
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2: Recent EPR Studies on the Bacterial Photosynthetic Reaction Centre 69

>
I I I I I

light frozen
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A
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! I

dark frozen

Zn-bRCs
Rb. 8 p h - m R-26

0 0.5 1.0 1.5 2.0


T / PS

Figure 5 Out-of-phase echo modulation pattern for protonated Zn-substituted RCs of Rb.
sphaeroides mutant HC(M266) frozen in the dark (lower trace) or under
illumination (upper trace) measured at X-band and at a temperature of 80 K
For details see196

and QX has been suggested to explain the prolonged P&Q* lifetime in


samples frozen under illumination. Transient EPR studies have shown that no
major reorientation of the QA acceptor for RCs frozen in the charge-separated
state can occur. Rather, small light-induced conformational changes in the
protein matrix were suggested, affecting the Q 'i affinity by changing
electron
the orientation of the methoxy substituents at the quinone ring. Zech and co-
workers have shown that dark-frozen and light-frozen samples exhibit the
same echo envelope modulation pattern resulting in virtually the same cofactor
distance between P& and Qk in both see Figure 5.
In contrast, Borovykh and c o - w ~ r k e r s have
' ~ ~ reported a slight increase of
the P;l;j5-4& cofactor distance of (0.4 f 0.2) A'97 Nevertheless, this small
displacement of QX seems not to be the major factor for the difference in
charge recombination of dark-frozen and light-frozen samples. Rather a
change in overall reorganisation energy, for example because of a frozen-in
rearrangement of polar groups involved in charge separation, was assumed to
be the major A similar explanation has been given by Zech and co-
workers who proposed that protein relaxation contributes substantially to the
reorganization energy. A suppression of relaxation modes by freezing could
occur slightly differently for samples frozen in the dark and under illumina-
tion. 196
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70 Electron Paramagnetic Resonance

6 Concluding Remarks

In conclusion it is fair to say that many questions about the structure and
function of the bacterial photosynthetic RC have been answered with the help
Published on 30 November 2000 on http://pubs.rsc.org | doi:10.1039/9781847553546-00043

of advanced EPR techniques. Nevertheless, some questions (still) remain


unanswered, one of which is the origin of the unidirectionality of the
photosynthetic electron transfer. However, with the potential of newly devel-
oped EPR techniques such as high-field EPR/ENDOR and pulsed EPR
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methods not yet fully exploited and with the availabilty of ever new types of
modified RCs, we can be certain that these remaining challenges will finally
also be tackled. And maybe, in one of the next reviews on bacterial photo-
synthesis, answers will be given to questions that today, at the beginning of the
third millennium, no-one ever dared to ask. . . .

Acknowledgements

It is a pleasure to thank my present and former teachers, Professor Klaus


Mobius (Free University of Berlin), Professor James R. Norris (University of
Chicago) and Professor Gerd Kothe (University of Freiburg), for helpful
discussions, encouragement and continuous support. I also want to express my
sincere gratitude to Dr. Christopher W. M. Kay (Free University Berlin) for
many stimulating discussions, fruitful teamwork and proof-reading. I am
indepted to Dr. Robert Bittl (Technical University of Berlin) and Dr. Stephan
Zech (Free University of Berlin) for providing me with Figure 5. Finally, I
would like to thank my wife Dr. Monika Koll-Weber for her help in preparing
this review. This work was supported by the Deutsche Forschungsge-
meinschaft (SFB 498).

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