Báo cáo Y học: Exploring the primary electron acceptor (QA)-site of the bacterial reaction center from Rhodobacter sphaeroides Binding mode of vitamin K derivatives pptx

Exploring the primary electron acceptor (Q
A
)-site of the bacterial
reaction center from
Rhodobacter sphaeroides
Binding mode of vitamin K derivatives
Oliver Hucke, Ralf Schmid and Andreas Labahn
Institut fu
¨
r Physikalische Chemie, Albert-Ludwigs-Universita
¨
t Freiburg, Germany
The functional replacement of the primary ubiquinone (Q
A
)
in the photosynthetic reaction center (RC) from Rh odo-
bacter sphaeroides with synthetic vitamin K derivatives has
provided a powerful tool to in vestigate the ele ctron transfer
mechanism. To investigate the binding mode of these qui-
nones t o t he Q
A
binding site we have determined the binding
free energy and c harge recombination rate from Q
A
–
to D
+
(k
AD
) of 29 different 1,4-naphthoquinone derivatives with
systematically altered structures. The most striking re sult

was that none of the eight tested compounds carrying methyl
groups in b oth positions 5 and 8 of t he aromatic r ing
exhibited functional binding. To understand the binding
properties of these quinones on a molecular level, the
structures of the reaction center-naphthoquinone complexes
were predicted with ligand docking calculations. All protein–
ligand structures show hydrogen bonds between the
carbonyl oxygens of the quinone and AlaM260 and
HisM219 as found for the native ubiqu inone-10 in t he X-ray
structure. The center-to-c enter distanc e between the naph-
thoquinones at Q
A
and the native ubiquinone-10 at Q
B
(the
secondary electron acceptor) is essentially the same, com-
pared to the native structure. A detailed analysis of the
docking calculations reveals that 5 ,8-disubstitution prohibits
binding due to steric clashes of the 5-methyl g roup with the
backbone atoms of AlaM260 and Al aM249. The experi-
mentally determin ed binding free energies were reproduced
with an rmsd of % 4kJÆmol
)1
in most cases providing a
valuable tool for the design of new artificial e lectron a cce p-
tors and inhibitors.
Keywords: ligand docking; structure activity relationship;
bacterial reaction centers; Rhodo bacter sphaeroides; infrared
spectroscopy.
The photosynthetic reaction center (RC) of the purple

bacterium Rhodobacter sphaeroides (R. sphaeroides)isan
intrinsic membrane protein complex that performs the
conversion of light energy into chemical energy. A
complex framework o f redox cofactors i s buried in the
protein matrix. The cofactors are arranged in two
branches, the active A-branch and the inactive B-branch,
showing nearly twofold symmetry (reviewed in [1,2]).
Following the absorption of a photon, an electron is
transferred within 200 ps from the bacteriochlorophyll
dimer, the p rimary donor D, via a bacteriochlorophyll
monome r (B
A
) and a bacteriopheophytin (F
A
)toatightly
bound ubiquinone molecule (Q
A
), forming the first stable
charge separated state D
+
Q
A
–
. The subsequent elec tron
transfer step from Q
A
–
to Q
B
proceeds on a slower time

scale (% 200 ls). After rereduction of the photooxidized
primary donor by a soluble cytochrome c
2
the light-
induced elec tron transfer leads to the formation of the
doubly reduced Q
B
and concomitant binding of two
protons from the cytoplasmic side of the membrane. The
ubiquinol dissociates from the RC and is reoxidized by
the cytochrome bc
1
complex releasing two protons to the
periplasmic side of the membrane. The net result of these
reactions is a transmembrane pH difference that drives
ATP synthesis.
In vitro, in the absence of both, an external reductant for
the primary donor and the secondary quinone, t he charges
on Q
A
–
and D
+
recombine with the r ate k
AD
. This reaction
was subject to numerous spectroscopic studies. One
important a pproach to investigate this electron transfer
mechanism in wild-type reaction centers was pioneered by
Okamura et al. [3,4]. They developed a method of

ubiquinone removal and readdition of ubiquinone or any
other synthetic quinone. Gunner et al. [5] measured the
temperature dependence of k
AD
in RCs with different
anthra-, benzo- and n aphthoquinone derivatives at Q
A
.
Most o f these compounds display a low m idpoint redox
potential in situ compared to the native UQ-10, leading to
an increase of the free energy d ifference between the D
+
Q
A
–
and t he ground state DQ
A
. E valuating t he free energy
dependence of k
AD
Gunner et al. [5] deduced that the
charge recombination from Q
A
–
in native RCs is a n
activationless p rocess. More recently, the quinone rep lace-
ment method was u sed to derive thermodynamic param-
eters f or that reaction [6] a nd to study the forward electron
Correspondence to A. Labahn, Institut fu
¨

r Physikalische Chemie,
Albert-Ludwigs-Universita
¨
t Freiburg, Albertstr. 23a, D-79104
Freiburg, Germany. Fax: + 49 761 203 6189,
Tel.: + 49 761 2 03 6188, E- mail:
Abbreviations: UQ-10, ubiquinone-10; NQ, 1,4-naphthoquinone; RC,
reaction center; B, Blastochloris;R,Rhodobacter;Q
A
, primary electron
acceptor; Q
B
, secondary electron acceptor; LDAO, lauryldimethyl-
amine-N-oxide; DAD, diaminodurene.
Note: web page available at />Andreas/homepage
(Received 1 3 August 2001 , revised 14 November 2001, a ccepted
23 Nov ember 2001)
Eur. J. Biochem. 269, 1096–1108 (2002) Ó FEBS 2002
transfer to the primary ubiquinone [7–9]. Ka
´
lma
´
n&
Maro
´
ti used the quinone reconstitution method to study
the proton b inding kinetics and stoichiometry associated
with the reduction of Q
A
[10,11]. It was shown that both

processes are controlled by protonatable residues in the
interior of the p rotein. Measuring the delayed fluorescence
of the excited dime r, D*, in R Cs with different quinones as
Q
A
Turzo
´
et al. [12] d erived an empirical relation betwee n
the in situ free energy of the primary quinone and the
charge recombination rate, providing the free energy levels
of the corresponding charge separated s tates. Similar
quinone reconstitution experiments h ave been performed
for the Q
B
site [13–15]. Palazzo et al. [14,15] incorporated
the reaction centers into lipid vesicles an d measured the
temperature dependence of the charge recombination rate
from Q
B
–
to D
+
. Based on a detailed analysis they
determined the binding free energy, enthalpy and entropy
of the ubiquinone to the Q
B
-site.
Methyl substituted 1,4-naphthoquinones have gained
substantial i nterest a s they bind tightly to the Q
A

site,
enabling its functional reconstitution while retaining the
native ubiquinone at Q
B
[16,17]. The difference in the
semiquinone anion spectra allows the direct monitoring of
the electron t ransfer f rom Q
A
to Q
B
in the V IS region with
transient absorption spectroscopy. In addition, these sub-
stitutions change the driving force of the electron transfer
reaction from Q
A
–
to Q
B
(and thus the electron transfer r ate
and e quilibriu m) but they affect neither conformational
changes nor the protonation rates or protonation equilibria
near Q
B
.Graigeet al.[18]andLiet al. [19,20] applied this
method to study the effect of the driving force on the fi rst
electron transfer t o Q
B
. Similarly, the m echanism of the
proton-coupled ele ctron transfer reaction [Q
A

–
Q
B
–
+H
+
fi
Q
A
(Q
B
H)
–
] was elucidated by Okamura and coworkers.
They showed that this reaction is a two-step process in
which fast protonation preced es rate-limiting electron
transfer [17,21,22]. Moreover, methyl substituted nap htho-
quinones play an important role in reaction centers of
other photosynthetic organisms. For instance, 2-methyl-
3-(isoprenyl)
(7)9)
1,4-naphthoquinone (menaquinone) was
identified as the primary electron acceptor in the reaction
centers from Blastochloris viridis [23], Chloroflexus auran-
tiacus [24] and in t he photosystem I of green plants [25–27].
Hence, a systematic variation of the redox potential of the
naphthoquinone compounds and a detailed knowledge of
their binding properties are of critical i mportance for the
investigation of electron t ransfer reactions in photosynthetic
reaction centers.

In this work , we i nvestigated the binding properties of 29
vitamin K de rivatives with respect to t he Q
A
site of the
reaction center from R. sphaeroides (see Table 1, Scheme 1).
Their midpoint redox potentials w ere altered by varying
both, the number and the p osition of methyl g roups at th e
ring system. I n some cases, additionally a hydrocarbon tail
was introduced in position 3 to improve the binding
affinity to Q
A
in analogy to ubiquinone [28]. Light-induced
FTIR difference s pectro scopy was u sed t o d etect structural
changes of the bind ing pocket upon binding of the
different quin ones. We measured the dissociation constants
K
d
of these compounds and compared our results with
those from ligand docking calculations. The calculated
structures of the quinone-protein complexes provide
insights into the aspects t hat govern the binding of
quinones to the Q
A
site, allowing to test whether their
positions at Q
A
are identical with that of ubiquino ne-10.
A preliminary account of this work has been presented
elsewhere [29].
MATERIALS AND METHODS

Quinone-depleted reaction centers
Reaction cente rs from the strain R. sphaeroides R-26 were
isolated and purified in lauryldimethylamine-N-oxide
(LDAO) from photosynthetically grown cells following
the p rocedure o f F eher & Okamura [ 30]. The ratio o f
absorbance ( A
280
/A
802
) o f the purified RCs was < 1.25. Q
A
and Q
B
were removed from RCs according to the method
of Okamura et al. [3]. The residual quinone content was
0.05–0.1 mol Q p er mol RC.
Quinones
1,4-Naphthoquinone, 2-methyl-1,4-naphthoquinone and
2-methyl-9,10-anthraquinone were purchased f rom Aldrich.
5,6,7,8-Tetramethyl-1,4-naphthoquinone was n ewly synthe-
sized from the D iels–Alder adduct of 2,3,4,5-tetramethyl-
thiophene-1,1-dioxide and 1,4-benzoquinone according t o a
Table 1. Overview of the 1,4-naphthoquinone compounds used in this
work. The structure of the naphthoquino nes is shown in S cheme 1.
Compound Abbreviation
Quinones without undecyl tail
1,4-Naphthoquinone NQ
2-Methyl-NQ 2MNQ
5-Methyl-NQ 5MNQ
6-Methyl-NQ 6MNQ

2,3-Dimethyl-NQ 23DMNQ
2,5-Dimethyl-NQ 25DMNQ
2,6-Dimethyl-NQ 26DMNQ
2,7-Dimethyl-NQ 27DMNQ
2,8-Dimethyl-NQ 28DMNQ
5,8-Dimethyl-NQ 58DMNQ
6,7-Dimethyl-NQ 67DMNQ
2,3,5-Trimethyl-NQ 235TMNQ
2,3,6-Trimethyl-NQ 236TMNQ
2,5,8-Trimethyl-NQ 258TMNQ
2,6,7-Trimethyl-NQ 267TMNQ
2,3,5,8-Tetramethyl-NQ 2358TeMNQ
2,3,6,7-Tetramethyl-NQ 2367TeMNQ
5,6,7,8-Tetramethyl-NQ 5678TeMNQ
2,5,6,7,8-Pentamethyl-NQ 25678PMNQ
2,3,5,6,7,8-Hexamethyl-NQ HMNQ
Quinones with undecyl tail
2-Undecyl-NQ 2UNQ
2-Methyl-3-undecyl-NQ 2M3UNQ
2,5-Dimethyl-3-undecyl-NQ 25DM3UNQ
2,6-Dimethyl-3-undecyl-NQ 26DM3UNQ
2,7-Dimethyl-3-undecyl-NQ 27DM3UNQ
2,8-Dimethyl-3-undecyl-NQ 28DM3UNQ
2,5,8-Trimethyl-3-undecyl-NQ 258TM3UNQ
2,6,7-Trimethyl-3-undecyl-NQ 267TM3UNQ
2,5,6,7,8-Pentamethyl-3-undecyl-NQ PM3UNQ
Ó FEBS 2002 Vitamin K derivatives at the Q
A
-site (Eur. J. Biochem. 269) 1097
literature procedure [31]. 5,6,7,8-Tetramethyl-1,4-naphtho-

quinone formed yellow needles,
1
H-NMR (250 MHz,
CDCl
3
, d
H
): 2.28, s, 6H; 2.53, s, 6H; 6.71, s, 2H; mp: 184–
185 °C; elemental analysis f or C
14
H
14
O
2
requires: C,
78.48% H, 6.57%; found: C, 78.43% H, 6.74%. The
synthesis of all other quinones was as described previously
[31]. Stock solutions of the quinones (1 l
M
)20 m
M
)were
prepared in dioxan and stored a t 4 °C.
Determination of dissociation constants,
K
d
,
and charge recombination rates,
k
AD

Reconstitution of the quinones into t he Q
A
-site was
accomplished by adding small amounts of the stock solution
(1 l
M
)20 m
M
) to quinone-depleted RCs (20–300 n
M
)sus-
pended in 10 m
M
Mops [3-(M-morpholino)propanesulfonic
acid], 50 m
M
KCl, 0.04% dodecyl-b-
D
-maltoside at
pH ¼ 7.2. The system was equilibrated for at least 25 min
[T ¼ (295 ± 2 ) (K)]. Transient a bsorbance changes were
recorded on a spectrometer of local design [32]. Charge
recombination kinetics were measured by monitoring the
change of the donor absorbance at 865 nm following a
single laser flash. The rate constant k
AD
was obtained from
a single exponential fit to the data using t he software
package
PEAKFIT

(version 4.0, SPSS Inc.) on an IBM-
compatible PC. The occupancy of the Q
A
-site corresponds
to the a mount of RCs i n the D
+
Q
A
–
state. Its v alue was
determined from th e amplitude of the charge recombination
kinetics at t ¼ 0 measured o n the t ime scale o f k
À 1
AD
relative
to the amount of bleach ing of RCs with a f ully occupied Q
A
site (see [4] for details). To account for the 5–10% RCs
where ubiquinone remained at Q
A
after quinone removal
the amplitude of the signal was c orrected accordingly.
The binding affinity of quinones to the Q
A
-site o f the
reaction center can be described with the model of ligand
binding to a population of single, noninteracting Q
A
-sites.
As naphthoquinone compounds bind functionally only to

the Q
A
site [5,16] the a pparent dissociation constant K
d
can
be obtained from Eqn. (1) in case of [Q]
0
) [QRC]:
DA
865
¼
DA
max
865
½Q
0
½Q
0
þ K
d
ð1Þ
where DA
865
corresponds to the concentration of bound
Q at t he RC, [QRC]. Its v alue was determined from the
absorbance changes of the donor recovery due to the
formation of D
+
Q
A

–
. The dissociation constant, K
d
,was
determined by fitting the absorbance change at 865 nm as a
function of the initial quinone concentration, [Q]
0
with
DA
max
865
and K
d
as adjustable parameters.
The condition of [Q]
0
) [QRC] essentially limits the
applicability of t he assay to K
d
> 100 n
M
. For smaller
values the amp litude of the charge reco mbination kinetics
can not be determined accurately with the experimental set
up described above. In this case 2-methyl-9,10-anthraqui-
none (0.01 m
M
) with the inhibition constant K
i
¼ 20 n

M
was
used as a competitive inhibitor similar as described previ-
ously [28]. This is suitable because charge r ecombination for
this anthraquinone occurs in the microsecond range and,
hence, does not interfere w ith the observation of th e D
+
Q
A
–
formation of the naphthoquinones. In this case the disso-
ciation constant was determined from a two p arameter least
squares fit of t he absorbance change vs. [Q]
0
according to
Eqn. (2):
DA
865
¼
DA
max
865
½Q
0
½Q
0
þ½I
0
K
d

=K
i
ð2Þ
where [I]
0
is the initial concentration of t he inhibitor.
The binding free energies
Based on the work by Warncke & Dutton [33], we applied a
correction m ethod to determine t he true binding free energy,
DG
0
bind
, a s a measure f or the direct interactions between the
quinones and the protein at the Q
A
site. The dissociation
constant, K
d
, is c orrelated to the apparent binding free
energy:
DG
0
app
¼ÀRT ln K
À1
d
ð3Þ
where T is the temperature and R the gas constant. The
apparent binding free energy, DG
0

app
, contains contri bu-
tions from specific interactions between the quinone and
the quinone binding site as well as unspecific hydrophobic
interactions between the quinone and the nonpolar protein
detergent micelles. Hence, this e nergy can be represented
as:
DG
0
app
¼ DG
0
bind
þ DG
0
trans
ð4Þ
The transfer fre e energy, DG
0
trans
, describes the free energy
change of the quinone transfer from the a queous bulk phase
to the nonpolar protein/detergent micelles. DG
0
trans
can be
approximated by the distribution of the quinone between
water a nd an apolar solvent, e.g. cyclohexane, which is given
by the partition coefficient P
cw

.
DG
0
trans
%ÀRT lnP
cw
ð5Þ
Scheme 1. The structure of the naphthoquinone compounds. The rota-
tional axis used for the description of the predicted RC-nap hthoqui-
none complexes are i ndicated.
1098 O. Hucke et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Hence, the true binding free energy, DG
0
bind
, is given by
Eqn. (6):
DG
0
bind
% DG
0
app
þ RT lnP
cw
ð6Þ
P
cw
values of quinones for the s ystem cyclohexane/water
were estimated u sing the software package
MOLECULAR

MODELING PRO
(Chem. SW, Fairfield, CA, USA) b ased on
the method of Ghose & Crippen [ 34,35].
Ligand docking calculations
The docking calculations were performed with
FLEXX
,a
program designed f or the docking of small to medium s ized
organic molecules into protein binding sites [ 36]. During the
docking procedure, the protein is considered as rigid,
whereas t he ligand conformation is flexible. This is realized
through allowing rotations around acyclic single bonds of
the ligand structure. Bond l engths a nd angles are k ept
constant as given i n the input structure. A relatively soft
atom model i s used by
FLEXX
to compensate f or the rigidity
of the binding site, i.e. small overlap of the ligand with the
receptor is tolerated by the p rogram.
The docking algorithm incorporated in
FLEXX
is based o n
the chemical interactions of ligand and receptor: For the
computation of ligand placements geometrically restrictive
interactions are used (mainly hydrogen bonds and s alt
bridges). Interaction geometries were deduced from the
analysis of crystallographic data. The computed placements
were optimized with respect to the empirical scoring
function of
FLEXX

, w hich estimates the binding free energy,
DG
0
bind
, of the ligand r eceptor complex:
DG
0
bind
¼ DG
0
translat
þ DG
0
rot
N
rot
þ DG
0
hb
X
neutral HÀbonds
fðDR; DaÞ
þ DG
0
io
X
ionic interactions
fðDR; DaÞ
þ DG
0

aro
X
arom interactions
fðDR; DaÞ
þ DG
0
lipo
X
lipoph: interactions
f
Ã
ðDRÞð7Þ
Here, t he terms DG
0
hb
, DG
0
io
, DG
0
aro
and DG
0
lipo
are t he
interaction energies for neutral hydrogen bonds, ionic
interactions, a romatic interactions (aromatic interactions
as considered by
FLEXX
are interactions of the electrostatic

quadrupole of aromatic r ings with permanent dipoles (for
example o f amide bo nds), the quadrupoles of other
aromatic rings and the induced dipoles of methyl groups)
and lipophilic contacts between ligand and receptor,
respectively, if i deal interaction g eometries are a ssumed.
The functions f (DR,Da)andf*(DR) penalize devi ations
from these geometries (DR, deviation from ideal distance;
Da, deviation from ideal angular geometry). The values of
DG
0
translat
and DG
0
rot
N
rot
consider the loss of translational
and rotational freedom of the entire ligand molecule a nd th e
freezing o f r otational d egrees of freedom of the ligand
structure upon binding, r espectively ( DG
0
rot
, loss of binding
energy due to fixation of rotation around one rotatable
bond; N
rot
, number of rotatable bond s).
Depending on the number o f possible ligand c onfor-
mations, the docking calculations resulted in a set of up to
% 200 possible protein ligand complexes per ligand. These

solutions were ranked according to the calculated binding
free energy. Unless stated otherwise, only the best
placement (Ôplacement 1Õ), displaying the smallest value
of the binding free energy, was considered for further
analysis.
The binding site of the primary quinone in the reaction
center protein from R. sphaeroides was determined with
the molecular modeling package
WHATIF
[37] based on the
X-ray structure from Stowell et al. [38] (RCSB PDB code
1AIJ). It included all amino acids with at least one atom
lying within a distance of 6.5 A
˚
from th e ubiquino ne-10
molecule in the X-ray structure (a larger binding site had
no effect on the results of the docking calculations). In
addition, four water molecules (numbers 64, 71, 409, 410
in the P DB file), the nonheme iron atom, parts o f the
bacteriochlorophylls and the bacteriopheophytins located
in the a ctive branch of the reaction center are found within
this cutoff distance and w ere t herefore considered in the
calculations.
FLEXX
uses an united atom model for all nonpolar
hydrogen ato ms, whereas polar protons are explicitly taken
into consideration. Where unambiguously clear, the posi-
tions of the protons in the protein binding site were
automatically assigned by
FLEXX

. In cases of ambiguities
(hydrogens of the h ydroxyl groups, the N-bound proton of
the h istidine side chain) they were determined with WhatIf.
The geometries of the ligand structures were optimized with
the MM+ force fi eld of the
HYPERCHEM
(Hypercube Inc.)
software prior to u se as input files for the docking
calculations.
Sample preparation for FTIR difference spectroscopy
To reconstitute the reaction center with 1,4-naphthoqui-
none derivatives a s primary electron acceptor, 3.5 nmol of
quinone-depleted RCs were dissolved in 3 mL buffer
containing 10 m
M
Mops, 50 m
M
KCl and 0.04% dode-
cyl-b-
D
-maltoside, pH ¼ 7.0 f ollowed by the addition of
20 lLofa10-m
M
stock s olution of t he corresponding
quinone in ethanol. The samples were incubated at room
temperature for a minimum period of 2 h and then
concentrated with Microcon YM-100 centrifugal filter
devices (Millipore Corp., cut-off molecular mass 100 kDa)
at 3000 g and 4 °C to two aliquots of % 50 lL volume each.
To avoid partial denaturation of the RCs due to high io nic

strength and detergent concentration upon the final
concentration step, the samples were diluted b y a f actor
of 10 with a buffer without detergent containing 1 m
M
Mops and 5 m
M
KCl at pH ¼ 7.0. The second concentra-
tion step yielded t wo highly concentrated samples of
reconstituted R Cs with a final volume of about 10 lL
each. One of them was used for FTIR difference spectro-
scopy (see next paragraph) whereas the remaining sample
was taken to determine the occupancy of the Q
A
-site and
the charge recombination kinetics with transient absorption
spectroscopy under the same conditions except that the
redox med iator diaminodurene (DAD) and sodium ascor-
bate were omitted.
Ó FEBS 2002 Vitamin K derivatives at the Q
A
-site (Eur. J. Biochem. 269) 1099
FTIR difference spectroscopy
FTIR difference spectroscopy was performed as described
by Breton et al. [39] with minor modifications. The sample
(% 10 lL) containing about 1.5 nmol reconstituted RC was
placed onto the depression of a CaF
2
window. After
addition of 5 lL of t he aqueous solution of the redox
mediator DAD (2.5 m

M
) and sodium ascorbate (1.25 m
M
)
as reductant for the primary donor, t he droplet was dried
under a smooth stream of nitrogen. Before complete
dryness, the RC film was sealed with a second CaF
2
window, yielding a sample with a thickness of a few
microns, minimizing the water a bsorption. The two win-
dows were fixed by a metal mounting and placed in the
sample chamber (T ¼ 2±0.2°C). FTIR spectroscopy
was performed with a Bruker IFS 66 V/S FTIR
spectrometer. Light-minus-dark FTIR d ifference s pectra
were obtained by recording the spectrum of the sample
under continuous illumination at a wavelength of 590 nm
and subtracting the spectrum measured in t he dark. For
each difference spectrum 1920 interferograms were accu-
mulated. To improve the signal to noise ratio, % 20
illumination c ycles of the sample w ere averaged. The FTIR
difference spectra were normalized with the vector normal-
ization method based o n t he spectral r egions b etween 1500
and 1560 cm
)1
and between 1670 and 1 750 cm
)1
. The
difference spectra obtained by this method (i.e. in the
presence of DAD/sodium ascorbate, leading to a fast
reduction of the primary donor after electron t ransfer to

Q
A
) show exclusively the absorption c hanges upon the
reduction of the primary quinone, designated ÔQ
A
–
/Q
A
difference spectraÕ.
RESULTS
Experimentally determined binding free energies
The methyl s ubstituted n aphthoquinones were character-
ized in terms of the midpoint redox potential E
1/2
,charge
recombination rate k
AD
and dissociation constant K
d
(see
Table 2). The in vitro midpoint redox potentials d ecrease
with increasing number o f alkyl substituents due to their
inductive effect. Similarly, the RCs with naphthoquinones
at Q
A
displayed low in situ redox potentials leading to an
increase of the free energy difference between the states
D
+
Q

A
–
and DQ
A
compared to RCs with native UQ-10
as Q
A
. Therefore, the charge recombination rates k
AD
were significantly faster for all RCs with at least two
methyl groups in the aryl ring due to the charge
recombination by a thermally activated route via the
D
+
/
A
–
state [5,40].
As previous studies revealed that even nonquinonic
compounds bind to the Q
A
site [33] indicating less specific
interactions between ligand and protein, the dissociation
constants of methyl substituted quinones are expected to be
dominated by t he quinone polarity which determines the
transfer free energy. However, the binding affinity is
strongly affected by the substitution pattern o f the aryl ring
(Table 2). The most striking result of our study was
obtained for all naphthoquinones with methyl groups in
position 5 and 8. These substituents drastically weaken the

association by at least a factor of 400 which represents the
current limit for K
d
determination.
To determine the actual interaction energy between
quinone and RC, the apparent binding free energies were
corrected for t he transfer free energy (Eqn. 6) by u sing
calculated values for the correspon ding partition coefficients
between cyclohexane and water ( Table 2). In case of the
naphthoquinone compounds without an undecyl chain in
position 3 the results agree fairly well with the e xperimental
values. However, the P
cw
values of the long-tail-de rivatives
are most likely overestimated by the Ghose–Crippen
method as for each CH
2
group of the aliphatic chain the
same increment was added to log P. The corresponding
binding free energies ranged from )13.1 to +6.2 kJÆmol
)1
for the compounds with an undecyl chain whereas the other
quinones exhibited values from )32.0 to )21.0 kJÆmol
)1
.
Rigid quinone binding site
A main assumption in the ligand-docking calculations was
that the structure of the b inding pocket does not change
upon binding of the different vitamin K derivatives
compared to the X-ray structure determined with UQ-10

as Q
A
. The FTIR Q
A
–
/Q
A
difference spectra show signals of
both, the quinone and t he adjacent region of the RC. With
isotope labeled ubiquinone and vitamin K
1
as Q
A
Breton
et al. [41] showed that the spectra in the range of 1750–
1670 and 1560–1500 cm
)1
are dominated by nonquinonic
contributions resulting from the response of the protein to
the Q
A
-reduction. Hence, these absorption bands are
indicative for the interaction between the quinone and t he
protein bind ing pocket. Therefore, structural changes upon
ligand binding are expected to alter specifically the
vibrational f requencies and/or intensities, m aking FTIR
difference spectroscopy an attractive method for probing
the effect of the different quinones on the structure of the
binding site.
Figure 1 shows the Q

À
A
=Q
A
difference spectra obtained
for three of the naphthoquinones which are objects of this
work compared to those of UQ-10 and vitamin K
1
.Inthe
nonquinonic regions, the band shapes and vibrational
frequencies exhibit a high similarity for all quinones. No
evidence for a change in the response of the protein due to
Q
A
–
formation w as found in these spectra supporting the
assumption of a rigid binding site.
Docked structure with UQ-10
To test the docking algorithm of
FLEXX
with our system, we
calculated the UQ-10/RC complex, as shown in Fig. 2.
With respect to the quinone head group the calculated
structure agrees very well with the X-ray structure [38] (rmsd
of the head groups, 0.29 A
˚
). In contrast, the positions o f the
isoprenoid chains differ significantly beyond the first two
isoprene units. As both, the binding affinity and the
midpoint redox potential of the quinone are mainly

determined by the head group [28,33], t hese results encour-
aged us to extend the calculations to the naphthoquinone
compounds.
Docked structures with naphthoquinone compounds
Using our set of 29 naphthoquinone compounds (Table 1,
Scheme 1) we computed the quinone-reaction center
complexes. For most naphthoquinones the ligand was
1100 O. Hucke et al.(Eur. J. Biochem. 269) Ó FEBS 2002
successfully docked into the binding pocket, e xcept for 7 o f
the 8 compounds containing methyl groups in both
positions 5 and 8.
Quinone orientation within the binding pocket
In all p redicted structures the 1 ,4-naphthoquinones share
essentially the s ame orientation of the naphthoquinone ring
system, i.e. the calculated protein–ligand complexes show
two hydrogen bonds between the carbonyl oxygens of the
quinones a nd the amide nitrogen of AlaM260 and the
imidazole nitrogen of t he HisM219 s ide chain as found for
UQ-10 in the X-ray structure. The position and the
orientation of the quinone rings are similar to those of
UQ-10 (Fig. 3). The aromatic r ings of the naphthoquinones
are directed towards the interio r of the binding pocket ( i.e.
towards M etM262 and AlaM245). Up to eight spec ific
interactions of the aromatic rings with side chain methyl
groups (AlaM248, AlaM249, AlaM260, C
c2
of ThrM222
and IleM265), aromatic rings (TrpM252) and backbone
amide bonds (AlaM248, A laM260, ThrM261) of s urround-
ing amino acids were a ssigned by

FLEXX
.
In case of the quinone oriented w ith two hydrogen bonds
of the carbonyl oxygens and the aromatic ring directed
towards t he in terior of the binding site, the ring system can
assume two orientations. They c an be matched by a rotation
Table 2. Comparison of the physicochemical properties of naphthoquinone c ompounds with different number and po sition of al kyl substituents i n the
ring system. Charge recombination rates from D
+
Q
A
–
to DQ
A
, k
AD
, were determined in one-quinone RCs monitored via t he absorbance change of
the rereduction of D
+
following a single laser flash. The dissociation constants K
d
were derived from a plot of the amount of b leaching vs. the initial
quinone c oncentration according t o Eqns. (1,2). From a n error analysis the accuracy of –l og K
d
was estimated to ± 0,2. Using the experimental
values for K
d
the appar ent bin ding fre e en erg ies, DG
0
app

, were calculated following E qn. 3 . T o account f or the d istribution of t he quinone between
the water and the apolar protein detergent micelles the cyclohexane water partition coefficients, P
cw
, were estimated with the Ghose–Crippen
method [34,35] and used for c orrectin g t he apparent bin ding free energies (Eqn. 6). The corresponding binding f ree e n ergies DG
0
bind
(Exp.) were
compared to the data from ligand docking calculations [designated DG
0
bind
(
FLEXX
)]. The predicted complexes were analyzed in terms of the distance
differences along the axis from the primary d on or D t o the primary electron acceptor Q
A
with r espect to t hat of 1,4-naph thoquin one [Dr(NQ)].
Experimental conditions: 2 0–300 n
M
quinone-depleted RCs, 30 n
M
–100 l
M
naphthoquinone, 10 m
M
Mops, 50 m
M
KCl, 0.04% dodecyl-b-
D
-

maltoside, pH ¼ 7.2 (T ¼ 293 K). See Table 1 for abbreviations. NF, no formation of Q
A
–
was detected; ND, not determined; NP, no acceptable
placement was found by
FLEXX
.
Quinone
E
1/2
a
(mV)
k
AD
(s
)1
)
Dr(NQ)
(A
˚
)
–log K
d
DG
0
app
(kJÆmol
)1
) log P
cw

log P
cw
b
DG
0
bind
(kJÆmol
)1
)
Exp.
FLEXX
D
d
NQ )1057 7.1 0.00 5.4 )30.3 1.21 1.26 )23.5 )23.9 )0.4
2MNQ )1146 6.2 )0.05 6.1 )34.2 1.80 1.88 )24.1 )25.8 )1.7
5MNQ )1150 10.2 0.08 5.6 )31.4 1.68 – )22.0 )23.4 )1.4
6MNQ )1091 10.6 )0.31 6.3 )35.3 1.68 – )25.9 )23.4 2.5
23DMNQ )1227 6.6 )0.45 8.1 )45.4 2.39 2.70 )32.0 )25.0 7.0
25DMNQ )1218 9.4 )0.38 6.0 )33.7 2.27 – )21.0 )22.3 )1.3
26DMNQ )1173 8.8 )0.44 7.2 )40.4 2.27 – )27.6 )25.5 2.1
27DMNQ )1179 14.4 )1.05 7.1 )39.8 2.27 – )27.1 )22.7 4.4
28DMNQ )1224 8.4 )0.02 6.6 )37.0 2.27 – )24.3 )25.3 )1.0
58DMNQ )1228 NF NF < 3 > )16.8 2.27 – ND )16.0
c
–
67DMNQ )1132 13.8 )0.49 7.0 )39.3 2.27 2.17 )26.6 )20.0 6.6
235TMNQ )1297 12.0 )0.17 7.7 )43.2 2.86 – )27.2 )23.9 3.3
236TMNQ )1279 11.2 )0.64 8.4 )47.1 2.86 – )31.1 )23.7 8.4
258TMNQ )1300 NF NF < 3 > )16.8 2.73 – ND NP –
267TMNQ )1209 15.0 )0.51 7.0 )39.3 2.73 2.72 )24.0 )22.0 2.0

2358TeMNQ )1386 NF NF < 3 > )16.8 3.33 – ND NP –
2367TeMNQ )1280 17.1 )0.69 7.8 )43.8 3.33 3.48 )25.1 )22.6 2.5
5678TeMNQ )1305 NF NF < 3 > )16.8 3.08 – ND NP –
25678PMNQ )1391 NF NF < 3 > )16.8 3.67 – ND NP –
HMNQ )1487 NF NF < 3 > )16.8 4.26 – ND NP –
2UNQ )1099 9.3 0.07 6.9 )38.7 5.76 – )6.4 )22.1 )15.7
2M3UNQ )1206 8.8 )0.08 8.7 )48.8 6.36 – )13.1 )21.4 )8.3
25DM3UNQ )1288 5.7 )0.50 5.7 )32.0 6.82 – 6.2 )14.9 )21.1
26DM3UNQ )1261 14.3 )0.76 8.5 )47.7 6.82 – )9.4 )19.8 )10.4
27DM3UNQ )1255 17.7 )0.76 7.8 )43.8 6.82 – )5.5 )16.6 )11.1
28DM3UNQ )1295 16.8 )0.05 7.5 )42.1 6.82 – )3.8 )20.6 )16.8
258TM3UNQ )1366 NF NF < 3 > )16.8 7.52 – ND NP –
267TM3UNQ )1294 24.8 )0.84 7.5 )42.1 7.52 – 0.1 )17.6 )17.7
PM3UNQ )1477 NF NF < 3 > )16.8 8.22 – ND NP –
a
In vitro midpoint redox potentials for the redox couple Q/Q
–
reported against ferrocene as internal standard measured in DMF [31,53].
b
Experimental data taken from [54].
c
Only one hydrogen bond between the carbonyl oxygen of the quinone and surrounding amino acid
residues was found.
d
D  DG
0
bind
ðFLEXXÞÀDG
0
bind

ðExp:Þ.
Ó FEBS 2002 Vitamin K derivatives at the Q
A
-site (Eur. J. Biochem. 269) 1101
of 180° on the x -axis ( Fig. 3) but are only distinguishable for
asymmetrical methyl substitution patterns. We arbitrarily
chose the naphthoquinone orientation with the hydrogen
bonds between the C
1
carbonyl and HisM219 and the
C
4
carbonyl and AlaM260 as reference (designated
reference orientation). In the calculated structures, f or all
naphthoquinones w ith this reference orientation the methyl
groups at a specific position o f the naphth oquinone ring
system have very similar environments which we used for
the definition of methyl group positions within the Q
A
binding site (Table 3). Each position i s defined by the
contacts that are observed in the predicted structures
between a specific methyl group and the RC atoms. It is
named a ccording to the number of t he C-atom of the
naphthoquinone to which the methyl group is bound. For
instance, the ¢position 5¢ is formed by all R C atoms showing
contacts to the 5-methyl group. A % 180° rotation on the
x-axis moves a methyl group from position 5, 6 and 2 to
position 8, 7 and 3, r espectively.
The detailed analysis of the placements of the tailless
naphthoquinones r eveals that the presence of specific methyl

substituents favors on e of t he two possible orientations
with respect to the x-axis: In all placements with 5- or
8-substitution (5MNQ, 25DMNQ, 28DMNQ, 235TMNQ)
the corresponding methyl group was found at position 8.
The best placements (with regard to t he
FLEXX
score of t he
binding free energy) of these quinones with t he methyl
group at position 5 in the binding pocket show binding free
energies which deviate from the optimal value correspond-
ing to placement 1 by 3.9, 1.7, 6.9 and 3.5 kJ Æmol
)1
,
respectively. In t he best placements of quinones w ith 6- or
7-methyl substitution (6MNQ, 26DMNQ, 27DMNQ,
236TMNQ) the methyl group was found at position 6.
Compared to these placements structures with the methyl
group at position 7 of the b inding site exhibit less favorable
energies of 3.1, 6.4, 0.1 and 2.4 kJÆmol
)1
, respectively. The
favored placement of 2 MNQ shows the methyl group at
position 2 and the corresponding binding free energy is
4.1 kJÆmol
)1
lowerthanthatofthebestplacementofthis
compound, rotated by approximately 180° on the x-axis
(which places the 2-methyl group at position 3). This also
accounts f or the small energy differences of 1.7 and
0.1 kJÆmol

)1
between the t wo rotated o rientations of
25DMNQ an d 2 7DMNQ, r espectively. Here, the placement
of the 5-methyl group to position 8 and the 7-methyl group
to position 6 leads to the unfavorable position 3 of the
2-methyl group.
Evaluating the docked structures of the naphthoquinones
with an undecyl tail we found for 2UNQ the rotated
orientation of the quinone head group with hydrogen
bonds between the C
1
-carbonyl group and AlaM260 and
Fig. 1 . Q
À
A
=Q
A
FTIR difference spectra of quinone-depleted reaction
centers from R. sphaeroides reconstituted with UQ-10, vitamin K
1
(Vit. K
1
), 2,3,5-trimethyl-1,4-naphthoquinone (235TMNQ), 2,8-
dimethyl-3-undecyl-1,4-naphthoquinone (28DM3UNQ) and 2,3,6-
trimethyl-1,4-naphthoquinone (236TMNQ). A total of 40 000
interferograms were averag ed for each spectrum. As shown for vitamin
K
1
and UQ-10, the regions of 1750–1670 cm
)1

and 1560–1500 cm
)1
are free of quinonic contributions (indicated as nonquinonic) [41].
Differences in the structure of the b inding site due to structural
differences of the primary quinone at Q
A
are expec ted to a lter the
vibrational frequencies and intensities of t he s p ectra i n these regions.
See text f or details of c ondition s. a.u. absorbance units.
Fig. 2. Comparison of the ubiquinone-10 (U Q-10) position a t Q
A
in the
photosynthetic reaction center from R. sphaeroides obtained from
docking calculations with the X-ray structure. Carbon atoms of the
UQ-10 from [38] and the docked quinone are depicted in black and
green, respectively. Amino acids (blu e) surrounding UQ-10 were t aken
from the crystal structure ( [38], PDB file 1AIJ). The nitrogen, o xygen
and hydrogen atoms are drawn in light b lue, red and white, respec-
tively. For sake of clarity t he isop renoid ch ains were trun cated. The
dashed lines indicate the hydrogen bonds between the ca rbo nyl oxy-
gens of the quinones and AlaM260 a nd HisM219. The quinone head
groups fit with an rmsd of 0.29 A
˚
.
1102 O. Hucke et al.(Eur. J. Biochem. 269) Ó FEBS 2002
the C
4
-carbonyl group and H isM219. However, in all six 2-
methyl-3-undecyl-1,4-naphthoquinone derivatives t he same
quinone head group orientation was observed as in the

reference o rientation of the tailless quinones. For these
compounds the rotated reference orientation is prohibited
as the presence of the 2-methyl group prevents a necessary
adjustment of the undecyl tail conformation directing the
hydrocarbon tail through the opening of the binding pocket
towards the protein exterior. For the same reason, in the
predicted structure with 25DM3UNQ the 5-methyl group is
not found at position 8 of the b inding site (as found for all
5-methylated tailless naphthoquinones, see a bove) but in the
less favorable position 5.
It should be mentioned t hat f or most of t he tailless
quinones placements with the aromatic rings directed
towards the opening of the binding pocket were also
computed. This orientation is obtained by a rotation of
% 180° on the y-axis r elative to the reference orientation,
designated Ôantireference orientationÕ). However, the bind-
ing free energies are significantly larger by a n average of
+5.4 kJ Æmol
)1
compared to the highest ranked place-
ments indicating that this orientation is less favorable. The
analysis of the differe nt free energy terms of the scoring
function reveals that the main contribution (% 47%) of
this increase in the binding free energy results from a loss
of aromatic interactions. O rientation of the aromatic
naphthoquinone rings to the opening of t he binding site
reduces the average number of these interactions from 5.4
to 1.9 suggesting t hat these contacts play an important
role for naphthoquinone binding to the Q
A

binding site.
Other contributions to the change in the binding free
energy include the loss of lipophilic contact area (% 15%)
and deviations from the i deal hydrogen bond geom etry
(% 38%).
Distances from the naphthoquinone at Q
A
to the
secondary ubiquinone Q
B
and the primary donor D
According to the Marcus theory the rate of electron
transfer between two molecules depends on three factors:
the overlap of the elec tron densities (wavefunctions) of
the two molecules, the difference in redox potential of the
molecules ( corresponding to the free e nergy d ifference ) and
the reorganization energy (reviewed in [42]). The most
critical parameter in determining the electron transfer rate
is the electron density overlap which was found to depend
exponentially on the distance o f the reactants [43]. The
center-to-center distances are m easured from the middle o f
the quinone rings and the center of the special pair
(defined as the middle of the line connecting the Mg
atoms). Due to different methyl s ubstitution patterns the
use of the edge-to-edge values produces incomparable
values. The distances between the different naphthoqui-
none compounds at Q
A
and the native ubiquinone at Q
B

range from 19.4 A
˚
to 20.0 A
˚
compared to 19.6 A
˚
as found
in the X -ray structure o f native RCs with ubiqu inone at
Q
A
. Thus, within t he resolution of the X-ray diffraction
data both structures are identical. However, i n terms of the
Table 3. Possible orientations of methylated 1,4-naphthoquinones in the
Q
A
binding site as determined with
FLEXX
. The methyl groups of the
different methylated 1,4-naphthoquinones occupy distinct positions
within the Q
A
binding s ite as defined by their contacts with amino acid
atoms of the reaction center. Each position number refers to the C
atom nu mber of the naphthoquinone r ing (see Scheme 1) to which the
corresponding methyl group is bound. It was assumed that the
naphthoquinone is located in the most comm on orientation with
the t wo hydro gen bonds between t he C
1
carbonyl and t he C
4

carbonyl
formed to HisM219 and A laM260, respectively (arbitrarily defined as
the Ôreference o rientationÕ).
Position within the
Q
A
site Amino acids Amino-acid atoms
2 HisM219 C
a
,C
d1
TrpM252 C
d1
,N
e1
IleM265 C
d1
,C
c1
,C
a
3 TrpM252 C
b
,C
c
,C
d1
MetM256 C
e
5 AlaM249 C

a
,C
b
,N
AlaM260 C, C
a
,N,O
6 AlaM245 C
b
,O
AlaM249 C
b
AlaM260 O
ThrM261 C, C
a
MetM262 N
HOH 64, 409 O
7 AlaM248 C
b
MetM262 C
a
,C
b
,C
c
,S
d
,C
e
8 HisM219 C

e1
IleM223 C
d1
MetM262 S
d
,C
e
IleM265 C
c2
Fig. 3. Calculated position of 2,6-dimethyl-3-undecyl-1,4-naphthoqui-
none (26DM3UNQ) in the Q
A
binding pocket as an example of the
predicted structures with 1,4-naphthoquinones as primary acceptors of
the RC from R. sphaeroides. The ring systems of both compounds
show a high s imilarity in terms of the position a nd orientation. x and y
denote rotational axis u sed f or the description of the q uinone o r ien-
tation within th e Q
A
binding s ite. See Fig. 2 for coloring.
Ó FEBS 2002 Vitamin K derivatives at the Q
A
-site (Eur. J. Biochem. 269) 1103
distance between the primary donor and Q
A
some naph-
thoquinones are further apart from the don or (up t o 1 .1 A
˚
in case of 5-methyl-1,4-naphthoquinone) than the native
ubiquinone affecting significantly the electron transfer rate

k
AD
(see Discussion).
Evaluation of binding free energies
The binding free energies of the protein-ligand complexes
were estimated with the
FLEXX
scoring function (Eqn. 7).
For all functionally binding naphthoquinones without an
undecyl t ail the values range from )25.8 to )20.0 kJÆmol
)1
.
The contributions of the hydrogen bonds, aromatic inter-
actions and lipophilic contacts amount to % 32, % 13 and
% 55%, r espectively. In case of 58DMNQ a protein-ligand
complex was predicted although with the charge recombi-
nation assay no binding was observed. However, the
binding free energy yielded a significantly higher value of
)16 kJÆmol
)1
mainly due to the loss of one of the two
hydrogen bonds. This c ompound was therefore disregarded
in all further analysis.
The calculated binding energies of the substituted
undecyl naphthoquinone derivatives range f rom )22.1 t o
)14.9 kJÆmol
)1
. Based on the different terms i n t he scoring
function we deduced that the m ajor component is the
lipophilic contact energy (% 67%) due to the large

hydrophobic surface of the alkyl chain. Smaller contribu-
tions arise from the hydrogen b onds (% 24%) and interac-
tions between the aromatic rings of the naphthoquinones
and the residues of the binding site (% 9%). A detailed
analysis reveals that the binding free energies of the quinone
head groups (methyl substituted r ing systems without
undecyl c hain) lie only slightly (on average 1.3 kJÆmol
)1
)
above the energies found for comparable naphthoquinones
without an alkyl chain, showing that the presence of the
undecyl t ail h as no significant effect on the interaction
between the quinone head group and the protein binding
site.
DISCUSSION
Binding or nonbinding?
The experimental values for the dissociation constants
manifested that 5,8-disubstitution of the naphthoquinone
system prohibits binding to the Q
A
site even in case of a
long tail in position 3. This result was rather surprising as
Warncke and Dutton found for 3-decyl substituted ubi-
quinone-0 and 2-methyl-1,4-naphthoquinone a decrease in
the dissociation constants by more than two orders of
magnitudes compared to the corresponding an alogues
with a h ydrogen at this position [ 28]. Our fi ndings agree
with previously published results obtained for 58DMNQ
[44]. As only functional binding is detected with the
charge recombination assay it cannot be decided whether

5,8-dimethyl-1,4-naphthoquinone compounds were not
bound at Q
A
or the structure of the RC-quinone complex
prohibits photoreduction. However, the experimental data
coincide strikingly w ith the results of the docking calcu -
lations: For none of these compounds an acceptable
protein–ligand complex was found which strongly su p-
ports the idea of nonbinding to Q
A
. This can be pinned
downtostericreasons:
As described above, the methyl groups of all 5-substi-
tuted quinones except that of 25DM3UNQ were found at
position 8 within the binding pocket in the calculated
complexes (Table 3). In t hese stru ctures, t he co ordinates o f
the methyl group were practically identical leading to the
same protein environment formed predominantly by the
side chains of HisM219, IleM223, MetM262 and IleM265
(Table 3). We have c onstructed hypothetical protein-ligand
complexes of the nonbinding compounds 58DMNQ,
258TMNQ, 2358TEMNQ and 258TM3UNQ based on
the p redicted structures with the corresponding binding
analogues 5 MNQ, 25DMNQ, 28DMNQ, 235TMNQ,
25DM3UNQ and 28DM3UNQ. For this purpose, the
ring systems of each of the 5,8-disubstituted naphthoqui-
nones and the corresponding monosubstituted quinone in
the computed protein-ligand complex were superimposed.
All resulting hypothetical structures share the same features
(Fig. 4 ). The additional m ethyl group shows a n intolerable

van der Waals overlap with either the backbone atoms of
AlaM260 and AlaM249 in case of 58DMNQ, 258TMNQ,
2358TEMNQ whereas for 258DM3UNQ steric clashes
with the side chains of HisM219, IleM223 and MetM262
are found. This can not be avoided by a displacement of
the quinone head group within the b inding site, as t he
methyl group of the 5- or 8-monosubstituted quinone
is already in close contact with the adjacent part of
the binding pocket restricting the positional freedom of the
quinone.
Naphthoquinone positions: implications
for the charge recombination rates
A main assumption for comparing the different naphtho-
quinone positions is that the original structure of the Q
A
binding pocket remains unchanged in view of the drastic
methods including the application of high concentrations of
Fig. 4. Constructed placement of 5,8-dimethyl-1,4-naphthoquinone
(58DMNQ) in the Q
A
binding site compared to the native s tructure. Fo r
reasons of clarity, the part of the binding site formed by IleM223 and
MetM262 is drawn s chematically as blu e curve. The pink dashed lines
symbolize steric c lashes of the 5-methyl g roup with (mainly backbone)
atoms of A laM260 and A laM249 prohibiting binding of this quinone.
See Fig. 2 for c olo r scheme.
1104 O. Hucke et al.(Eur. J. Biochem. 269) Ó FEBS 2002
ionic detergent (LDAO) and inhibitor (o-phenanthroline) to
remove t he native ubiquinone from the b inding site. B reton
et al.[45]measuredtheQ

A
–
/Q
A
FTIR d ifference s pectra with
native RCs havin g UQ-10 as Q
A
and compared the result
with that of quinone–depleted RCs after reconstitution of
the Q
A
-binding pocket w ith UQ-10. Within the n o ise level,
the t wo spectra were practically ide ntical. More r ecently,
Kuglstatter et al. [ 46] d etermined the X-ray structure of the
photosynthetic RC from R. sphaeroides reconstituted with
9,10-anthraquinone as Q
A
to 2.4 A
˚
resolution. Quinone-
depleted RCs were prepared under the same conditions as
described in this work. Within the resolution limit no
structural changes o f the Q
A
binding pocket w ere observed.
From our predicted placements it follows that the
position o f 1,4-naphthoquinones w ithin the Q
A
binding
pocket of the reaction center varies depending on the

substitution pattern of t he naphthoquinone. This s lightly
affects the distances of Q
A
to other cofactors involved in
electron transfer r eactions which may influence the rates of
these reactions. The differences are neglectable for the
forward electron transfer from Q
A
–
to Q
B
whereas the
deviations are more critical with respect to the distance
from the primary donor to Q
A
. According to the Marcus
theory, a t r oom temperature the charge r ecombination rate
k
AD
depends on the reorganization energy, the standard
reaction free energy and t he electronic coupling matrix
element (designated V
R
at the distance R between the
reactants). To estimate the effect of quinone relocation in
the c alculated c omplexes on the r ate k
AD
we use in a
simple m odel t he expression for t he distance dependence o f
the electronic coupling m atrix element ( Eqn. 8) by ignoring

any possible changes in the reorganization energy, the
driving force and the electron transfer pathway upon
substitution.
V
2
R
¼ V
2
0
expðÀbRÞð8Þ
Here, V
0
is the maximum electronic coupling matrix
element, R is the distance between the reactants and b is
the transmissional coefficient. For this quantity Moser et al.
[43] have empirically determined a value of b ¼ 1.4 A
˚
)1
.
The maximum difference with respect to the donor-
quinone distance was found between 5MNQ and 27DMNQ
(Table 2). The position of the latter compound was % 1.1 A
˚
closer t o the donor leading to an approximate fivefold
increase in the charge recombination rate k
AD
. With respect
to u biquinone as found in the X-ray structure a mean
displacement of % 0.6 A
˚

away from the donor was
determined for the different naphthoquinone compounds.
This d isplacement may account for a % 2.4-fold decrease in
the rate k
AD
.
Evidence for position-dependent influences on the rate
k
AD
was previously reported by Warncke et al. [28]. They
studied both menaquinone and ubiquinone compounds
with systematically altered hydrocarbon tail structures.
Using the empirical relation for the distance dependence
of the electron transfer rate in proteins of Moser et al . [43]
the relocation of the quinones was estimated to 0.8 A
˚
and
0.6 A
˚
along the line connecting t he quino ne and the primary
donor, respectively. Similar values w ere derived from
FLEXX
calculations on quinones with s ystematically altered hydro-
carbon chain length (data not shown). Gunner et al.[5]
proposed positional differences of about 1 A
˚
to explain a
three to fourfold increased recombination rate for
b-comparedtoa-substituted 9,10-anthraquinones. More-
over, in the X-ray structure of the reaction center from

R. sphaeroides with 9,10-anthraquinone as Q
A
[46] its
position was found to be % 1A
˚
displaced compared t o that
of ubiquinone. The docked anthraquinone-reaction center
structure exhibits v ery s imilar r esults (data not shown).
Comparison of experimental and calculated
binding free energies
1,4-Naphthoquinones without undecyl chain. According
to our model, the f ree energies of the quinone tran sfer from
the aqueous solu tion to the hydrophobic d etergent and
protein-detergent micelles were estimated by calculating the
free energies for the transfer from water to cyclohexane. The
experimental binding free energies were corrected for these
transfer energies to account for the tendency o f the
hydrophobic quinone compounds to accumulate w ithin
the h ydrophobic micellar phase. Althou gh this correction is
based on a relatively simple model, we achieved a
reasonable agreement b etween theoretical and experimental
binding free energy values (Table 2). The standard deviation
of the two data sets amounts t o only 3 .9 kJÆmol
)1
.
1,4-Naphthoquinones with undecyl chain. The experimen-
tal binding energies of the naphthoquinones with a
3-undecyl chain display on average an offset of 14.4 kJÆmo l
)1
compared to the p redicted values. Warncke & Dutton [33]

found empirically that the binding free energies ( DG
0
bind
)of
many quinones to t he Q
A
site can be c orrected with respect
to their hydrophob ic transfer free energies (DG
0
trans
)by
applying a simple linear r elationship y ielding a measure for
the direct inte ractions of the p rotein with the ligand. In case
of ubiquinones w ith more t han two isoprene u nit tails,
corresponding to a linear c hain l ength of eight carbon
atoms, this correction me thod failed [28]. This was explained
with the t hird and subsequent isoprene units being not
completely removed from contact with the solvent. There-
fore, DG
0
trans
is expected to be overestimated for naphtho-
quinones w ith a hydrocarbon chain of 11 carbon atoms with
our me thod as well. Other possible sources of error include
inaccuracies of the lipophilic contact energy by the scoring
function of
FLEXX
and of the calculated partition coefficients
P
cw

. Under our experimental conditions the apolar phase
was not cyclohexan but consists o f both, detergent micelles
and mixed micelles of detergent and protein.
Assuming that a systematic error in estimating the
effective e nergy f or the transfer o f t he undecyl-naphthoqui-
none compounds from water to the mixed p rotein-detergent
micelles accounts for the discrepancies, a simple offset
correction based on the average values of the experimental
and predicted values matched the two data sets w ith a
standard deviation of 4.1 kJÆmol
)1
(data not shown).
Aromatic interactions of the naphthoquinone
compounds with the Q
A
binding site
From our calculations it follows that the interactions
between the aromatic rings of the tailless naphthoquinone
derivatives and the protein play an important role with res-
pect to the quinone orientation within the Q
A
binding site.
The binding free energy associated with these interactions
Ó FEBS 2002 Vitamin K derivatives at the Q
A
-site (Eur. J. Biochem. 269) 1105
amounts to only % 13% of t he total e nergy, representing the
smallest of the different co ntributions (Eqn. 7). In contrast
to this finding, the aromatic interactions represent the major
component with resp ect to the orientation of the quinones.

Approximately 46% of the energy difference between the
best placements of the reference and the antireference
orientation arises from these interactions.
A structural alignment of the reaction centers from
B. viridis and R. sphaeroides shows t hat t he positions of all
groups of the Q
A
binding site, involved in the aromatic
interactions are highly conserved . This indicates, that
aromatic interactions may be important for t he binding of
the physiological primary electron acceptor of the reaction
center from B. viridis, vitamin K
1
, which is a naphthoqui-
none derivative.
The aromatic interactions of the n aphthoquinone with
the Q
A
binding site in contrast to ubiq uinone might be the
reason for the failure of ubiquinone to replace naphthoqui-
none at this site. This i s the prerequisite for the preparation
of RCs with naphthoquinone compounds at Q
A
and
ubiquinone at Q
B
(Ôhybrid RCsÕ), enabling mechanistic
studies of the forward electron t ransfer from t he primary to
the s econdary quinone [ 18,19] and of the d irect charge
recombination process from Q

B
–
and D
+
[16].
Accuracy of the calculated binding free energies
To estimate the binding free energies of the place ments, we
used the empirical s coring function introduced by Bo
¨
hm
[47], with m inor modifications. This function was tested by
evaluating the X-ray structures of 82 different protein-
ligand complexes displaying e xperimentally determined
binding free energies in the range from )8to)80 kJÆmol
)1
.
These data were reproduced with a standard deviation of
9.5 kJÆmol
)1
[48]. However, a much better result was
obtained in a test case which is similar to our docking
study of naphthoquinone compounds: Five different,
structurally closely related i nhibitors of dihydrofolate
reductase were docked by hand into the binding pocket o f
the enzyme by use of computer graphics [47]. The scoring
function was applied to the modeled protein-ligand com-
plexes, y ielding a standard deviation f rom the experimental
binding energies of only 4.0 kJÆmol
)1
.

This value obtained for a water soluble protein is nearly
identical with the rmsd of the calculated and experimentally
determined binding free energies of the different naphtho -
quinones to t he Q
A
site of the R. sphaeroides RC indicating
that our m ethod to correct the a pparent binding energies for
the hydrophobic transfer energies works well (at least for the
tailless n aphthoquinones). I n addition, the scoring function
seems t o be well suite d for the characterization of the
interactions between the naphthoquinones and the Q
A
binding site. To the best of our knowledge this is the first
application of
FLEXX
to a m embrane protein with a binding
site being inaccessible from t he aqueous phase.
The good agreement of the predicted with the experi-
mentally observed naphthoquinone binding properties
shows that docking calculations with
FLEXX
provide a
powerful tool for the rational design of new artificial
electron acceptors and inhibitors of other quinone binding
proteins in t he context of t he recently d e termined structures
of photo system II [49], mitochondrial bc
1
complex [50] and
fumarate reductase [51,52].
ACKNOWLEDGEMENTS

We thank Ursula Friedrich for growing th e b acteri al cul tures, i so lating
and purifying th e reaction c enters and P eter Gra
¨
ber for general support
which made t his w ork possible. This work was supported b y a grant
from the D eutsche Forschungsgemeinschaft ( La 816/3–3).
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