Defining the Q
P
-site of Escherichia coli fumarate reductase
by site-directed mutagenesis, fluorescence quench
titrations and EPR spectroscopy
Richard A. Rothery
1
, Andrea M. Seime
1
, A M. Caroline Spiers
1
, Elena Maklashina
2,3
,
Imke Schro
¨
der
4
, Robert P. Gunsalus
4
, Gary Cecchini
2,3
and Joel H. Weiner
1
1 CIHR Membrane Protein Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
2 Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, CA, USA
3 Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
4 Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA
Escherichia coli, when grown anaerobically with fuma-
rate as the respiratory oxidant, develops a respiratory
chain terminated by a membrane-bound menaqui-

2
analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indi-
cate that the Q
P
site is defined by residues from FrdB, FrdC and FrdD. In
FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K
d
of
2.5 nm, and the following mutations significantly increase this value: FrdB-
T205H (K
d
¼ 39 nm); FrdB-V207C (K
d
¼ 20 nm); FrdC-E29L (K
d
¼
25 nm); FrdC-W86R (no detectable binding); and FrdD-H80K (K
d
¼
20 nm). In all titrations performed, data were fitted to a monophasic bind-
ing equation, indicating that no additional high-affinity HOQNO binding
sites exist in FrdABCD. In all cases where HOQNO binding is detectable
by FQ titration, it can also be observed by EPR spectroscopy. Steady-state
kinetic studies of fumarate-dependent quinol oxidation indicate that there
is a correlation between effects on HOQNO binding and effects on the
observed K
m
and k
cat
values, except in the FrdC-E29L mutant, in which

differences exist between the membrane-intrinsic
domains of the two enzymes [1,7]. The membrane-
intrinsic domain of SdhCDAB coordinates a single
heme b (b
556
) that is sandwiched between the SdhC
and SdhD subunits [8,9]. Quinone binding and reduc-
tion is believed to take place in the region between the
heme and the [3Fe-4S] cluster of SdhB [1,6]. In the case
of FrdABCD, the membrane-intrinsic domain does not
contain heme, but instead contains two menaquinones
at discreet sites in the crystallized form of the enzyme
[3,4]. In both enzymes, despite the available structures,
the number of functional quinone ⁄ quinol binding sites
has yet to be unequivocally determined.
The menaquinones identified in the crystal structure
of FrdABCD [3] are located at sites towards the inner
(cytoplasmic) and outer (periplasmic) sides of the mem-
brane-intrinsic domain of the enzyme (FrdCD). One
site, the Q
P
site (the proximal Q-site), is located in the
interface region between the FrdCD subunits and
the [3Fe-4S] cluster coordinating region of FrdB on the
cytoplasmic side of the membrane. The other site, the
Q
D
site (the distal Q-site) is located approximately 25 A
˚
from the Q

Menaquinol (MQH
2
) oxidation by FrdABCD has
been studied using a combination of site-directed muta-
genesis, enzymology, EPR spectroscopy and X-ray crys-
tallography. Initial mutagenesis studies suggested that
there may be two Q-sites present – a polar Q
B
site
(equivalent to the Q
P
site), and an apolar Q
A
site (equiv-
alent to the Q
D
site) [13–15]. Investigation of the steady-
state kinetics of quinol-dependent fumarate reduction
by FrdABCD suggests that MQH
2
binding and oxida-
tion occur at a single site [16]. Kinetic studies carried
out in the presence of HOQNO or alkylated dinitro-
phenol derivatives also support the presence of a single
MQH
2
oxidation site [17]. By exploiting the fluorescent
properties of HOQNO in fluorescence quench (FQ)
titrations, we determined that this inhibitor binds at a
single high-affinity site within FrdABCD [18,19]. EPR

P
-site
in the structure of FrdABCD: T205, F206, Q225 and
K228 from FrdB; R28, E29, W86, L89 and A93 from
FrdC; and W14, F17, G18, H80, R81 and H84 from
FrdD [3,4,10]. Site-directed mutants of some of these res-
idues have been generated and partially characterized,
including the following: FrdC-E29L [14,20], FrdC-
W86R, FrdD-H80K and FrdD-H84K [14]. In the con-
text of this study, mutants of the following residues
located at a slightly greater distance from the Q
P
site are
also potentially of interest: FrdB-V207 (% 8A
˚
from Q
P
,
a FrdB-V207C mutant) [21], and FrdC-A32 (% 9A
˚
from Q
P
, a FrdC-A32V mutant) [14]. At an even greater
distance away from the Q
P
site is FrdC-F38 (% 18 A
˚
), a
mutation at this position (FrdC-F38M [14]), would be
expected to have little effect on MQH

characterize the Q
P
site of FrdABCD.
FQ titrations of HOQNO binding to mutant
FrdABCD
HOQNO is a close structural analog of MQH
2
⁄ MQ
and is a very potent inhibitor of FrdABCD [16,18].
When excited at 341 nm, free HOQNO in aqueous
solution fluoresces with an emission wavelength of
479 nm. Its fluorescence is completely quenched when
bound to FrdABCD and certain other E. coli respirat-
ory chain enzymes (including dimethylsulfoxide reduc-
tase and nitrate reductase A [18,19,22–24]). This
enables its binding to a Q-site to be analyzed by FQ
titration. Figure 2 shows representative titrations of
membranes containing the wild-type and mutant
enzymes studied herein. Data for all of the mutants is
presented in Table 1. DW35 membranes lacking
FrdABCD (Fig. 2A) do not exhibit high-affinity
HOQNO binding. The following FrdABCD mutants
bind HOQNO with K
d
values equivalent to that of the
wild-type enzyme (K
d
¼ 2.5 nm; Fig. 2B): FrdC-A32V
(2.5 nm; not shown), FrdC-F38M (2.5 nm; not shown)
and FrdD-H84K (3.0 nm, not shown). At the opposite

FrdC and FrdD have labels starting with ‘B-’,
‘C-’, and ‘D-’, respectively.
R. A. Rothery et al. Quinol binding to E. coli fumarate reductase
FEBS Journal 272 (2005) 313–326 ª 2004 FEBS 315
these observations and the FrdABCD structure [3,4], it
is clear that residues from FrdB, FrdC and FrdD play
important roles in defining the Q
P
site. In every case
where binding is detected, the data can be fitted to an
equation (Eqn 1) describing noncooperative binding at
a single site within FrdABCD.
Table 1 shows the calculated specific concentration
of HOQNO binding sites for each mutant in which
binding is detected by FQ titration. It also shows the
concentration of FrdABCD calculated by EPR spin
quantitation of both the [2Fe-2S] and [3Fe-4S] clusters.
In each case, the estimated number of Q-sites per
enzyme is very close to unity, indicating that HOQNO
binding occurs at a single site within FrdABCD. Based
on enzymes that bind HOQNO, 1.02 ± 0.12 sites were
observed per [3Fe-4S] cluster and 1.05 ± 0.09 sites
were observed per [2Fe-2S] cluster.
Detection of HOQNO binding by EPR
spectroscopy
Figure 3 shows the effect of HOQNO on the EPR
spectrum around g ¼ 2.0 of ferricyanide-oxidized
HB101 membrane samples containing wild-type and
mutant FrdABCD. EPR spectra of membranes lacking
overexpressed FrdABCD exhibit low-intensity features

)1
)andK
d
values (nM): (A) background, 0.36, > 500; (B) wild-type, 3.54, 2.5; (C) FrdB-T205H, 3.13, 39; (D) FrdC-E29L,
3.26, 25; (E) FrdC-W86R, negligible binding; (F) FrdD-H80K, 3.61, 20. Note that in the cases of the background and FrdC-W86R mutant
membranes, the data presented represent insignificant binding.
Quinol binding to E. coli fumarate reductase R. A. Rothery et al.
316 FEBS Journal 272 (2005) 313–326 ª 2004 FEBS
FrdB-T205H mutant in the absence of inhibitor
(g
xy
¼ 2.0 in the presence of inhibitor rather than at
1.98). Figure 3D shows the spectrum of oxidized mem-
branes containing overexpressed FrdB-V207C mutant
enzyme. In agreement with Manadori et al. [21], little
or no [3Fe-4S] cluster is assembled into this mutant
enzyme (compare Fig. 3A and D), and therefore
HOQNO binding cannot be detected by its perturba-
tion of the EPR spectrum of the oxidized enzyme (see
below).
In contrast to the results of Ha
¨
gerha
¨
ll et al. [20], the
EPR experiments reported herein indicate that HO-
QNO elicits an effect on the EPR line-shape of the
[3Fe-4S] cluster of the FrdC-E29L mutant enzyme
(Fig. 3E). This result is consistent with the observation
of HOQNO binding by FQ titration (Fig. 2D and

of HOQNO (Fig. 4Ai) and that recorded in its pres-
ence (Fig. 4Aii). The spectrum of reduced membranes
containing overexpressed wild-type FrdABCD recor-
ded in the absence of HOQNO has an intense peak at
g ¼ 2.02 (g
z
) and a peak-trough at g ¼ 1.93 (g
xy
)
(Fig. 4Bi). These comprise the EPR spectrum of the
[2Fe-2S] cluster of FrdB [27]. The EPR spectrum of
the [4Fe-4S] cluster manifests itself as a very broad,
rapidly relaxing signal underlying that of the [2Fe-4S]
cluster [21,26] with peaks at g ¼ 2.18 and troughs at
g ¼ 1.82 and g ¼ 1.66. No significant effect is elicited
on this spectrum by HOQNO (compare Fig. 4Bi and
Bii).
Figure 4C shows similar spectra recorded of mem-
branes containing the overexpressed FrdB-V207C
mutant that contains a [4Fe-4S] cluster in place of the
[3Fe-4S] cluster of the wild-type enzyme [21]. In this
case, the broad underlying spectrum arises from the
Table 1. Effect of the FrdABCD mutations on HOQNO binding determined by FQ titrations and EPR spectroscopy in E. coli strain DW35.
The concentration of the dithionite-reduced [2Fe-2S] cluster was estimated by double integration of EPR spectra recorded at 40 K under
nonsaturating conditions using a CuEDTA concentration standard [47]. The concentration of the ferricyanide-oxidized [3Fe-4S] cluster was
estimated by double integration of EPR spectra recorded at 9 K under nonsaturating conditions using a Cu-EDTA concentration standard
[47]. The effect of HOQNO on the [3Fe-4S] cluster EPR line-shape was determined using E. coli HB101 membranes. Samples and EPR con-
ditions were as described for Figs 3 and 4. ND, not detected.
Membrane
preparation

b
Yes
c
FrdC-E29L 25.0 3.26 2.74 3.11 1.19 1.05 Yes
FrdC-A32V 2.5 2.97 2.96 3.16 1.00 0.94 Yes
FrdC-F38M 2.5 3.26 3.14 3.44 1.04 0.95 Yes
FrdC-W86R ND ND 2.37 2.34 ND ND No
FrdD-H80K 20.0 3.61 3.15 3.47 1.15 1.04 Yes
FrdD-H84K 3.0 3.68 3.09 3.47 1.19 1.06 Yes
a
Features clearly attributable to either a [2Fe-2S] cluster or a [3Fe-4S] are not detected in spectra of reduced and oxidized membrane sam-
ples from E. coli strain DW35.
b
The FrdB-V207C mutant contains a [4Fe-4S] cluster in place of the [3Fe-4S] cluster of the wild-type enzyme.
c
In this case, the effect of HOQNO was determined by analyses of spectra of dithionite-reduced samples recorded as described in the
legend to Fig. 4.
R. A. Rothery et al. Quinol binding to E. coli fumarate reductase
FEBS Journal 272 (2005) 313–326 ª 2004 FEBS 317
spin-coupled pair of [4Fe-4S] clusters and comprises a
peak at g ¼ 2.29, and troughs at g ¼ 1.87 and 1.67.
Addition of HOQNO causes the appearance of a peak
at g ¼ 1.98 (compare Figure 4Ci and ii). Overall, these
data are consistent with there being a perturbation of
the engineered [4Fe-4S] cluster in the FrdB-V207C
mutant by HOQNO, and with there being no pertur-
bation of the [4Fe-4S] cluster of the wild-type enzyme.
Fig. 4. Effect of HOQNO on the engineered [4Fe-4S] cluster EPR
spectrum of FrdB-V207C FrdABCD in HB101 membranes. Mem-
branes were incubated in the absence of (i) or presence of (ii)

oxidoreductase activity of FrdABCD
In order to gain a broader understanding of the effects
of the mutants on the physiological quinol oxidation
reaction catalyzed by FrdABCD, we studied their
effects on the steady-state kinetics of the quinol:
fumarate oxidoreductase reaction using the MQH
2
analog lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-
naphthoquinone; LPC]. When reduced, this substrate
(LPCH
2
) has significant structural similarity to MQH
2
,
and in its oxidized form has a convenient absorbance
peak in the visible region at 481 nm in aqueous solu-
tion [16]. Figure 5 shows representative Eadie–Hofstee
plots describing the steady-state kinetic behavior of
wild-type and a subset of the mutants of FrdABCD in
DW35 membranes. The wild-type enzyme has a K
m
for LPCH
2
of approximately 225 lm and a k
cat
of
approximately 71 s
)1
. The FrdB-T205H and FrdD-
H80K mutants have increased K

in the FrdC-E29L mutant
The FrdC-E29L mutant is unusual because it retains
high-affinity HOQNO binding (Table 1 and Fig. 2),
but demonstrates no fumarate-dependent LPCH
2
oxi-
dation. It has been demonstrated previously by redox
potentiometry to stabilize a menasemiquinone radical
Fig. 5. Determination of steady-state kinetic parameters for wild-
type and mutant FrdABCD. e, wild-type, K
m
¼ 225 lM, k
cat
¼
71 s
)1
. h, FrdAB
T205H
CD; K
m
¼ 355 lM, k
cat
¼ 68 s
)1
. s,
FrdABCD
H80K
, K
m
¼ 670 lM, k

, high-affinity HOQNO binding, supports growth; 3, no quinol oxidation,
high-affinity HOQNO binding, does not support growth. NA, not applicable. Membranes from the background strain, E. coli DW35, do not
contain FrdABCD. ND, not detected.
Membrane preparation K
m
a
(lM) k
cat
a
(s
)1
) Growth on GF HOQNO Binding Group
Background ND ND No No NA
FrdABCD 225 ± 25 71 ± 3 Yes Yes 1
FrdB-T205H 355 ± 34 68 ± 4 Yes Yes 1
FrdB-V207C 203 ± 17 31 ± 1 Yes Yes 2
FrdC-E29L ND ND No Yes 3
FrdC-A32V 115 ± 6 31 ± 1 Yes Yes 2
FrdC-F38M 385 ± 40 82 ± 5 Yes Yes 1
FrdC-W86R ND ND No No 0
FrdD-H80K 670 ± 47 67 ± 3 Yes Yes 1
FrdD-H84K 451 ± 34 75 ± 3 Yes Yes 1
a
Kinetic parameters were determined from Eadie–Hofstee plots such as those presented in Fig. 5.
R. A. Rothery et al. Quinol binding to E. coli fumarate reductase
FEBS Journal 272 (2005) 313–326 ª 2004 FEBS 319
anion [20]. Thus, a plausible explanation for the lack
of quinol:fumarate oxidoreductase activity is that this
mutant becomes trapped in a state in which a mena-
semiquinone radical anion is bound to the Q

mined by EPR spin quantitation of the [2Fe-2S] and
[3Fe-4S] clusters (Table 1). Where modulation of the
K
d
for HOQNO is detected, the FQ data can be fitted
to a binding equation describing noncooperative bind-
ing at a single site within FrdABCD. These observa-
tions are consistent with the presence of a single
redox-active dissociable Q-site in FrdABCD, and indi-
cate that this site coincides with the Q
P
site observed
in the crystal structures of Iverson et al. [3,4]. The
HOQNO binding data agree with the structure of
FrdABCD incubated in the presence of HOQNO, in
which the inhibitor is bound exclusively at the Q
P
site.
We previously reported the effect of HOQNO on
the EPR line-shape of the [3Fe-4S] cluster of FrdB,
and showed that a point mutation in FrdC, FrdC-
H82R, eliminated both this effect and HOQNO bind-
ing detected by FQ titration [18]. However, the posi-
tion of FrdC-H82 within the hydrophobic core of
FrdC (> 5 A
˚
away from Q
P
), along with the relatively
severe Arg substitution, warranted re-examination of

the FrdC-W86R mutant), we were able to study a
range of mutations that are more likely to have local
effects within the protein. Overall, there is a good cor-
relation between the location of the mutated residues
and the severity of the observed effects on HOQNO
binding (compare Figure 1 and Table 1).
An effect on the EPR spectrum of the [3Fe-4S] clus-
ter is clearly observed in each case where HOQNO
binding is detected by FQ titration. In addition, we
were able to observe that this effect is not propagated
beyond the location of the [3Fe-4S] cluster (Fig. 4).
The FrdB-V207C mutant contains a [4Fe-4S] cluster in
place of the [3Fe-4S] cluster of the wild-type enzyme,
so that the mutant enzyme contains two [4Fe-4S] clus-
ters coordinated by a motif similar to those found in
the bacterial 8Fe ferredoxins [21]. In this mutant, the
converted cluster is paramagnetic in its reduced state,
but its spectroscopic analysis is complicated by spin–
spin interactions with the other two reduced clusters of
the enzyme (Fig. 4). Despite this, we were able to dem-
onstrate that HOQNO elicits a line-shape change on
the EPR spectrum of the fully reduced FrdB-V207C
mutant. Overall, the combination of FQ and EPR data
confirm that the Q
P
site is defined by residues from
FrdB, FrdC and FrdD.
Our observation that the Q
P
site is closely coupled

HQONO binding, able to support growth. Members:
the wild-type enzyme, the FrdB-T205H, FrdC-F38M,
FrdD-H80K and FrdD-H84K mutants.
2 – normal or modulated K
m
, decreased k
cat
, high-
affinity HOQNO binding, able to support growth.
Members: the FrdB-V207C and FrdC-A32V mutants.
3 – no quinol oxidation, high-affinity HOQNO bind-
ing, unable to support growth. Member: the FrdC-
E29L mutant.
Overall, the kinetic data presented herein are consis-
tent with the occurrence of simple Michaelis–Menten
kinetics, with LCPH
2
binding and oxidation occurring
at a single Q-site (Fig. 5). However, it is notable that
mutants that appear to have little effect on HOQNO
binding can modulate the observed steady-state kinet-
ics of the enzyme. For example, the FrdC-A32V
mutant significantly decreases the observed k
cat
.A
possible explanation for this is that the increased bulk
of the hydrophobic sidechain is able to stabilize qui-
nol ⁄ quinone species at the Q
P
site, decreasing the rate

to the MQ at the Q
P
site than that of FrdD-H80, the
axis of the His-84 imidazole points slightly away from
the MQ naphthoquinone bicycle, whereas that of the
His-80 imidazole appears to be pointing at least parti-
ally towards it. Thus, it is more likely that the side-
chain of the Lys substitution of FrdD-H80 elicits an
effect on HOQNO binding and LPCH
2
oxidation than
the Lys substitution of FrdD-H84. Although this
explanation appears plausible, it should be noted that
it is based on structural data of fairly low resolution
(3.3 A
˚
) [3,4].
The FrdB-T205H mutant is of interest in establish-
ing the role of FrdB in defining the Q
P
site. As men-
tioned previously (Results), this mutation was chosen
because of the location of FrdB-T205H with respect to
the Q
P
site, the [3Fe-4S] cluster and the interface
between FrdB and the membrane anchor subunits.
With the exception of the FrdC-W86R mutant, the
R. A. Rothery et al. Quinol binding to E. coli fumarate reductase
FEBS Journal 272 (2005) 313–326 ª 2004 FEBS 321

acceptor during enzyme turnover [3,4]. Furthermore, it
is widely believed that HOQNO represents a good ana-
log of the menasemiquinone radical intermediate
[32,33]. Our observation of a radical when enzyme turn-
over is attempted indicates that the mutant is only able
to accept a single electron from MQH
2
, resulting in a
bound and stabilized menasemiquinone intermediate,
thus explaining the observed binding of HOQNO and
the lack of quinol:fumarate oxidoreductase activity.
In addition to the E. coli complex II homologs
(FrdABCD and SdhCDAB), a high-resolution struc-
ture is available for one additional bacterial complex
II homolog. This is the Wolinella succinogenes fuma-
rate reductase (FrdCAB) [34,35] which belongs to a
distinct class of complex II homologs that includes the
Bacillus subtilis succinate dehydrogenase (SdhCAB)
[33]. These enzymes have a single membrane anchor
subunit (FrdC and SdhC, respectively) that contains
two hemes. The structure of the W. succinogenes Frd-
CAB [35] reveals that one heme is proximal to the
membrane-extrinsic dimer (heme b
P
), whilst the other
is distal to it (heme b
D
). It has been demonstrated that
a point mutation (FrdC-E66Q) that eliminates MQH
2

site. This is supported by
theoretical models of through-protein electron transfer
which indicate that the 25 A
˚
distance between the Q
P
and Q
D
menaquinones identified in the protein struc-
ture is too far to allow for physiologically relevant
electron transfer between these sites [11]. Our prelimin-
ary investigations of mutants (such as FrdD-F57V and
FrdC-V35A) surrounding the MQ
D
observed in the
protein structure indicate that these have no effect on
the HOQNO binding detected by FQ titration and by
EPR; and have little effect on quinol:fumarate oxidore-
ductase activities. A full description of these mutants
will appear in a later communication (E Maklashina,
RA Rothery, JH Weiner and G Cecchini, unpublished
data). Thus, it is likely that the Q
D
site plays no direct
role in menaquinol oxidation.
Overall, by using a range of FrdB, FrdC, and FrdD
mutants, we have demonstrated that in every case
where HOQNO binding is detected, it occurs at a sin-
gle site within FrdABCD. In agreement with the struc-
tural data of Iverson and coworkers [3,4], we provide

mutants were originally generated and partially character-
ized using plasmid pDW100 (frdC
+
D
+
) in combination
with a second plasmid, pFRD23 (frdA
+
B
+
) [14]. In order
to express high levels of FrdABCD, it was necessary to sub-
clone the mutated frdCD genes of pDW100 as a DraIII-
XhoI fragment into appropriately cut pH3. FrdB mutants:
FrdAB
V207C
CD was encoded by pH3-V207C [21]. A plas-
mid encoding FrdAB
T205H
CD (pH3-T205H) was generated
by site-directed mutagenesis using the methodology des-
cribed by Cecchini et al. [44].
Cell growth
DW35 strains
E. coli DW35 and its transformants were grown overnight
in 5 L batches in a B. Braun Biostat B fermenter (B. Braun
Biotech International, Melsungen, Germany) at 37 °Cin
the presence of 100 lgÆmL
)1
streptomycin (Amresco, Solon,

being stored at )70 °C.
Isolation of cytoplasmic membranes
Crude membranes were prepared by French pressure cell
lysis and differential centrifugation at 150 000 g for 1.5 h at
4 °C in 100 mm Mops ⁄ KOH and 5 mm EDTA (pH 7.0)
which contained the protease inhibitor phenylmethanesulfo-
nyl fluoride (0.2 mm) [29]. Cytoplasmic membranes were
isolated from resuspended crude membranes by layering
them on top of a 55% (w ⁄ v) sucrose step (made up in buf-
fer) in an ultracentrifuge tube. Following centrifugation at
40 000 r.p.m. for 1.5 h in a Beckman 50.2Ti rotor
(150 000 g at 4 °C), the floating band enriched in the cyto-
plasmic membrane fraction was removed, diluted in buffer,
and subjected to a further centrifugation. Finally, to ensure
complete removal of residual sucrose, the pellet was resus-
pended in buffer and recentrifuged [24]. Membranes were
then resuspended in buffer to a protein concentration of
approximately 30 mgÆmL
)1
, flash frozen in liquid nitrogen,
and stored at )70 °C until use.
FQ titrations with HOQNO
The affinity of FrdABCD for HOQNO (Sigma-Aldrich,
Oakville, Ontario, Canada) was determined using FQ titra-
tions performed as described previously using a Perkin
Elmer (Norwalk, CT) LS-50B luminescence spectrometer
[18,23,45]. Fluorescence intensities were measured using an
excitation wavelength of 341 nm and an emission wave-
length of 479 nm. All experiments were carried out at room
temperature (23 °C) and pH 7.0 in 100 mm Mops ⁄ KOH

tot

ð1Þ
with
Q ¼
1
2
Âð½I
tot
þK
d
þ n
s
½E
tot
Þ ð2Þ
and
½I
tot
¼½I
bound
þ½I
free
ð3Þ
These equations are from reference [45]. The specific fluo-
rescences of the bound and free inhibitor are f
bound
and
f
free

were incubated in the pres-
R. A. Rothery et al. Quinol binding to E. coli fumarate reductase
FEBS Journal 272 (2005) 313–326 ª 2004 FEBS 323
ence of 0.5 mm HOQNO for 5 min before being oxidized
with 0.2 mm ferricyanide for % 2 min. Samples were trans-
ferred to 3 mm internal diameter quartz EPR tubes prior to
being frozen and stored as described above. To investigate
the effect of HOQNO on the EPR line-shape of fully
reduced FrdABCD and FrdAB
V207C
CD, 150 lL membrane
samples at 30 mgÆmL
)1
were incubated with 0.5 m m HO-
QNO, then reduced with 5 mm dithionite for 5 min under
an argon atmosphere before being frozen and stored as des-
cribed above. For EPR spin quantitations, reduced samples
were prepared by incubation with 5 mm dithionite for
5 min, and oxidized samples were prepared by incubation
with 0.2 mm ferricyanide for 2 min. To investigate the
appearance of menasemiquinone radical species, samples
were reduced with 5 mm dithionite for 2 min and then trea-
ted with 25 mm fumarate for 30 s or 1 min before being
frozen as described above.
EPR spectroscopy
EPR spectra were recorded using a Bruker ESP 300 spec-
trometer (Bruker Biospin, Rheinstetten, Germany)
equipped with an Oxford Instruments (Abingdon, Oxon,
UK) ESR-900 flowing helium cryostat and a Hewlett Pack-
ard 5350B microwave frequency counter (Hewlett Packard,

mined by generating Eadie–Hofstee plots (v vs. v ⁄ s), and the
protein concentration was between 0.016 mgÆmL
)1
and
0.056 mgÆmL
)1
.
Structural alignment and molecular graphics
Protein structures of MQ-bound and HOQNO-bound
FrdABCD (PDB files 1L0V and 1KF6, respectively [4])
were manipulated using the program pymol (version 0.97,
Delano Scientific LLC, ). Prior
to generating the views presented in Fig. 1, the structures
(all subunits) were aligned with a root mean square devi-
ation of 0.17 A
˚
for the superposition of 1021 C-a atoms
between the two forms.
Acknowledgements
The authors wish to thank: Delilah Mroczko for her
assistance with the B. Braun Biostat B fermentation
system, and Monica Palak for the preparation of mem-
brane samples.
This work was funded by the Canadian Institutes of
Health Research and the Canada Foundation for
Innovation. A M.C.S. and A.M.S. were supported by
Alberta Heritage Foundation for Medical Research
Summer Studentships. J.H.W. holds a Canada
Research Chair in Membrane Biochemistry. Further
funding was provided by National Institutes of Health

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