Characterization of mutations in crucial residues around
the Q
o
binding site of the cytochrome bc
1
complex from
Paracoccus denitrificans
Thomas Kleinschroth
1
, Oliver Anderka
1
, Michaela Ritter
2
, Andreas Stocker
1,2
, Thomas A. Link
2
,
Bernd Ludwig
1
and Petra Hellwig
3
1 Institut fu
¨
r Biochemie der Johann Wolfgang Goethe Universita
¨
t, Molekulare Genetik, Biozentrum, Frankfurt am Main, Germany
2 Institut fu
¨
r Biophysik der Johann Wolfgang Goethe Universita
¨

cules, Universite
´
Louis Pasteur 4,
rue Blaise Pascal, 67000 Strasbourg, France
Fax: +33 390 241431
Tel: +33 390 241273
E-mail:
(Received 31 March 2008, revised 14 June
2008, accepted 28 July 2008)
doi:10.1111/j.1742-4658.2008.06611.x
The protonation state of residues around the Q
o
binding site of the cyto-
chrome bc
1
complex from Paracoccus denitrificans and their interaction
with bound quinone(s) was studied by a combined electrochemical and
FTIR difference spectroscopic approach. Site-directed mutations of two
groups of conserved residues were investigated: (a) acidic side chains
located close to the surface and thought to participate in a water chain
leading up to the heme b
L
edge, and (b) residues located in the vicinity of
this site. Interestingly, most of the mutants retain a high degree of catalytic
activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On
the basis of electrochemically induced FTIR difference spectra, we suggest
that E295 and D278 are protonated in the oxidized form or that their
mutation perturbs protonated residues. Mutations Y302, Y297, E81 and
E295, directly perturb signals from the oxidized quinone and of the protein
backbone. By monitoring the interaction with the inhibitor stigmatellin for

FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4773
with covalently bound c-type heme, cytochrome b with
two b-type hemes (b
L
and b
H
), and the Rieske iron sul-
fur protein with a [2Fe–2S] cluster. Crystal structures
of several mitochondrial complexes that contain addi-
tional subunits have been reported [2–5]. Recently, a
new crystal structure for a bacterial complex has been
solved [6].
The enzyme couples the electron transfer from
ubiquinol to cytochrome c to the translocation of pro-
tons across the membrane. Both bacterial and mito-
chondrial bc
1
complexes follow the same catalytic
mechanism, the so-called Q-cycle [7–9], which relies on
two separate binding sites for quinones, Q
o
and Q
i
.
The Q
o
site is located close to heme b
L
and the [2Fe–
2S] cluster, and the Q

1
complex with stigma-
tellin bound at the Q
o
site [2] shows tight and specific
binding of the inhibitor. The position of the conju-
gated trienes is stabilized by several van der Waals
interactions with cytochrome b residues. The chromone
headgroup is oriented by numerous nonpolar and a
few polar interactions, including a hydrogen bond
from the carbonyl group (4-C = O) to His155 (His188
in yeast), one of the [2Fe–2S] cluster ligands of the
Rieske protein, which is thereby fixed in a cyto-
chrome b docking position [2] (unless otherwise indi-
cated, numbering of the amino acids corresponds to
the Paracoccus denitrificans bc
1
complex). On the heme
b
L
facing side of the inhibitor, the 8-hydroxy group is
within hydrogen-bonding distance of the side chain of
cytochrome b residue Glu295 (272 in yeast). Bound
stigmatellin is thought to mimic an intermediate of
ubiquinol oxidation [2]. Based on published structures
and biochemical characterization of variants, Glu295
has been proposed to be part of the proton exit path-
way for ubiquinol oxidation [2,16].
The cytochrome bc
1

Results
Site-directed mutations in the Q
o
binding site
Mutations in conserved positions of cytochrome b at
the Q
o
site were constructed (Fig. 1). The three subun-
its of the P. denitrificans bc
1
complex are expressed in
all mutants and assembled into a stable complex that
corresponds to the wild-type enzyme as determined
by SDS–PAGE and Western blot analysis. After
Fig. 1. 3D representation of the Q
o
site environment of the cyto-
chrome bc
1
complex based on the structure obtained from Rhodob-
acter sphaeroides [46]. Cytochrome c
1
is shown in blue,
cytochrome b in red, and the Rieske protein in green. The iron–sul-
fur cluster is shown in purple and yellow, and the bound inhibitor
stigmatellin is shown in turquoise. Heme is shown in light purple,
and the heme iron is shown in purple. Mutations of conserved
amino acids introduced in seven positions of the P. denitrificans
enzyme are indicated as follows: 1, D71 ⁄ 86 (mitochondrial ⁄ bacte-
rial complex); 2, E66 ⁄ 81; 3, D255 ⁄ 278; 4, Y132 ⁄ 147; 5, E272 ⁄ 295;

value. A
distinct increase of the IC
50
value was observed for the
E295Q and Y147F mutant enzymes.
FTIR difference spectra of mutations in the Q
o
binding site
Figure 2 shows an overview of the oxidized-minus-
reduced FTIR difference spectra of the E295Q,
D278N, E81Q and D86N mutant enzymes in compari-
son with wild-type. The redox-induced FTIR difference
spectra include contributions from reorganization of
the cofactors, heme b
L
, b
H
and c
1
, the bound quinones,
individual amino acids, the backbone and coupled pro-
tonation reactions. All purified mutants retained their
bound quinones, as their spectra include the character-
istic contributions that dominate the overall spectrum
of the P. denitrificans bc
1
complex, such as the typical
contribution of the methoxy side chain at 1264 cm
)1
,

b
E81Q 120 3.5
D86N 66 1.4
D278N 105 1.3
E295Q 10 4.6
Y147F 55 5.2
Y297F 90 2.0
Y302F 95 2.1
a
100% indicates a turnover number of 327 s
)1
based on one cyto-
chrome b (per monomer).
b
1 indicates an IC
50
value for the wild-
type of 131 ± 7 n
M under our experimental conditions.
1800 1700 1600 1500 1400 1300 1200
1455
1559
1474
1560
Δ Abs
0.001
1656
1654
1746 1746
1726

E295Q
E81Q
1743
Fig. 2. Overview of the oxidized-minus-reduced FTIR difference
spectra of wild-type and acidic side-chain mutant cytochrome bc
1
complexes from P. denitrificans obtained for a change in potential
from )0.292 to +0.708 V. The inset shows double difference spec-
tra obtained by subtracting the wild-type red-ox difference spec-
trum from that of each mutant.
T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q
o
site
FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4775
acidic residues were perturbed. The decrease is shown
in the inset to Fig. 2, showing double difference spec-
tra obtained by subtracting the spectrum of the
E295Q, D278N and E81Q variants from that of the
wild-type. Both D278N and E295Q show a decrease in
the mode at 1746 cm
)1
associated with the oxidized
form, without a complete loss of the signal (see Fig. 4
below), so both residues may contribute to this signal
or indirectly influence the contributing C = O group.
In the case of the D86N mutant enzyme, the negative
mode at 1724 cm
)1
is decreased. In contrast, the E81Q
mutation does not induce changes in this region. In

and Y302F mutant enzymes in comparison with wild-
type. The wild-type spectrum shows contributions in
the spectral range around 1516 and 1500 cm
)1
that are
characteristic of tyrosine side chains. In previously
reported model spectra of the protonated tyrosine, the
signal at approximately 1518 cm
)1
was attributed to
the m
19
(CC) ring mode. At 1249 cm
)1
, a signal com-
posed of the m
7’a
(CO) vibration and the d(COH) vibra-
tion is expected, and the position is sensitive to the
hydrogen-bonding environment [23,25,28,29]. For
deprotonated tyrosine in solution, the m
8a
⁄
8b
(CC) ring
mode was identified at 1560 cm
)1
and the m
19
(CC) ring

)
as
and 1522 cm
)1
for d(NH
3
+
)
s
[23,25]. For the Y302 mutant, perturbations were seen
at 1666, 1626 and 1522 cm
)1
.
Contributions of the quinones and the protein
backbone
In redox-induced FTIR difference spectra of quinones
in solution, the positive signals between 1670 and
1540 cm
)1
, as well as at 1610, 1288, 1264 and
1204 cm
)1
, correlate with the neutral quinone, while
the negative signals at 1490, 1470, 1432 and 1388 cm
)1
represent the reduced and protonated quinol form. The
mode between 1670 and 1640 cm
)1
was previously
assigned to the C = O vibration of the quinone, and

1644
1644
1644
1658
1656
1746 1746
1746
1644
1550
1508
1498
1520
1630
1658
1746
0.001
wt
Y297F
Y302F
Y147F
Wavenumber (cm
–1
)
Fig. 3. Overview of the oxidized-minus-reduced FTIR difference
spectra of wild-type and tyrosine side-chain mutant cytochrome bc
1
complexes from P. denitrificans obtained for a change in potential
from )0.292 to +0.708 V.
Infrared spectroscopic characterization of mutations in the Q
o

(1266 cm
)1
in the H ⁄ D-exchanged
sample) remain unperturbed (Fig. 3). As an alternative
explanation for the loss of signal intensity, e.g. for the
E81Q mutation, the dependence of the m(C = O) signal
for up to 50% of its intensity on the orientation of the
methoxy side chains in relation to the position of the
quinone ring should be noted, as previously reported
[36]. The change in intensity was confirmed in the
H ⁄ D-exchanged sample, for which the signals at 1655
and 1639 cm
)1
both strongly decrease due to the muta-
tion. This may indicate a change of the quinone envi-
ronment in some of the mutants. In addition, we note
some broadening of the m(C = O) signals, for example
in the case of the E295Q mutation. This may be due to
the loss of a hydrogen-bonding partner, allowing
greater rotational freedom of the C = O groups. In
order to differentiate between the effects on the protein
backbone and on the quinones, further experiments on
isotopically labeled quinones are necessary.
Wild-type FTIR difference spectra in the presence
of stigmatellin
Figure 5 shows the oxidized-minus-reduced FTIR
difference spectra of the wild-type cytochrome bc
1
complex from P. denitrificans obtained for a potential
step from )0.292 to +0.708 V, in comparison with

1726
1751
1746
1724
1693
1612
1644
1658
1746
Wavenumber (cm
–1
)
1800
1700 1600 1500 1400 1300 1200
0.001
Δ
Abs
D86N
E81Q
D278N
E295Q
WT
1448
1639
1560
1540
1266
1692
1448
1655

the unbound inhibitor. Double difference spectra were
obtained by subtracting wild-type spectra from those
obtained in the presence of a 2-fold excess of stigmatel-
lin to further elucidate the observed shifts (Fig. 5).
Large variations were seen over the full spectral
range. The spectral region between 1760 and
1710 cm
)1
is characteristic of variations in the
m(C = O) mode for protonated acidic residues
[26,27,37]. A new positive feature appears at 1723 cm
)1
,
and a small decrease of the signal at 1744 cm
)1
is seen.
This is in line with a previous study on the yeast bc
1
complex [18]. These difference signals include contribu-
tions from several acidic residues (Fig. 5). Shifts at
approximately 1540 cm
)1
as well as at 1447 and
1428 cm
)1
indicate possible variations of a deprotonat-
ed acidic residue, like, for example, amino acid side
chains and heme propionates [39]. Further significant
shifts, not arising from contributions of the inhibitor
itself, are seen in the amide I range, i.e. at 1635, 1646

the mutants with respect to the spectroscopic binding
characteristics were seen in the double difference spec-
tra obtained by subtracting the oxidized-minus-reduced
FTIR difference spectra of the mutants recorded in the
presence and absence of stigmatellin (Fig. 7).
The redox-induced FTIR difference spectrum of the
E295 mutant in the presence of stigmatellin displays
most of the typical signals of the inhibitor binding,
except for the spectral range specific for protonated
acidic residues around 1744 cm
)1
. No obvious varia-
tion was seen here. Interestingly, a new signal arose at
1560 cm
)1
, reflecting changes in the binding pocket.
Additional variations were seen around 1637 cm
)1
in
the amide I region, possibly due to displacement of the
differently bound quinone. The signal seen at
1744 ⁄ 1723 cm
)1
in the wild-type spectrum can thus be
attributed to the E295 side chain.
1800
1700 1600 1500 1400 1300
1200
1800
1700 1600 1500 1400 1300

4
0
7 1
C
3 1 5 1
4 9
2 1
6 4
3 1
3 8
3 1
4
4 4 1
7
6
4 1
5 3 5 1
3 6 5 1
0 0 6 1
2 2 6 1
3
9 6 1
4 4 6 1
0 7 6 1
2 5 2 1
Wavenumber (cm
–1
)
Wavenumber (cm
–1

ing of the inhibitor. On the basis of the up-shift of the
differential signals by about 6–4 cm
)1
in comparison
with wild-type, weaker hydrogen bonding or a more
hydrophobic environment of the C = O group of the
E295 side chain can be deduced. Differential features
in the spectral range for deprotonated acidic residues
at 1588 ⁄ 1565 cm
)1
and 1446 ⁄ 1428 cm
)1
were lost in
the double difference spectra of the D278N mutant as
highlighted by arrows. The signals in the amide I range
are clearly shifted in comparison to wild-type. D278
appears to be deprotonated in the stigmatellin-bound
form, and this residue obviously influences the stigma-
tellin binding site.
In the redox-induced FTIR difference spectra of the
Y302F variant in the presence of stigmatellin (Fig. 6),
only a small amount of inhibitor is observed, but most
of the typical shifts are observed. Interestingly, the
negative signals at 1668 and 1702 cm
)1
are not
decreased as seen for wild-type and the D278N and
E295Q mutant enzymes, and instead only a broad shift
at 1707 cm
)1

6461
88
5
1
2051
20
5
1
21
5
1
5361
3371
4471
0
571
0
6
5
1
8241
644
1
4351
7441
8
2
41
5
651

0.001
Δ
s
bA
1651
6461
4471
6
471
2471
Y302F
E295Q
D278N
WT
Wavenumber (cm
–1
)
Fig. 6. Oxidized-minus-reduced FTIR difference spectra for the
D278N, E295Q and Y302F mutants of the cytochrome bc
1
complex
from P. denitrificans obtained for a change in potential from )0.292
to +0.708 V in the presence of stigmatellin.
T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q
o
site
FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4779
discussed below in the light of current views on the
role of the so-called PEWY loop.
Residues E81 and D86 are positioned close to the

cating an interaction between these acidic residues and
the Q
o
binding site. The observed shifts may be a sec-
ondary-order effect induced by perturbation of the
water chain that leads to the heme b
L
edge and resi-
dues of the PEWY loop, including the E295 and Y297
residues studied here.
E295 is a heavily discussed position in close proxim-
ity to the quinone binding site, as suggested by
site-directed mutagenesis [10,13,16,41–45] and X-ray
crystallography [1–3,46]. All crystallographic data were
obtained in the presence of stigmatellin under the
assumption that the inhibitor remains oxidized. In the
FTIR spectroscopic analysis of the E295 mutant in
the absence of inhibitor, signals characteristic of pro-
tonated acidic residues in the fully oxidized form are
partially lost in direct comparison to the wild-type.
Table 2. Summary of tentative assignments for the oxidized-
minus-reduced FTIR difference spectra of the P. denitrificans bc
1
complex based on recent data from potential titrations [18] and
site-directed mutants in this study. A positive symbol (+) indicates
the oxidized state, a negative symbol ()) indicates the reduced
state. In case of a composite signal, the main peak is given.
Band position (cm
)1
)

)
1670 (+) Stigmatellin when added in excess
Perturbed m(C = O) heme propionates
1658 (+) Amide I (a-helical, unordered)
m(C = O) quinone
1646 ⁄ 1635 (+) Amide I
m(C = O) quinone
1644 (+) m(C = O) quinone
m
37
heme c
1
1628 ()) Amide I (Rieske b-sheet)
m(CN
3
H
5
) Arg (cytochrome b
H
)
1612 (+) m(C = C) quinone
1592 (+)
1570 (+) Amide II
m
37
heme b
L
m
38
heme c

m(COO
)
)
as
Asp ⁄ Glu (cytochrome b
H
)
m(COO
)
)
as
heme propionates b
L
, b
H
1520 (+) Y297, Y302
m
19
(CC) ring mode, protonated Tyr
1516 ()) Y297, Y302
m
19
(CC) ring mode, protonated Tyr
1508 (+) Amide II (Rieske)
1496 ()) Quinone ring
1470 ()) Quinone ring
1447 (+) 1447 (+) m(COO
)
)
s

1264 (+) m(C–O) methoxy group, quinone
m
42
heme c
1
1240 ()) m
42
heme b
H
1204 (+) Quinone
Infrared spectroscopic characterization of mutations in the Q
o
site T. Kleinschroth et al.
4780 FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS
On this basis, we suggest that the side chain is proton-
ated in the oxidized form (signal at 1746 cm
)1
) and de-
protonated in the reduced form (signal at 1561 cm
)1
).
In the presence of inhibitor, the residue remains pro-
tonated in the oxidized form, but exhibits stronger
hydrogen bonding (signal at 1723 cm
)1
). In the
reduced form, however, it is possibly deprotonated
(signal at 1565 cm
)1
). The redox-induced FTIR differ-

studies may therefore be considered complementary.
However, this may not be the only conflicting evi-
dence regarding mutations at position 295. Recently,
the stigmatellin resistance of yeast mutations at this
position has been studied by various groups: whereas
conservative replacements lead to increased stigmatel-
lin resistance [48], more pronounced exchanges had no
noteworthy effects [6]. Indeed, none of the mutations
completely abolished the prominent signals characteris-
tic for protonated acidic residues. We suggest that resi-
dues D278 and E295 both contribute to the signal of
the oxidized form. Contributions from other acidic res-
idues within the enzyme cannot be excluded. The
observation that several acidic residues participate in
this spectral feature is in line with the elaborate pH
dependency previously described [19].
The tyrosine mutations appear rather unperturbed
in comparison with wild-type, despite the close prox-
imity of the tyrosines to the Q
o
binding site. Most of
the mutants studied here alter the spectral features of
the quinone, indicating a variation of the hydrogen-
bonding environment and ⁄ or structure within the
binding site. This observation is not surprising in the
light of previous data showing that mutations on the
Y302 site induce noticeable conformational changes,
perturb kinetics, and affect inhibitor as well as quinone
binding [30].
A second quinone has been discussed to be located

)1
appears and the original sig-
nal decreases [18]. These results are not unambiguous,
especially in light of currently discussed mechanisms
and experimental observations suggesting that E295 is
deprotonated upon inhibitor binding [2,43]. Certainly,
the suggested proton transfer via residue E295 within
the hydrogen-bonding network of a water channel
could also occur with a protonated E295 residue
[2,43,51]. The binding of quinol to the protonated resi-
due, however, is difficult to substantiate. We note that
binding of stigmatellin was previously suggested to
mimic the interaction with the quinone radical [52] and
the stable intermediate that involves binding of the
Rieske iron sulfur protein [53]. According to the cur-
rent view, stigmatellin displaces a quinol molecule [51],
and the spectra shown here (Fig. 7) reflect this interac-
tion. We suggest that the high pK seen here for E295
in the oxidized form (> 7) may shift during the cata-
lytic cycle, allowing deprotonation and thus stabiliza-
tion of the quinol.
The redox activity of the stigmatellin reported previ-
ously [18] poses a challenge for data interpretation, as
the structure of the reduced form is not clear. A recent
study [54] has suggested reduction of the C = O group
T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q
o
site
FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4781
in the stigmatellin ring to a hydroxyl group, with the

[45], into which a StuI site was introduced between the fbcF
and fbcB open reading frames at residue 1024, and subcl-
oned into the vector pSL1180. For mutations E81Q, D86N
and Y147F, mutagenesis was performed using an NcoI ⁄
SmaI cassette from the wild-type fbc operon introduced
into the pUC18 vector.
The following primers were used: bE81Q, 5¢-CGCC
TCGGTCCAGCATATCATGCG-3¢; bD86N, 5¢-GCATA
TCATGCGCAACGTGAACGGCGGCTAC-3¢; bY147F,
5¢-GCCTTCATGGGCTTCGTGCTGCCCTGG-3¢; bD278N,
5¢-CTCGATATAGTTGTTGGGATGGCCCAG-3¢; bD295Q,
5¢-CATATCGTGCCGCAATGGTATTTCGTG-3¢; bY297F,
5¢-GTGCCGCAATGGTTCTTCCTGCCCTTC-3¢; bY302F,
5¢-GGTATTTCCTGCCCTTCTTCGCCATCCTGCG-3¢.
These were phosphorylated with T4 kinase (Fermentas, St
Leon-Rot, Germany) as specified by the manufacturer.
Mutations E81Q, D86N, Y147F and Y302F were intro-
duced into the wild-type fbc operon using the ‘Quik
Change’ mutagenesis kit from Stratagene (La Jolla, CA,
USA). The mutated cassettes were reinserted into the fbc
operon. Mutations E295Q, Y297F, and D278N were
introduced using the Altered Sites system (Promega, Man-
nheim, Germany). All mutations were confirmed by DNA
sequencing.
fbc operons encoding the wild-type or mutated P. deni-
trificans bc
1
complex were cloned into the HindIII ⁄ SacI
sites of the vector pRI2 [55]. The resulting plasmids were
conjugated into MK6, a chromosomal fbc deletion mutant

found to be better than 80% as determined from the shift
of the amide II mode (data not shown). For inhibition of
the Q
o
site, the concentrated samples were incubated for
1 h on ice in the presence of a 2-fold molar excess of
stigmatellin.
Activity assay
Ubihydroquinone–cytochrome c oxidoreductase activities
for the isolated wild-type and mutant preparations were
measured using decyl-ubihydroquinone (80 lm) and horse
heart cytochrome c (25 lm) as substrates in a buffer contain-
ing 50 mm Mops ⁄ NaOH pH 7.5, 1 mm EDTA, 1 mm KCN
and 0.04% DDM. The reduction of cytochrome c was fol-
lowed at 550 nm. Dilutions of the concentrated samples for
the activity measurements were made in a buffer containing
50 mm Mops ⁄ NaOH pH 7.5, 100 mm NaCl, 0.04% DDM,
5% glycerol and 0.05% BSA. To inhibit enzyme activity,
stigmatellin from a stock solution of 10 mm in ethanol was
added to a final concentration of 2 lm.
The IC
50
value was determined under activity test condi-
tions, but stigmatellin (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 lm
final concentration from 10 mm stock in ethanol) was
added before the addition of the enzyme. V
max
was plotted
against the common logarithm (log 10) of the stigmatellin
concentration and fitted non-linearly. The IC

range and a dispersive spectrometer
for the 400–900 nm range as reported previously [58,59].
The protein was equilibrated at an initial electrode poten-
tial, and a single-beam spectrum was recorded. Then the
final potential was applied, and a single-beam spectrum was
again recorded after equilibration. Equilibration generally
took less than 4 min for the full potential step from )0.292
to +0.708 V. The difference spectra presented here were
calculated from two single-beam spectra, with the initial
spectrum taken as reference. Typically, 128 interferograms
at 4 cm
)1
resolution were co-added for each single-beam
spectrum, and Fourier-transformed using triangular apodi-
zation and a zero filling factor of 2. Eight to ten difference
spectra were averaged. To account for differences in sample
concentration and path length, the FTIR difference spectra
were normalized to the difference signals of the a-band in
the UV ⁄ visible spectrum (not shown).
Acknowledgements
We are grateful to A. Herrmann (Institute of Biochem-
istry, University of Frankfurt) for excellent technical
assistance and W. Ma
¨
ntele (Institute of Biophysics,
University of Frankfurt) for his support at an early
stage of the experiments, and thank T. Thieme and
A. Klein (Institut fu
¨
r Biochemie, Universita

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