Photosynthetic reaction centers of purple
bacteria
C Roy D Lancaster and Hartmut Michel
in
Handbook of Metalloproteins
Edited by
Albrecht Messerschmidt, Robert Huber, Thomas Poulos and Karl Wieghardt
© John Wiley & Sons, Ltd, Chichester, 2001
Photosynthetic reaction centers of purple
bacteria
C Roy D Lancaster and Hartmut Michel
Max-Planck-Institut fuÈr Biophysik, Frankfurt am Main, Germany
FUNCTIONAL CLASS
Photosynthetic reaction center (RC).
In a light-driven electron transfer reaction, the RC
stabilizes the separation of charged species across the
photosynthetic membrane. It thereby catalyzes the twoelectron reduction of a quinone to a hydroquinone, also
termed quinol (quinone photoreductase activity). The
electrons are replenished by the one-electron oxidation of
reduced cytochrome c2 (ferrocytochrome c2 photooxidase
activity).
O C C U R R E N CE
Purple bacteria find their ecological niche in deeper layers
of stagnant bodies of water.1 In all purple bacteria, the
photosynthetic pigments and the photosynthetic apparatus
3D Structure
Schematic representation of the structure of the Rp. viridis RC showing the heterotetramer of C, L, M, and H subunits
as Ca traces in green, yellow, blue, and purple, respectively, plus the fourteen cofactors. In addition, these fourteen cofactors are drawn
separately on the right for clarity. Also for the sake of clarity, the quinone tails are truncated after the first isoprenoid unit and the phytyl
side chains of the bacteriochlorophyll and bacteriopheophytin molecules have been omitted, as have those atoms of the carotenoid
molecule which were not observed in the electron density and assigned zero occupancy. PDB code: 2PRC. Prepared with programs
molscript147 and raster3d.148
HANDBOOK OF ME T A LL OP ROT E I NS
119
Photosynthetic reaction centers of purple bacteria
Figure 1 Light-induced cyclic electron flow and the generation and utilization of a transmembrane electrochemical potential in the
purple bacterium Rp. viridis. Modified from reference 58. See text for details.
are located within a more or less extended system of
invaginated intracytoplasmic membranes.2
BI O L O G I C A L F U N C T I O N
The function of the reaction center is to convert solar
energy into biochemically amenable energy (Figure 1). The
absorption of two photons of light leads, via the
stabilization of charged separated states, to cytochrome
c2 oxidation in the periplasm and to the reduction of a
Table 1 References to representative amino acid
sequence information of RC subunits derived from
nucleic acid sequences and partial peptide sequencing
…n:d: ˆ not determined)
Subunit
C
Purple Bacteria
Rhodospirillaceae
Rhodopseudomonas viridis
22
Rhodobacter capsulatus
±
Rhodobacter sphaeroides
±
Rhodospirillum rubrum
±
Rhodospirillum molischianum
134
Rubrivivax gelatinosus
135
Chromatiaceae
Chromatium vinosum
136
Ectothiorhodospiraceae
Ectothiorhodospira shaposhnikovii
137
Filamentous Green Bacteria
Chloroflexus aurantiacus
138
Obligate Aerobic, Anoxygenic Bacteria
Roseobacter denitrificans
141
120
L
M
H
20
131
132
133
134
135
20
131
132
133
134
135
21
131
132
n.d.
n.d.
n.d.
136
136
n.d.
137
137
n.d.
139
140
-
141
141
n.d.
H AN D B OOK OF M ETAL LOP RO TEI NS
quinone to a hydroquinone (quinol), which is coupled to
the uptake of two protons from the cytoplasm. The quinol
then leaves its binding site, diffuses in the photosynthetic
membrane and is reoxidized by a second membrane
protein complex, the cytochrome bc1 complex, which
results in proton release to the periplasm. The electrons are
transferred to re-reduce the cytochrome c2 in the periplasm. This net proton transport produces a transmembrane electrochemical gradient that can drive ATP
synthesis3 through a third membrane-spanning complex,
the ATP synthase.
A M I N O A C I D SE Q U E N C E I N F O R M A T I O N
Most bacterial reaction centers contain four protein
subunits, referred to as H, M, L, and C (a tetraheme
cytochrome c). Some, however, such as the RCs of
Rhodobacter (Rb.) sphaeroides, Rhodobacter capsulatus,
and Rhodospirillum (Rs.) rubrum, contain only the H, M,
and L subunits. The related RC of the green aerobic
thermophilic bacterium Chloroflexus (Cf.) aurantiacus
lacks the H subunit. References to representative amino
acid sequence information of RC subunits are listed in
Table 1. The gene for the H subunit lies on a different
operon than those for the other subunits and is less
frequently examined.
PROTEIN PRODUCTION, PURIFICATION,
AND MOLECULAR CHARACTERIZATION
Generally, RCs from purple bacteria have been isolated
and characterized from Rhodopseudomonas (Rp.) viridis,4
recently referred to as Blastochloris viridis, Rb. sphaer-
Photosynthetic reaction centers of purple bacteria
Table 2 Assignment of visual and near-infrared RC absorbance bands from Rp. viridis and Rb. sphaeroides. Values are for
room temperature. In the case of significant deviations, values for 5 K are included in parentheses. Data compiled from
references 6, 29, 67, 75, 142±144
Rp. viridisb
l (nm)
535
545
552
554
556
559
600
790/808
830
960 (990)
1 (mM
21
21
cm
)
D1 red-ox (mM
21
21
cm
)
34
26.5
25
27.5
300
123
1300
Rb. sphaeroidesa
l (nm)
1 (mM21 cm21)
535
545
600
760
800
865 (890)
1016
1260
288
128
Band type
Assignment
Qx
Qx
a
a
a
a
Qx
Qy
Qy
Qy
Qy
Qy
fB
fA
heme4
heme1
heme2
heme3
D, BA, BB
fA,B, fB/fA
BA, BB, Dh
Dl
BA2
D+
a
Contains BChl a.
Contains BChl b.
h
Higher energy exciton band of the primary donor dimer.
l
Lower energy exciton band of the primary donor dimer.
b
oides,5 Rb. capsulatus and a number of other purple
bacteria.5,6 Mutant RCs have been isolated and characterized from Rb. capsulatus,7±9 Rb. sphaeroides,10,11 and Rp.
viridis.12±15 The methods for isolation (and crystallization)
of the RCs from Rb. sphaeroides and Rp. viridis are the
subject of a recent review.16 The purification procedures
consist of disrupting the bacteria by ultrasonication,
isopycnic centrifugation of the chromatophores in a
sucrose gradient, and solubilization of the RCs with the
detergent N,N-dimethyldodecylamine-N-oxide (LDAO) at
concentrations of 6% (Rp. viridis) and of 0.5% (Rb.
sphaeroides), respectively. The RCs are further purified by
a combination of column chromatography steps. In the
case of Rp. viridis RCs, molecular sieve chromatography is
used exclusively.17 For the RCs of Rb. sphaeroides, various
modifications of a combination of anion exchange
chromatography and molecular sieve chromatography18
have been employed. A procedure for the rapid isolation
using Ni2+-nitrilotriacetic acid (NTA) affinity chromatography of Rb. sphaeroides RCs with an engineered polyhistidine tag fused to the C terminus of the M subunit has
been published, but no successful crystallization of the
isolated material has been reported.19 Recently, a procedure with an engineered His6-tag fused to the C-terminus
of the C subunit of recombinant Rp. viridis RC has yielded
material which could be crystallized.15
The L, M, and H subunits of the Rp. viridis RC contain
273, 323, and 258 amino acid residues …Mr ˆ 30:5 kDa;
35.9 kDa, 28.3 kDa), respectively.20,21 The C subunit of
Rp. viridis (336 residues, Mr ˆ 40:5 kDa†22 is a lipopro-
tein and is anchored in the membrane by a diacylglycerol
moiety, which is covalently bound to the N-terminal Cys
side chain via a thioether bond.23 A recognition site for the
covalent attachment of a diglyceride and removal of the
signal peptide by signal peptidase II is present in Rp. viridis
and Rv. gelatinosus but not in Cf. aurantiacus.
METAL CONTENT AND COFACTORS
RC preparations have a non-heme iron and four magnesium-containing bacteriochlorophyll cofactors per RC,5 as
measured by AA spectroscopy.24 In Rb. sphaeroides and
Rp. viridis, these are bacteriochlorophyll a and bacteriochlorophyll b, respectively. Those preparations with a
tightly bound C subunit have four iron-containing heme
groups which are covalently bound to the protein. Apart
from these four hemes, all other cofactors are noncovalently bound by the L and M subunits. In addition
to the metal-containing cofactors, these comprise two
bacteriopheophytin groups, a carotenoid, and two quinones. In Rb. sphaeroides, these are bacteriopheophytin a,
spheroidene, and ubiquinone-10, respectively, whereas Rp.
viridis contains bacteriopheophytin b, 1,2-dihydroneurosporene, menaquinone-9 and ubiquinone-9.
A further metal-binding site has been reported for Rb.
sphaeroides RCs, which were found to stoichiometrically
and reversibly bind Zn2+ in addition to the non-heme
iron.25 The suggested binding site is not conserved in Rp.
viridis RCs.
HANDBOOK OF ME T A LL OP ROT E I NS
121
Photosynthetic reaction centers of purple bacteria
Table 3 Carbonyl RR bands of bacteriochlorophylls and
pheophytins in cm21 (adapted from reference 29)
Rp. viridis
Rb. sphaeroides
1628
1635
1665
1678
1703
Assignment
DM ac¼Tyr M195
DL ac¼His L168
DL K¼Thr L248
fA K¼Glu L104
fB K free
1637
1678
1703
S PE C T R O S C O P Y
Apart from the availability of high resolution crystal
structures, one major reason why, despite its complexity,
the purple bacterial RC has become the `hydrogen atom of
protein electron transfer'26 (see also references 27 and 28)
is the richness of its characterization by optical absorption,
EPR, electron-nuclear double resonance (ENDOR), Fourier transform infrared (FTIR), RR, fluorescence, Stark
effect, and other types of spectroscopy, comprehensively
reviewed in references 29±34.
Optical spectroscopic properties of the Rp. viridis and
the Rb. sphaeroides RC are listed in Table 2. The
assignment of the spectral bands given in Table 2 is now
reasonably well established, but is an oversimplification, as
is shown by a more quantitative analysis by Knapp and
coworkers.35 The origin of the slightly different optical Qx
bands of fA and fB is due to an additional hydrogen bond
provided by the protein to fA, which is discussed below.
Since the Rp. viridis RC contains a bacteriochlorophyll b
(with a 4-ethyliden group), its Qy bands are at considerably
Table 4
54)
longer wavelengths than those of the Rb. sphaeroides RC,
which contains bacteriochlorophyll a (with a 4-ethyl
group). This is most extreme for the primary donor
band, which upon cooling, shifts to longer wavelengths
and becomes much narrower. Long before the X-ray
structure of the RC complex was known, the primary
donor had been suggested to be a dimer of bacteriochlorophyll, based on the observation of narrowed linewidths in
the EPR spectra of the Rb. sphaeroides RC.36
Spectroscopic properties and assignments of EPR and
ENDOR spectra of RCs have been tabulated29,37,38 as have
those of FTIR difference spectra.29,39,40 Another technique
to selectively investigate protein±cofactor interactions is
RR spectroscopy, by e.g. probing the coordination number
of the bacteriochlorophyll Mg ion (reviewed by Hoff and
Deisenhofer29) and determining the characteristic effects of
ligation to the bacteriochlorophyll a, b 9-keto and 2-acetyl
groups (see Table 3).
X-RAY STRUCTURES OF RCS FROM
PU R PL E BA CT E R I A
Crystallization
Crystals of the Rp. viridis RC were first grown by vapor
diffusion from a protein droplet containing 1.5 M
(NH4)2SO4, 0.1% N,N-dimethyldodecylamine-N-oxide
(lauryl-N,N-dimethyl-N-oxide, LDAO) and 3% heptane1,2,3-triol against a reservoir containing 2±3 M
(NH4)2SO4.17 They are tetragonal, space group P43212
c ˆ 113:6 A
41 and one molecule per
with a ˆ b ˆ 223:5 A;
asymmetric unit. Using these crystals, the structure of the
Globally well-defined structures of photosynthetic RCs from purple bacteria (modified and updated from reference
PDB entry
Rp. viridis RC
1PRC
2PRC
3PRC
4PRC
5PRC
6PRC
7PRC
1DXR
Rb. sphaeroides RC
1PCR
1MPS
1QOV
High resol. limit (AÊ)
R (%)
RfreeT(%)
2.30
2.45
2.40
2.40
2.35
2.30
2.65
2.00
19.3
18.2
17.8
19.1
19.2
18.5
19.0
19.4
n.d.
22.9
21.5
24.1
23.4
22.1
23.0
21.8
2.38
1.89
2.07
1.82
2.20
2.43
1.73
4.40
43,44
94
94
94
58
58
58
145,152
2.65
2.55
2.10
18.6
19.4
16.9
n.d.
21.7
18.6
1.91
1.92
4.27
55
101
52,153
nobs/npar
Reference
Criteria for inclusion in this table are as outlined in reference 54: (1) Structures are considered sufficiently well determined if the number of
independent observations, nobs, is larger than the number of parameters required to define the atomic model (npar). (2) Structural models are
considered to accurately represent the experimental diffraction data if the free crystallographic residual value, RTfree ,146 is less than 25%. In those
(older) cases, when RTfree was not determined, an R value of less than 20% is used as criterium instead.
122
H AN D B OOK OF M ETAL LOP RO TEI NS
Photosynthetic reaction centers of purple bacteria
Rp. viridis RC was solved by MIR41,42 and refined to a
crystallographic R-factor of 19.3% up to a resolution of
2.3 AÊ.43,44 More recent crystals diffract to at least 1.8 AÊ
resolution (CRD Lancaster and H Michel, unpublished
observations), and the structure has been refined with
Ê resolution (see Table 4 below).
complete data to 2.0 A
Three kinds of well-diffracting crystals have been
obtained of the Rb. sphaeroides RC (as reviewed by
Fritzsch16). They are orthorhombic,45±48 trigonal49 and
tetragonal.50 Orthorhombic crystals are grown in the
presence of 10±12% polyethylene glycol 4000
(PEG4000), 0.06% LDAO and 3.5±3.9% heptane-1,2,3triol or 0.8% n-octyl-b-d-glucopyranoside against a
reservoir buffer containing 18±25% PEG4000. The space
group is P212121. The best resolution is 2.8 AÊ in the
direction of the long axis, but worse in the other directions.
Using a partially refined coordinate set of the Rp. viridis
RC for molecular replacement, three different groups used
these orthorhombic crystal forms with slightly different
cell dimensions to determine the structure of the Rb.
sphaeroides RC. For all RC structures based on these
orthorhombic crystals, the number of observed unique
reflections is less than the number of parameters required
to define the model, as discussed earlier.51
Trigonal crystals can be obtained in the presence of 0.5±
1.0 M potassium phosphate, pH 6.5±7.5, 0.06±0.15%
LDAO and 1.8±3.0% heptane-1,2,3-triol against a reservoir buffer containing 1.4±1.7 M potassium phosphate.
The space group is P3121. The best crystals diffract to
beyond 2.4 AÊ.16 Substitution of potassium phosphate with
trisodium citrate (0.5 M in the droplet against 1.1 M in the
reservoir) and 10 mM Tris±HCl buffer, pH 8.0, and the
use of lower detergent concentrations for solubilization,
Ê resolution.52 To
yields crystals which diffract to 2.1 A
date, this is the only crystal form of the Rb. sphaeroides
RC which has yielded globally well-defined structures (cf.
Table 4).
Tetragonal crystals are grown in the presence of 6%
PEG4000, 0.85% n-octyl-b-D-glucopyranoside, 2.5%
heptane-1,2,3-triol and 0.4% benzamidine hydrochloride
against a reservoir solution containing 32% PEG4000.50
Crystals belong to the space group P43212 with two RCs
per asymmetric unit. Data from these crystals have been
collected to 2.2 AÊ resolution.53
Very recently, crystallization of the four-subunit RC
from the thermophilic bacterium Thermochromatium
tepidum in the presence of n-octyl-b-D-glucopyranoside,
PEG4000, and NaCl has yielded a structure refined at
2.2 AÊ resolution.154
Overall description of the structure
Table 4 lists the coordinate sets of those RC structures
which are globally well-defined, according to criteria
discussed earlier54 and detailed also in Table 4. Only
those Rb. sphaeroides RC structures based on the trigonal
crystal form satisfy these criteria, so we will refer to these
when comparing the RC structure from this species to that
of Rp. viridis. The structure of the four-subunit Rp. viridis
RC is shown schematically in the representation of the 3D
Structure. The RC from Rb. sphaeroides would appear
almost identical except for the cytochrome subunit at the
top, which would be missing. A detailed comparison of the
Rb. sphaeroides and Rp. viridis RC structures has been
performed previously.55,56
The Rp. viridis RC has an overall length of 130 AÊ in the
direction perpendicular to the membrane. Parallel to the
membrane, the maximum width is about 70 AÊ. The central
core of the RC is formed by the L subunit and the M
subunit, which possess five membrane-spanning segments
each. Both subunits are closely associated and noncovalently bind 10 cofactors as detailed above and
shown in the representation of the 3D Structure. Large
parts of the L and M subunits and their associated
cofactors are related by a two-fold axis of symmetry
perpendicular to the plane of the membrane. The H
subunit is anchored to the membrane by a single
membrane-spanning helix and is attached to the LM core
on the cytoplasmic side. On the periplasmic side, the C
subunit with its four covalently bound heme groups is
attached. The N-terminal diacylglycerol moiety is not
visible in the electron density map.
The pigments form two symmetry-related branches,
also shown in the representation of the 3D Structure, each
consisting of two bacteriochlorophylls, one bacteriopheophytin and one quinone, which both cross the membrane
starting from the `special pair' D of two closely associated
bacteriochlorophylls near the periplasmic side, followed by
the `accessory' bacteriochlorophyll, B, one bacteriopheophytin, f, and a quinone, Q. As shown in Figure 1, only
the branch more closely associated with L subunit is used
in the light-driven electron transfer. It is called the Abranch, the inactive one the B-branch. The active branch
ends with the primary quinone QA, the inactive one with
the secondary quinone QB. Halfway between both
quinones, a non-heme iron is located. The carotenoid,
which has a cis double bond at the 15±15 0 position in its
RC-bound state,57,58 is in van der Waals contact with BB
and disrupts the two-fold symmetry. In both species the
crystallographic temperature factors, which are a measure
for the rigidity of the structure, are considerably higher
along the B-branch than along the A-branch.
The Ca trace of the L subunit of the Rp. viridis RC is
shown in Figure 2(a). The dominant features are the five
long membrane-spanning helices (A±E). They are 21 (helix
A), 24 (helices C and E), or 28 (helices B and D) residues
long.43 On the periplasmic side, the connection of
transmembrane helices C and D contains a helix (`cd') of
eleven residues and the connection between transmembrane helix E and the C-terminus a helix (`ect') of nine
residues. On the cytoplasmic side, the connection of
HANDBOOK OF ME T A LL OP ROT E I NS
123
Photosynthetic reaction centers of purple bacteria
Figure 2
Ca Traces of the Rp. viridis L subunit (panel a), M
subunit (b) and H subunit (c). The letters `A' to `E' designate the
five transmembrane helices in the L and M subunits. The
additional helices `ab', `cd', `dde', `de', and `ect' are detailed in
the text. In the H subunit, residues H47 to H53 (on the right of
panel c) are not observed in the electron density. This region is
included as a very thin line in order to facilitate chain tracing.
124
H AN D B OOK OF M ETAL LOP RO TEI NS
transmembrane helices D and E contains a helix (`de') of
twelve residues. This region of the structure forms the
binding site of the secondary electron acceptor QB, which
is also included in Figure 2(a). In projection, viewed from
the top of the membrane, the transmembrane helices form
a semicircular arrangement in the order A, B, C, E, and
D.43 Transmembrane helices A, B, and D are straight, helix
E is smoothly curved, and helix C possesses a kink of more
than 308. When the L subunits from Rp. viridis and Rb.
sphaeroides are compared, an additional eight amino acid
residues are found at the C-terminus in the Rb. sphaeroides
RC.56
The M subunit of the Rp. viridis RC is displayed in
Figure 2(b). As indicated already by the sequence identity
of around 30% between the L and M subunits, the overall
protein fold is very similar. The five transmembrane helices
of the M subunit possess a length of 24 (C), 25 (A,E), 26
(D) or 27 (B) residues. The connecting helices `cd' (twelve
residues) and `ect' (seven residues) on the periplasmic side
as well as `de' (fourteen residues) on the cytoplasmic side,
forming part of the QA site, are also present. Accompanied
by an insertion of seven amino acids (compared to the L
subunit), short additional helices are found in the connections of transmembrane helices A and B (helix `ab', seven
residues) on the periplasmic side, and between transmembrane helix D and the connecting helix `de' on the
cytoplasmic side (helix `dde', six residues).
On the cytoplasmic side, the L and M subunits are
tightly interwoven. When the L and M subunits are
compared, the M subunits are 26 (Rp. viridis) or 25 (Rb.
sphaeroides) residues longer at the N-termini than the L
subunits. At the C terminus, the M subunit from Rb.
sphaeroides is nine amino acids shorter than the L subunit.
The M subunit from Rp. viridis possesses an additional 18
amino acids at the C-terminus which interact with the C
subunit (see also 3D Structure).
The N-terminus of the H subunit (see Figure 2(c)) is
located on the periplasmic side of the membrane. Residues
H12 to H35 form a membrane-spanning helix, which is an
a-helix at its beginning but a p-helix at its very end. The
next 70 residues are preferentially in contact with the LM
complex. A globular region follows that contains an
extended system of antiparallel and parallel b-sheets, and
an a-helix close to the C-terminus.
The structure of the tetraheme cytochrome or C subunit
as shown in Figure 3 has been described in detail.44 It is not
related to other known tetraheme protein structures and
consists of five segments, an N-terminal segment (C1±
C66), the first heme-binding segment (C67±C142), a
connecting segment (C143±C225), a second heme-binding
segment (C226±C315), and the C-terminal segment
(C316±C336). Apart from an a-helix (C25±C34) in the
N-terminal segment, the three non-heme-binding segments
contain little regular secondary structure. The four hemes
and the two heme-binding segments make up the core of
the cytochrome subunit. The first heme-binding segment
Photosynthetic reaction centers of purple bacteria
site (Figure 4(b)). The average His N1 ±Fe distance is 2.0 AÊ,
the average distance between the porphyrin N atoms and
Ê . The average Met Sd ±Fe
the respective Fe2+ is 2.05 A
Ê
distance is 2.3 A.
Non-heme-iron site geometry
Figure 3 The C subunit of the Rp. viridis RC. The protein
backbone is shown as a Ca trace, with the N-terminal segment
drawn in blue, the first heme-binding segment in green, the
connecting segment in yellow, the second heme-binding segment
in red, and the C-terminal segment in purple. The cofactor heme
groups and the side chains of their ligands are displayed as atomic
models.
contains the binding sites for heme1 (c554) and heme2
(c556), the second those for heme3 (c559) and heme4 (c552).
Each heme-binding site consists of an a-helix that runs
parallel to the heme plane, a loop, and the heme
attachment site with the sequence Cys-X-Y-Cys-His.
Heme-iron site geometries
The first four ligands to the six-coordinated heme iron are
provided by the porphyrin ring nitrogen atoms. The Cys
residues of the heme attachment site sequence Cys-X-YCys-His (C87±C91; C132±C136; C244±C248; C305±
C309) form thioether bonds with the heme groups and the
His is the fifth ligand to the heme iron. The Met residues
C74, C110, and C233 in the respective parallel helices are
the sixth ligands to heme1, heme2, and heme3 (Figure
4(a)), whereas the sixth ligand to heme4 is His C124,
which is located in the loop region of the heme2 binding
The environment of the six-coordinated non-heme ferrous
iron (see Figure 4(c)) is that of a distorted octahedron, the
base plane of which is formed by the three N1 atoms of His
L190, His L230, and His M217, and by one carboxyl O1
of Glu M232. The apices of the octahedron are formed by
the N1 atom of His M264 and the second carboxyl O1
atom of Glu M232. Such a distorted octahedral coordination had been predicted from MoÈssbauer and EXAFS
results, as reviewed by Feher and Okamura.59 The average
The four His ligands
ligand±Fe distances are 2:2 ^ 0:2 A:
are located four to eight residues away from the
cytoplasmic ends of transmembrane helices D and E of
the L and M subunits. The Glu ligand is situated at the Nterminal end of the cytoplasmic helix `dde', which is only
present in the M, but not in the L subunit (see above). The
flanking residues Asp M230, Arg M231, Glu M234, are
important constituents of the `QB cluster', a group of
electrostatically strongly interacting, protonatable residues
calculated60 to be important for proton uptake and
transfer to the QB site coupled to quinone reduction. The
His ligands M217 and L190 also provide, with their Nd
atoms, the proximal hydrogen bonding partners to the
quinones QA and QB, respectively. The non-heme Fe2+ ion
can be removed and replaced with Fe2+, Mn2+, Co2+, Ni2+,
Cu2+, and Zn2+ in the RC of Rb. sphaeroides61 and with
Zn2+ in the RC of Rp. viridis.62 Apparently, neither Fe2+
nor any divalent cation is required for rapid electron
transfer from QA2 to QB.61 However, the presence of a
metal ion in the Fe site appears to be necessary to establish
the characteristic electron transfer properties of QA.61
Bacteriochlorophyll-magnesium site geometries
The first four ligands to the five-coordinated bacteriochlorophyll magnesium are provided by the bacteriochlorin ring nitrogen atoms, and the fifth ligand is
provided by the N1 atom of a His side chain. In the case
of the `special pair' bacteriochlorophylls, these His residues
(L173 and M200) are located close to the N-terminal ends
of the L and M subunit transmembrane helices D,
respectively. The His ligands for the accessory bacteriochlorphylls, L153 and M180, are situated close to the Nterminal end of the L and M subunit periplasmic helices
`cd', respectively. The average His N1 ±Mg distance is
Ê , as is the average distance between the bacterio2.1 A
chlorin N atoms and the respective Mg2+.
HANDBOOK OF ME T A LL OP ROT E I NS
125
Photosynthetic reaction centers of purple bacteria
Figure 4 Iron site geometries in the Rp. viridis RC: (a) the binding site of heme3 as an example for a His-Met ligated heme iron. The
binding sites for heme1 and heme2 are similar except for the close proximity of Arg C264, which has been shown both theoretically71 and
experimentally15 to strongly modulate the redox potential of heme3; (b) the binding site of heme4 as a His±His ligated heme iron; (c) the
structural role of the non-heme iron.
Apart from binding the Mg2+ ion, the protein displays a
number of hydrogen bonding interactions with the bacteriochlorophyll molecules, as deduced from the structure,63
126
H AN D B OOK OF M ETAL LOP RO TEI NS
shown in Figure 5, and also listed in Table 3. The hydrogen
bonding of the fA 9-keto group to the glutamic acid side
chain of Glu L10463 (see Figure 6) has been shown to be
Photosynthetic reaction centers of purple bacteria
Figure 5
Bacteriochloropyhll±Mg site geometries in the RCs of (a) Rp. viridis and (b) Rb. sphaeroides. For clarity, C atoms are not
drawn explicitly, and O atoms are drawn as small white spheres.
responsible for the 10 nm red shift (see Table 2) of the fA Qx
band compared to the fB Qx band,64 but is not a dominant
contributor to the directionality of electron transfer in RCs.
The corresponding residue for fB is Val M131, which cannot
form such a hydrogen bond.
Figure 6
The region of the bacteriopheophytin molecule fA in
the Rp. viridis RC. For clarity, C atoms are not drawn explicitly,
and O atoms are drawn as small white spheres. Selected
phenylalanine residues are labeled with subunit designator and
residue number only.
FUNCTIONAL ASPECTS
Oxidation±reduction potentials
The redox midpoint potentials of the four heme groups
follow the order low, high, high, low in the sequence (see
Table 2) or low, high, low, high if the hemes are ordered
with decreasing distance (see Figure 7) from the primary
electron donor D.65±70 The application of continuum
electrostatics to the crystal structure of the cytochrome
subunit has provided quantitative estimations of the
factors contributing to the equilibrium EM values of the
four hemes.71 Specific residues and the propionic side
chains on the hemes are calculated to strongly modulate
the EM values. The correct division into low and high
potential hemes can be obtained by taking only the protein
into account. Consideration of heme±heme interactions is
required to reproduce the experimental data quantitatively.
The redox potentials of the other Rp. viridis RC
cofactors are also included in Figure 7. The potential of
D* is derived from the free energy difference between the
lowest vibrational levels of D and D*. Estimated from the
wavelength of the absorption maximum, the dimer of
bacteriochlorophyll b in Rp. viridis (cf. Table 2) provides
an energy of 1240 meV between D and D* and that of the
bacteriochlorophyll a dimer in Rb. sphaeroides an energy
of 1380 meV.72 The redox potential of Rb. sphaeroides D
has been increased from 505±765 mV by the introduction
of three additional hydrogen bonds to the special pair
bacteriochlorophylls by site-directed mutagenesis,73 thus
destabilizing the oxidized state of the donor, D+.
The redox potential of QA is higher in the Rb.
sphaeroides RC than in the Rp. viridis RC because of the
different chemical nature of the quinones (ubiquinone vs.
menaquinone). However, that of Rb. sphaeroides QA is still
HANDBOOK OF ME T A LL OP ROT E I NS
127
Photosynthetic reaction centers of purple bacteria
Figure 7 Equilibrium oxidation-reduction potentials of the Rp. viridis RC cofactors as reported in references 14, 67, 72, 149±151 as a
function of inter-cofactor distance. The soluble electron donor protein cytochrome c2 has been included as suggested by references 91 and
109. Reaction halftimes indicated are taken from the references cited in the text. The photochemical excitation is indicated by a dashed
arrow and unphysiological charge recombination reactions are shown as dotted arrows.
67 mV lower than that of Rb. sphaeroides QB,74 even
though both cofactors are chemically identical, thus
requiring a role of the protein in tuning the in situ redox
potentials of the quinones.
Kinetics
Figures 1 and 7 show schemes of the electron transfer steps
that occur in the purple bacterial reaction center. Light
absorption leads to an excited primary donor D*, from
which an electron is transferred via the monomeric
bacteriochlorophyll BA75 (reaction halftime: 2.8 ps) and
the bacteriopheophytin fA (700 fs) to QA in 200 ps,76,77
leading to the formation of D+QA2QB (see references 78±
80 for reviews). Re-reduction of D+ by cytochrome c559
(heme 3)68 occurs in 320 ns.67 These processes are much
faster than the subsequent proton uptake and inter-
128
H AN D B OOK OF M ETAL LOP RO TEI NS
quinone electron transfer reactions. Therefore, the first
step of quinone reduction in the RC can be viewed as a
`photochemical cytochrome oxidation', giving rise to the
radical state DQA2QB. Re-reduction of cytochrome c559+
by cytochrome c556 (heme 2) occurs in 1.7 ms.81 The
second step of quinone reduction involves the transfer of
the first electron to QB (in 17±25 ms,82±84 resulting in the
state DQAQB2. The one-electron reduction of QB is not
associated with direct protonation and the semiquinone
species is anionic. However, the QA2QB $ QAQB2 equilibrium constant85 is pH-dependent, as electron transfer is
accompanied by substoichiometric proton uptake due to
the protonation of amino acid residues.86±90. Cytochrome
c556+ is re-reduced by cytochrome c2 in 40±60 ms.91 In
species that lack the tightly bound tetraheme-cytochrome c
subunit, e.g. Rb. sphaeroides, the photoxidized special pair
is directly re-reduced by cytochrome c2 in a biphasic
reaction. The fast phase of ,1 ms is attributed to
Photosynthetic reaction centers of purple bacteria
Figure 8
Derivatives at the QB site of the Rp. viridis RC: (a) Comparison of distal (1PRCnew, black) and proximal (2PRC, gray)
ubiquinone-binding sites;94 (b) comparison of QB-depleted (3PRC, black) and ubiquinone-2-occupied (2PRC, gray) QB sites;94 (c)
comparison of stigmatellin binding (4PRC, black) and ubiquinone-2 binding (2PRC, gray);94 (d) atrazine binding (5PRC, black) compared
to distal (1PRCnew, light gray) and proximal (2PRC, gray) ubiquinone-binding sites.58
intermolecular electron transfer, the slow phase of 100 ms
is limited by docking and reorientation of the cytochrome
c2-RC complex.92 After a second `photochemical cytochrome oxidation', the diradical state DQA2QB2 is formed
at rates similar to those for the first electron transfer.
Coupled transfer of the first proton and the second electron
to QB2 leads to the monoprotonated, doubly reduced state
DQA(QBH)2. Transfer of the second proton for the
formation of QBH2 is kinetically indistinguishable from
the first proton transfer in the wild-type RC and can only
be resolved in the case of mutants with significantly
retarded second proton transfer rates.93
FUNCTIONAL DERIVATIVES
In the original Rp. viridis RC structure, the QB site was
poorly defined because it was only partially occupied with
the native ubiquinone-9 in the standard RC crystals.
However, ubiquinone-2-reconstitution experiments have
yielded crystals with full quinone occupancy of the QB
site.94 Subsequent X-ray diffraction analysis and refine-
ment has led to a well-defined QB-site model (PDB code:
2PRC), with the quinone bound in the `proximal' position,
i.e. close to the non-heme iron (hydrogen-bonded to its
ligand His L190, see Figure 8(a)). In the RC structure with
a QB-depleted QB site (3PRC), refined at 2.4 AÊ, apparently
five, possibly six, water molecules are bound instead of the
ubiquinone head group, and a detergent molecule binds in
the region of the isoprenoid tail.94 Using the structures
2PRC and 3PRC as references, the original data set
1PRC44 was re-examined. While not excluding the
presence of a minor fraction of the quinone in the proximal
site, this resulted in the suggestion94 of a `distal' dominant
QB-binding position for the native ubiquinone-9
(1PRCnew), not hydrogen-bonded to His L190 and further
away from the non-heme iron (see Figure 8(a)). A more
quantitative analysis54 of the original data resulted in 20%
of the QB sites being occupied with quinone in the
proximal site, 30% having quinone bound in the distal
site, and half of the QB sites being empty or having the
quinone unaccounted for. A further structure, the RC
complex with the inhibitor stigmatellin (4PRC), refined at
Ê , indicates that additional hydrogen bonds stabilize
2.4 A
HANDBOOK OF ME T A LL OP ROT E I NS
129
Photosynthetic reaction centers of purple bacteria
the binding of stigmatellin over that of ubiquinone-2 (see
Figure 8(c)). The binding pattern observed for the
stigmatellin complex can be viewed as a model for the
stabilization of a monoprotonated reduced intermediate
(QBH or QBH2).51,94,95 This indicates that the QB site is
not optimized for QB binding, but for QB reduction to the
quinol.95 In combination with the results of electrostatic
calculations, these crystal structures can provide models
for intermediates in the reaction cycle of ubiquinone
reduction to ubiquinol, as discussed below.
It had previously been established by X-ray crystallography that the triazine herbicide terbutryn binds to the
QB site.63 However, the exact description of protein±
triazine interactions has had to await the refinement of
high resolution structures of complexes of the RC with
atrazine (PDB code: 5PRC) and two chiral atrazine
derivatives (PDB entries: 6PRC and 7PRC) at 2.35 AÊ,
Ê , and 2.65 AÊ resolution, respectively. In addition to
2.30 A
two previously implied hydrogen bonds, a third hydrogen
bond, binding the distal side of the inhibitors to the
protein, and four additional hydrogen bonds, mediated by
two tightly bound water molecules are apparent (Figure
8(d)).58 The structures provide explanations for the relative
binding affinities of the three atrazine-based compounds
and thus also for the enantioselectivity of the QB site for
the chiral derivatives.
Mutants
Work on site-directed mutagenesis of photosynthetic
reaction centers started with the RC from Rb. capsulatus
(see references 7 and 8 for early reviews). This species is
genetically very well characterized and able to grow nonphotosynthetically under aerobic conditions, as well as
under anaerobic conditions using e.g. dimethylsulfoxide as
an electron acceptor. Most importantly, under these latter
conditions the photosynthetic apparatus is fully induced.
Unfortunately, the RC from Rb. capsulatus could not be
crystallized, thus thwarting proper inspection for structural
changes.
The closely related Rb. sphaeroides can be grown under
similar non-photosynthetic conditions, so that site-directed
mutagenesis is also straightforward. As detailed above, this
RC is amenable to inspection by X-ray crystallography for
structural changes. Many amino acids which were considered to be of importance for pigment binding or electron
transfer63 were changed in Rb. sphaeroides RCs. Most
outstanding is the mutation Tyr M210 ! Phe: In the
mutant RC the rate of initial electron transfer is slowed
down by a factor of 4±6.96,97 The X-ray crystallographic
analysis98 using the orthorhombic crystal form did not
reveal any significant structural changes except for the
absence of the O atom, which appears to be the reason for
the decreased observed rate of electron transfer. Other
mutations such as those involving protonatable `QB
130
H AN D B OOK OF M ETAL LOP RO TEI NS
cluster' residues Glu L212 ! Gln and Asp L213 ! Asn;
and a ligand to the non-heme iron His M219 ! Cys
(corresponding to residue M217 in Rp. viridis, Figure 4(c)),
also do not lead to detectable structural changes.98
However, the resolution of the latter data set was limited
to 4 AÊ. When the residues His L173 and His M202
(corresponding to M200 in Rp. viridis, Figure 5) liganded
to the special pair bacteriochlorophylls DL and DM, are
replaced by Leu residues, bacteriopheophytins are incorporated as DL and DM respectively. Vice versa, in the Leu
M214 ! His mutant, corresponding to residue M212 in
Rp. viridis (Figure 6), a bacteriochlorophyll, termed b, is
incorporated as fA instead of a bacteriopheophytin.
Using the trigonal crystal form, the reaction center
mutants Trp M252 ! Phe; Trp M252 ! Tyr; and Thr
M222 ! Val99 have been analyzed. In these mutants the
electron transfer from fA to QA has been slowed down by
a factor of three. The structural analysis100 yields no
detectable structural changes in the Trp M252 ! Phe and
Thr M222 ! Val variants, but there are some structural
changes, involving also a movement of QA, in the Trp
M252 ! Tyr mutant. The structure of a Rb. sphaeroides
RC double mutant Phe M197 ! Arg=Tyr M177 ! Phe
has also been determined.101 The mutant complex shows
an unexpected change in the structure, with a reorientation
of the new arginine, the incorporation of a new water
molecule into the structure, and the rotation of the DM 2aacetyl group. The structure of a Phe M197 ! Tyr mutant
has recently been described.102 In combination with
electrochemically induced FTIR spectra, there is clear
evidence for the existence of a newly established hydrogen
bond between Tyr M197 and the DM 2a-acetyl group. In
addition to mutagenesis, cofactors may be removed or
replaced chemically with a wide range of similar compounds, reviewed by Gunner72 in the case of quinones and
by Scheer and Struck103 for bacteriochlorins. Very recently,
the structure of an Ala M260 ! Trp variant RC has been
described at 2.1 AÊ resolution (PCB code 1QOV).153 This
mutation leads to the exclusion of QA from the RC.
Site-directed mutagenesis of the structurally best characterized RC from Rp. viridis is possible104 but more
difficult. Rp. viridis can grow only under photosynthetic
and, very slowly, under microaerophilic conditions. However, under microaerophilic conditions, the photosynthetic
apparatus is not induced and photosynthetic growth
conditions exert a selection pressure for revertants and
suppressor mutants if the RCs are functionally impaired.
On the other hand, very interesting herbicide-resistant
mutants were obtained by classical selection procedures,
with mutations some of which would not have been made
by site-directed mutagenesis.105,106
Some of these herbicide resistant mutants of the Rp.
viridis RC have also been analyzed by X-ray crystallography. In the double mutant Arg L217 ! His=Ser
L223 ! Ala; the side chain of Asn L213, which is
hydrogen-bonded to Ser L223 in the wild type, is rotated
Photosynthetic reaction centers of purple bacteria
very uphill electron transfer from heme2 to heme4 (see
Figure 7).15
Very recently, the structure of a His L168 ! Phe variant
Ê resolution (PDB code
RC has been described at 2.0 A
152
This mutation leads to a drastic acceleration of
1DXR).
the initial rate of electron transfer from D* to D1BA2. This
effect is due to a 208 rotation of the ring I acetyl group of
DL upon loss of the hydrogen bond donated by His L168 in
the wild-type enzyme (see Figure 5(a)), bringing the acetyl
oxygen 1.1 AÊ closer to the Mg21 of DM.
I N T E R - A N D I N TR A M O L E C U L A R
ELECTRON AND PROTON TRANSFER AND
CATALYTIC MECHANISM
Figure 9
Reduction of the photo-oxidized tetraheme C
subunit of the Rp. viridis RC by Rp. viridis cytochrome c2.
Docking as suggested by Osyczka and coworkers109. The three
pairs of interacting residues Glu C67, C93, C79 of the RC and Lys
11, 71, 78 of cytochrome c2 are indicated by dashed lines between
their Ca positions.
towards the cavity which is created by the replacement of
Arg L217 by the smaller His.107 At the same time, QB
becomes more firmly bound.105 The mutation Tyr L222 !
Phe unexpectedly leads to resistance against the herbicide
terbutryn. In the wild type, Tyr L222 forms a hydrogen
bond with the peptide carbonyl oxygen of Asp M43. Since
this hydrogen bond is now missing, a stretch of the M
subunit (M25±50) moves into a new position. The side
chain of Phe L222 rotates by 908 into the herbicide binding
site (see above), thereby preventing the binding of
terbutryn by steric hindrance.108
Using site-directed mutagenesis, the highly conserved
Tyr L162, positioned halfway between D and the proximal
heme3 (cytochrome c559) in the Rp. viridis RC, was
exchanged against a number of amino acids. All mutants
grew photosynthetically. The redox potentials of D and
c559 were changed by the mutations. The structures of two
mutants (Tyr L162 ! Phe and Tyr L162 ! Thr† were
determined and found not to differ significantly from the
wild-type structure.13 Analysis of the kinetics of electron
transfer led to the conclusion that the tyrosine residue at
position L162 is not required for fast electron transfer from
c559 to D+.13
A recent mutation of Arg C264 ! Lys decreases the
midpoint potential of heme3 (cytochrome c559) from
+380 mV to +270 mV, i.e. below that of heme2
(+320 mV, see Figures 4(a) and 7).15 In the structure of
the mutant RC at 2.46 AÊ resolution, no remarkable
differences were found apart from the mutated residue
itself.15 The halftime of electron transfer between heme2
and heme3 was the same as in the wild-type, indicating
that the observed reaction rate is limited by the
All four hemes of the Rp. viridis RC tetraheme C subunit
are located close enough to the surface of the protein to
accept electrons from soluble cytochrome c2. Site-directed
mutagenesis in Rvi. gelatinosus has led Osyczka and
colleagues109 to identify of a patch of acidic residues
immediately surrounding the distal low-potential heme1 of
the tetraheme C subunit that apparently forms an
electrostatically favorable binding site for soluble cytochromes. Thus all four hemes in the C subunit seem to be
directly involved in the electron transfer towards the
photo-oxidized special pair. Based on these findings, a
model was proposed for the transient cytochrome c2-RC
complex for Rp. viridis (see Figure 9).
The kinetics of light-induced electron transfer via QA to
QB and of the re-reduction of the special pair have been
detailed above. The reasons for the unidirectionality of
electron transfer along the active A branch and not along
the inactive B branch, despite the two-fold pseudosymmetry of the LM core of the RC, have been the subject of
numerous theoretical and experimental investigations.
Slight differences in geometry, differences in rigidity, and
differences in the amino acid composition of the L and M
subunits have been suggested to contribute to unidirectionality.43 The latter aspect was specified by the theoretical
identification of a large electrostatic field favoring charge
separation along the A branch.110 A major contributor to
the potential gradient is Arg L103, whose positive charge is
stabilized by different sections of the polypeptide backbone. This dipolar stabilization leads to a much longerrange effect of the positive charge than if it were stabilized
by a counter ion.
An experimental observation consistent with this
electrostatic analysis is the finding that a site-directed
double mutant in Rb. capsulatus RCs (Leu L212 ! His;
Gly L201 ! Asp† appears to show significant electron
transfer to fB.111 The first mutation leads to the
incorporation of the b bacteriochlorophyll in the fA
position (see above) and the second mutation introduces
a negative charge close to BA, thus making electron transfer
down the A branch less favorable.
HANDBOOK OF ME T A LL OP ROT E I NS
131
Photosynthetic reaction centers of purple bacteria
Figure 10
Mechanistic implications of the structures 2PRC, 3PRC, 4PRC,94 and the revised model 1PRCnew for the events at the QB
site within the reduction cycle of quinone to quinol. For clarity, C atoms are not drawn explicitly, and H atoms are drawn as small white
spheres. Dashed arrows symbolize quinone movements, black solid arrows proton and electron transfer events, and grey solid arrows
highlight important interactions. Panel (a) is drawn from co-ordinate set 1PRCnew, panels (b) and (c) from 2PRC, panel (d) is derived from
4PRC, panel (e) is derived from panel (d), and panel (f) is a combination of 3PRC and 1PRC.55 In panel (f), the ring plane of Phe L216
stacks directly above the QBH2 ring system. Figure modified from references 94 and 95; made with molscript.147
Both QA and QB sites are deeply buried within the
photosynthetic reaction center complex, approximately
15 AÊ from the cytoplasmic surface. Proton transfer to the
reduced quinone within the QB site could occur by protons
moving along a chain of proton donors and acceptors
by a `proton wire', or hydrogen-bonded chain mechanism.112±114 Possible proton donors and acceptors are
protonatable amino acid residues and water molecules. A
number of the protonatable residues between the QB site
and the cytoplasmic surface have been shown to be
functionally relevant to the proton transfer process by
analysis of site-directed mutations,11,115 and second site
revertants.7,116 The observed effects can be due to the
modification of the kinetics and thermodynamics of
electron or proton transfer. Electrostatic calculations on
the RCs of Rb. sphaeroides117±119and Rp. viridis60,120
led to the identification of residues that can contribute
to the changes in equilibrium distributions of protons
in the different redox states of the protein, thus helping
to determine the role of the functionally important
residues.
132
H AN D B OOK OF M ETAL LOP RO TEI NS
In combination with the results of electrostatic calculations,60 the crystal structures 3PRC, 2PRC, 4PRC, and
1PRCnew, discussed above (cf. Figure 8) can provide
models for intermediates in the reaction cycle of ubiquinone reduction to ubiquinol (see Figure 10).94,95 The
binding of the incoming QB to the distal site displaces some
of the water molecules present in the `empty' pocket
(Figure 10(a)). The quinone ring is flipped around the
isoprenoid tail and further water molecules are displaced
for the QB to occupy the proximal position (Figure 10(b)).
This is the position in which neutral QB accepts an electron
from QA2. The hydrogen bonds donated to the quinone
will automatically lead to a tighter binding of the
negatively charged semiquinone QB2 compared to the
neutral QB. Additionally, the side chain of Ser L223 can
reorient by rotation of its x2 (Ca ±Cb ±Og ±Hg) torsional
angle, thus establishing an additional hydrogen bond to
QB2. Coupled to the transfer of the second electron, the
first proton is transferred (Figure 10(c)), possibly via a
transiently protonated Ser L223±OH2+,60 thus forming the
monoprotonated, doubly reduced intermediate QBH2
Photosynthetic reaction centers of purple bacteria
(Figure 10(d)). After transfer of the second proton,
movement of the quinol from the proximal (Figure 10(e))
to the distal position (Figure 10(f)) may be facilitated by
increased stacking interactions of the aromatic ring
systems with the Phe L216 ring and the diffusion of
water molecules back into the pocket. The structures of
these intermediates provide explanations for their relative
binding affinities, as required for proper enzymatic
function of the QB site. A rearrangement of hydrogen
bonds, most prominently the reorientation of the Ser L223
side chain for QB reduction, as suggested by the scenario in
Figure 10, is also calculated to be necessary to make QB
reduction more favorable than QA reduction.119 These
local rearrangements may constitute the conformational
changes deduced to be required for function by a variety of
experiments.25,121±123
RELEVANCE TO PHOTOSYSTEM II
Based on the determined structure of the purple bacterial
RC, very specific sequence homologies, and azidoatrazine
labeling, the RC core of higher plant photosystem (PS) II
was proposed to be similar to the LM core of the bacterial
RC, with the D1 and D2 proteins corresponding to the L
and M subunits, respectively.124±128 This proposal could
be verified experimentally.129 Recently, suitably designed,
modified bacterial RCs have been shown to mimic tyrosine
oxidation in PS II.130 In the absence of a high-resolution
structure of the photosystem II RC, the purple bacterial RC
still serves as the basis for models of PS II.
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