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INTRODUCTION |
Succinate-ubiquinone oxidoreductases (SQR1; succinate
dehydrogenase, complex II) and
menaquinol-fumarate reductase (QFR; fumarate reductase) are
membrane-bound complexes that play critical roles in cellular
metabolism in prokaryotic and eukaryotic organisms. The enzymes
catalyze the reversible transfer of two electrons and two protons
between succinate-fumarate and the quinol-quinone couples (1); however,
they normally are only expressed in aerobic (SQR) or anaerobic
(QFR) environments (2, 3). SQR directly connects the Krebs
cycle with the aerobic respiratory chain by transferring reducing
equivalents via quinone, whereas QFR is a terminal reductase in
anaerobic respiration, where it oxidizes low potential quinols and
reduces fumarate as the final electron acceptor (2, 4-6).
Two distinct operons encode the subunits of SQR (sdhCDAB)
(7, 8) and QFR (frdABCD) (9, 10) in Escherichia
coli. X-ray structures for membrane-bound QFR from E. coli (11) and Wolinella succinogenes (12) have recently
become available. Both QFR and SQR are composed of a conserved
catalytic domain that consists of the two largest subunits
(flavoprotein and iron-sulfur protein subunits). The flavoprotein
subunit (SdhA, FrdA, or Fp) contains covalently bound FAD and
the dicarboxylate binding site. The iron-sulfur protein subunit (SdhB,
FrdB, or Ip) contains three distinct linearly arranged iron
sulfur clusters ([2Fe-2S]2+,1+,
[4Fe-4S]2+,1+, and [3Fe-4S]1+,0). The
soluble dehydrogenase fragment (FpIp) binds to the hydrophobic anchor
domain to form a membrane-bound complex (complex II) that is able to
carry out electron transfer with quinone-quinol. The hydrophobic
membrane anchor subunit pairs (SdhC, FrdC and SdhD, FrdD) are
essential for forming the quinone binding sites and assembly of the
whole complex (13-16). The complex II membrane anchor subunits also
coordinate one or two b-type hemes; however, E. coli QFR is a type of complex II that contains no heme (4). The
single heme in E. coli SQR (17), as for the heme(s) in other complex IIs, has been shown to have bis-histidine axial ligation by EPR
and near infrared magnetic circular dichroism (18, 19), and from the
x-ray structure (12). The complex II membrane anchor domains of
different species share little similarity in amino acid sequence;
however, the overall structure suggests a similar arrangement of the
trans-membrane helices. A structural model for the membrane anchor
domain of complex II has been proposed (20, 21), and the recent high
resolution structure of the diheme W. succinogenes QFR (12)
is consistent with this model including the arrangement of the heme
moieties. The redox properties of the heme b in complex II
from different organisms also vary over a range of ~200 mV. The
single heme in bovine heart complex II is not readily reducible by
succinate due to its low redox potential at pH 7.0 (Em,7 =
185 mV) (22). Bacillus subtilis complex II contains two b hemes, with the
higher potential heme bH
(Em,7 = +65 mV) reducible by succinate,
whereas the lower potential heme bL
(Em,7 =
95 mV) is not (23). Succinate
is able to reduce the single heme b556 in
E. coli SQR (Em,7 = +36 mV)
(24). The varied presence and reducibility of the heme in complex IIs
raises questions about whether catalysis is linked to the redox
properties of cytochrome b. The heme, where present, has
been shown to have an important role for proper assembly of complex II.
In heme-deficient mutants of B. subtilis, the apocytochrome of SQR is made and inserted into the membrane, whereas the catalytic domain (FpIp) of the enzyme is accumulated in the cytoplasm (25). Similar results have also been reported for E. coli SQR when
expressed in cells deficient in heme synthesis (26). The heme axial
ligands for E. coli SQR have been shown to bridge the two
membrane anchor subunits. SdhC His84 and SdhD
His71 were identified as the heme ligands, and it was shown
that succinate-quinone reductase activity was retained in the mutant
enzymes despite the apparent absence of the heme
b556 (27). Herein, we present spectral and
kinetic characterization of the SdhC His84 and SdhD
His71 mutants of E. coli SQR that retain heme.
The results show that an altered heme b does assemble in the
isolated mutant enzyme although with significantly lowered redox
potential. The data also show that the heme moiety is near the quinone
binding domain.
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MATERIALS AND METHODS |
Bacterial Strains and Plasmids--
E. coli
strain DW35 (
frdABCD, sdhC::kan) and its
recA derivative GV141 have been previously described (15,
27). The deletion in the frd operon and the insertional
mutation in the sdh operon eliminates strain background
expression of any enzyme capable of succinate oxidase activity. Plasmid
pSDH15
(sdhC+D+A+B+)
and the plasmid derivatives encoding mutations in the heme histidyl ligands (pSdhC H84L and pSdhD H71Q) have been described previously (27). Plasmids pFAS and pFGS (1) contain a frd promoter
fusion to sdh
(PFRDsdhC+D+A+B+)
such that SQR can be expressed anaerobically in E. coli with plasmid pFAS giving the highest expression level. In order to express
the mutant enzyme anaerobically, the 0.57-kilobase pair EcoRI-KpnI fragment from pSdhC H84L or the
fragment from pSdhD H71Q was inserted into the equivalent site in pFAS
or pFGS to create plasmids pFAS or pFGS SdhC H84L
(PFRDsdhCH84LD+A+B+)
and pFAS or pFGS SdhD H71Q
(PFRDsdhC+DH71QA+B+).
Growth Conditions--
Aerobically grown cultures were grown
overnight in Luria-Bertani (LB) medium with appropriate antibiotics,
and 150 ml was used as inoculum for a 10-liter fermentor (New Brunswick
Scientific, Edison, NJ) containing the medium previously described (1) supplemented with 0.05% (w/v) casamino acids, 0.2% (w/v) tryptone, 0.1% (w/v) yeast extract, 0.2 mM MgCl2, 5 µM CaCl2, 20 µg/ml
Fe2(SO4)3, and 50 mM
sodium succinate. Ampicillin (35 µg/ml) and kanamycin (50 µg/ml)
were included in all media. Cultures were grown with high aeration and
harvested at late exponential phase. Anaerobically grown cultures were
grown overnight in the same medium described above (minus succinate)
with 50 mM glycerol and 50 mM fumarate as
electron donor and acceptor, respectively (1). The heme-deficient strain, E. coli SASX41B (HfrP02A hemA41 metB1
relA1), a
-aminolevulenic acid (ALA) auxotroph (28), was
transformed with appropriate plasmids and grown in 50 ml of LB medium
in the presence of ampicillin (50 µg/ml) and ALA (50 µg/ml). Cells
were collected by centrifugation (10 min at 500 × g)
and gently resuspended in 20 ml of LB medium without ALA, and 1 ml was
used to inoculate 1 liter of LB medium. Cultures were then grown
aerobically in LB with ampicillin (35 µg/ml) or anaerobically in the
same medium with 50 mM glucose. When necessary, the medium
was supplemented with 100 µM ALA.
Preparation of Membrane Fraction and Enzyme
Purification--
Cells were collected by centrifugation, and the
membrane fraction enriched in SQR was isolated as previously described
(1) with the exception that the cells were disrupted by one passage with an EmulsiFlex-C5 homogenizer (Avestin, Inc., Ottawa, Ontario, Canada) at 18,000 p.s.i. at 4 °C. Membranes containing wild type or
mutant SQR were resuspended in 50 mM potassium phosphate,
0.2 mM EDTA (pH 7.2) to ~30 mg of protein/ml and frozen
at
70 °C. To purify wild type and mutant SQR, the membranes were
extracted with 2% (w/v) of the nonionic detergent Thesit
(polyoxyethylene 9-dodecyl ether) (Roche Molecular Biochemicals) as
previously described (1). The solubilized extract was then applied to a
HiLoad 26/10 Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech)
and eluted using a 600-ml linear gradient of 0.1-0.25 M
NaCl in 10 mM potassium phosphate (pH 7.2) with 0.05%
Thesit according to published procedures (24, 27). The brownish
fractions containing succinate dehydrogenase activity were pooled and
concentrated with Centriprep-30 concentrators (Amicon Inc., Beverly,
MA). The enzyme was washed with 100 mM potassium phosphate
(pH 7.0), and reconcentrated to 15-20 mg of protein/ml and stored at
70 °C.
Measurement of Enzyme Activity--
Activity measurements for
the succinate oxidase reaction were measured in the presence of 10 mM succinate in the assay cuvette as previously described
(29). The succinate-phenazine ethosulfate (PES) reaction in the
presence of dichlorophenolindophenol (DCIP) (
600 = 21.8 mM
1
cm
1, pH 7.8) was measured with 1.5 mM PES and 50 µM DCIP. To measure kinetic
parameters of succinate-quinone reductase activity of wild type and
mutant SQR, 30 µM Wurster's Blue (
612 = 12 mM
1
cm
1) was used as final electron acceptor with
varied amounts of quinone as previously described (29, 30).
Spectrophotometric Measurements--
Absorption spectra were
recorded at 25 °C with a Hewlett Packard 8453 diode array
spectrophotometer (Palo Alto, CA) in a 2-ml anaerobic cuvette. The
spectrum and concentration of cytochrome b556
attributed to purified and membrane-bound SQR enzymes was determined as
previously described (1, 24, 27). Spectra were routinely recorded of
membranes suspended in 50 mM potassium phosphate (pH 7.0),
0.2 mM EDTA at a protein concentration of 0.25 mg/ml.
Anaerobiosis was achieved by vacuum evaporation and saturation of the
buffer with oxygen-free argon.
EPR Spectroscopy and Redox Potentiometry--
EPR spectra were
recorded using a Bruker ESP300 spectrometer equipped with an Oxford
Instruments ESR-900 flowing helium cryostat. Samples were prepared as
described in Fig. 4. Potentiometric titrations were carried out
as previously described with 150-µl samples being extracted from the
tritrations into 3-mm internal diameter quartz EPR tubes (31, 32).
Titrations were carried out on membranes enriched in wild-type and
mutant enzymes at a protein concentration of ~30 mg/ml in 100 mM MOPS and 5 mM EDTA (pH 7.0). EPR spectra were recorded as described in Fig. 6 legend.
Analytical Methods--
Protein content in membranes was
determined by the Biuret method and in isolated enzymes by the method
of Lowry in the presence of 1% (w/v) SDS with bovine serum albumin as
a standard. The protoheme IX content of cytochrome b was
determined from the pyridine hemochromogen difference spectra
(dithionite-reduced minus oxidized) (
558-540 = 23.98 mM
1
cm
1) as described (33). The histidyl-flavin
concentration in purified SQR enzymes was determined as follows.
Purified protein (0.5-0.7 mg) was precipitated with 1 ml of cold
acetic acetone (8 µl of 6 M HCl per 1 ml of acetone) to
remove protoheme IX and the iron-sulfur clusters and then centrifuged
for 30 s in a microcentrifuge. The yellowish pellet was washed
three more times with the same volume of acidic acetone and suspended
in 0.8 ml of 0.1 M sodium phosphate (pH 7.0) with 1% (w/v)
SDS, and the precipitated protein was solubilized after 2 h at
38 °C. The spectrum of the resulting solution shows two peaks at 354 and 445 nm attributed to histidyl-riboflavin. The covalent flavin
concentration was determined using
445 = 12.0 mM
1 cm
1
for histidyl-riboflavin (34).
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RESULTS |
Anaerobic Expression of SQR Mutants--
It has been previously
shown that aerobic overexpression of SQR can be achieved in E. coli from plasmids that encode wild type SQR (24). Nevertheless,
it has been shown that anaerobic expression of SQR driven from the
frd promoter (PFRD) enables even higher levels
of SQR to be produced in the membranes of E. coli (1).
Therefore, to facilitate expression of site-directed mutant forms of
SQR and to aid in purification of the enzymes, constructs were cloned
into plasmid pFGS so expression could be driven by the PFRD
promoter. E. coli strain DW35, when transformed with pFGS,
is capable of growth under anoxic conditions on glycerol-fumarate minimal medium, indicating that wild type SQR can replace fumarate reductase in the anaerobic respiratory chain (1). It has been shown
that E. coli GV141 expressing SdhC H84L or SdhD H71Q mutant enzyme is able to grow aerobically on succinate minimal medium, indicating a functional complex II is formed (27). To test whether anaerobic growth is possible with these mutant SQR enzymes, DW35 containing either pFGS SdhC H84L or pFGS SdhD H71Q was grown
anaerobically on glycerol-fumarate minimal medium as previously
described (1). Both mutants supported anaerobic growth on
glycerol-fumarate minimal medium in E. coli DW35 with a
doubling time of 3.8 h for pFGS SdhC H84L and 3.2 h for pFGS
SdhD H71Q as compared with 3.0 h for wild-type SQR (data not
shown). These results indicate that a functional complex competent in
catalysis for the menaquinol-fumarate reductase reaction in
vivo is expressed from both the wild-type and mutant plasmids.
Properties of Isolated Mutant Membranes--
Previous studies with
aerobically grown E. coli cells encoding the SdhC H84L and
SdhD H71Q SQR mutants suggested that these substitutions resulted in
formation of catalytically active membrane-associated complexes that
lacked heme (27). Therefore, it was surprising that membranes isolated
from anaerobically grown DW35 cells transformed with both mutants
plasmids had an intense color. Membranes from SdhC H84L were brownish
red in color, similar to those from cells transformed with wild type
SQR plasmids. The membrane fraction from the SdhD H71Q mutant was
brownish green in color. The absorption spectra of membranes from
strain DW35 enriched in wild-type or mutant SQR complex show a
significant absorbance at the Soret region compared with membranes
obtained from an E. coli control strain (MC4100) transformed
with pBR322. MC4100, the parent strain of DW35 (15), contains
chromosomal copies of the sdh and frd operons and
under the anaerobic conditions used for growth does not express SQR.
Under the anoxic conditions used for growth, the heme containing
bd-oxidase is expressed (35) and contributes somewhat to the
absorbance at the Soret region in the membrane fraction. Fig.
1 shows the dithionite-reduced minus
air-oxidized difference spectra of membranes enriched in wild-type and
mutant SQR complexes as well as membranes from anaerobically grown
cells of E. coli MC4100. The spectra show a significant
-absorption at 558 nm and a broader
-absorption between 526 and
528 nm as well as the Soret absorption (~425 nm) characteristic of
room temperature difference spectra for cytochrome
b556 from SQR (36). Analysis of protoheme IX
content of the membranes by the pyridine hemochromogen method showed
similar cytochrome b concentrations for wild-type SQR and
both mutant SQRs (Table I). Mutant forms, however, show lower amplitudes at the Soret absorption and also changes
in the line shape of the spectrum (Fig. 1). Both mutants demonstrate
succinate-PES reductase and succinate-Q1 reductase activity; however, SdhC H84L showed at least two times lower
succinate-PES reductase activity than the wild-type or SdhD H71Q mutant
membranes. Moreover, the succinate-quinone reductase activity was even
lower in the SdhC mutant, and the ratio of Q1/PES
activities indicated that this mutant is significantly impaired in its
ability to interact with quinones.

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Fig. 1.
Light absorption difference
(dithionite-reduced minus air-oxidized) room temperature spectra of
membranes from E. coli strains grown under anaerobic
conditions. E. coli strain GV141 was transformed with
wild type and mutant SQR-encoding plasmids, and control strain MC4100
was transformed with pBR322 and grown anaerobically under the same
conditions. The spectra were determined in a 2-ml cuvette containing 1 mg of membrane protein/ml in 50 mM potassium phosphate (pH
7.2), and then 1-2 mg of solid sodium dithionite was added to reduce
the membrane suspension. The horizontal line for
each spectrum indicates zero absorbance for the individual spectra,
with the absorbance units shown on the vertical
axis on the left.
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Spectral Properties of Isolated SdhC H84L and SdhD H71Q
Mutants--
Previous data had suggested that the SdhD H71Q SQR
complex was less stable during purification. In the present studies,
however (using slight modifications of the original protocol; see
"Materials and Methods"), the chromatographic profiles for both SQR
mutants and the wild-type enzyme were identical. A single brownish peak with succinate dehydrogenase activity appears at the end of the 0.1-0.25 M NaCl gradient (data not shown). On the basis of
SDS-PAGE analysis, the purity of the wild-type and mutant SQRs also
appear identical (data not shown). Table
II indicates the protoheme IX content of
the isolated SQR enzymes. Comparing the ratio of covalent FAD to
protoheme IX content of the purified enzymes, it appears that the SQR
mutants show a 10-15% deficiency in protoheme IX compared with the
ratio of wild-type enzyme.
The absorption spectra of the wild-type and mutant SQR enzymes isolated
from the anaerobically grown E. coli cells are shown in Fig.
2. The oxidized cytochrome in wild-type
SQR shows a broad absorption at the
- and
-regions with the Soret
absorption at 412 nm. Incubation of wild-type enzyme with sodium
dithionite reduces the cytochrome completely within half a minute, and
an
absorption at 558 nm and a broad
absorption at 528 nm appear along with a sharp symmetrical Soret absorption at 424 nm. The air-oxidized spectrum for the SdhC H84L mutant SQR shows a broad absorption at 540 and 580 nm unlike wild-type SQR; however, the Soret
absorption shows a maximum at 411 nm with similar intensity to the
wild-type cytochrome b556. Complete reduction of
the cytochrome by dithionite in this mutant takes 4-5 min. The reduced
enzyme displays an
absorption at 559 nm and a broad
absorption
at 528 nm. The Soret absorption in the SdhC H84L mutant enzyme exhibits a maximum at 426 nm and a discernible shoulder at 445 nm, and its
absorption intensity is some 2-fold lower compared with wild-type SQR
(Fig. 2). The SdhD H71Q mutant SQR differs noticeably in color from
wild type or the SdhC H84L mutant SQR; it is less reddish and more
green-brown. As shown in Fig. 2, the oxidized spectrum of the SdhD H71Q
mutant SQR shows no pronounced peak in either the
or
regions,
whereas the Soret absorption is similar to wild-type and the SdhC
mutant SQR, although the maximum is shifted to 407 nm. The SdhD H71Q
enzyme could be slowly reduced with dithionite, similar to the results
with the SdhC mutant. The spectrum of the reduced SdhD mutant shown in
Fig. 2 shows an
absorption at 560 nm, a broad absorption at the
region, and a Soret absorption at 423 nm similar in intensity to that
of SdhC H84L.

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Fig. 2.
Visible light absorption spectra of purified
wild-type and mutant SQR complex. The oxidized spectrum (as
isolated) trace is shown by the dashed line and
the reduced enzyme is shown by the solid line.
The spectra of the oxidized enzymes were scanned at 25 °C in a 1-ml
cuvette in 50 mM potassium phosphate, 0.05% (w/v) Thesit
at a protein concentration of 2.65 µM heme b.
The dithionite-reduced (solid line) spectra were
recorded following reduction of the enzyme with 1-2 mg of solid sodium
dithionite after incubation for 5 min. The and absorbance
(500-700 nm) on the right is shown at 3 times the
gain of the Soret absorbance (380-500 nm).
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Reduced cytochrome b556 from E. coli
SQR or the isolated SdhCD domain does not react with carbon monoxide
(36), typical of low spin hexacoordinated hemes. As shown in Fig.
3, carbon monoxide causes noticeable
alterations in the absorption spectra of both the SdhC and SdhD
mutants. The Soret absorption is shifted to 422 nm in SdhC H84L and 423 nm in the SdhD H71Q mutant with a comparable increase in the absorption
intensity and a more symmetrical shape to the Soret absorption. The
absorption was unaffected in the SdhC mutant, whereas a decrease in
absorption intensity is found in the SdhD mutant similar to that seen
in the isolated cytochrome domain of beef heart succinate dehydrogenase
(22). One interpretation of this data is that both mutations result in
a change of ligation of the heme b556 from hexa-
to pentacoordinate (viz., from low to high spin).

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Fig. 3.
Effect of carbon monoxide on the visible
absorption spectra of purified SdhC H84L and SdhD H71Q SQR
enzymes. The dithionite-reduced (solid line)
and dithionite-reduced with carbon monoxide (dashed
line) visible absorption spectra are shown. Conditions are
the same as in Fig. 2 with the cuvette flushed with carbon monoxide gas
for 2 min. CO has no effect on the wild type with the spectrum
identical to that seen in Fig. 2 (top
panel).
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In order to further investigate potential spin state changes elicited
by the SdhC H84L and SdhD H71Q mutations, we subjected oxidized
membranes enriched in these mutant enzymes to EPR analysis. Fig.
4A shows EPR spectra around
g = 2 that arise primarily from [3Fe-4S] clusters. Comparison of
the spectrum shown in Fig. 4A (i) (background
strain, DW35) and those of Fig. 4A (ii-iv)
(overexpressing wild-type and mutant enzyme) indicates that high levels
of the SdhB [3Fe-4S] cluster can be detected in the overexpressed
wild-type and mutant enzymes. Fig. 4B shows equivalent
spectra recorded around g = 6. Noticeable in the spectrum of the
background strain (Fig. 4B (i)) is a typical high
spin heme spectrum that probably arises from pentacoordinate hemes such
as those found in cytochromes bo3 (37) and
bd (28). In membranes containing overexpressed wild-type SQR
(Fig. 4B (ii)), there is a diminution of the
g = 6 signal compared with that observed in the background strain. This is likely to be due to the dilution of the proteins responsible for the background signal of Fig. 4B (i) by the
overexpressed wild-type SdhCDAB, which contains no high spin heme. The
spectrum of membranes enriched in SdhC H84L (Fig. 4B
(iii)) is essentially identical to that of membranes
containing wild-type SdhCDAB. The spectrum of membranes enriched in
SdhD H71Q, however, has an intense signal at g = 6.0, indicative
of the presence of elevated amounts of pentacoordinate heme. Given that
wild-type SdhCDAB contains hexacoordinate heme
b556, it is likely that the intense g = 6 signal arises from the loss of one of the histidine imidazole ligands
of this heme, in agreement with the optical data presented herein.
Given that no significant increase in g = 6.0 signal intensity is
observed in the SdhC H84L mutant, we also looked for low spin heme
spectra in samples containing wild-type and mutant enzymes (Fig.
4C). Noticeable in the spectra of membranes lacking
overexpressed enzyme (E. coli DW35, Fig. 4C
(i)) is a peak at gz = 3.3 similar to that
assigned to heme b558 observed in spectra of
membranes containing the cytochrome bd ubiquinol oxidase
(28, 38). A broad peak is observed between g = 3.65 and g = 3.50 in spectra of membranes containing overexpressed wild-type enzyme
(Fig. 4C (ii)). This feature appears to be
essentially identical to the spectrum reported for the gz
feature of low spin heme b556 in purified
E. coli SQR (36). The spectrum of the SdhC H84L mutant lacks
the g = 3.33 and g = 3.76-3.50 features and instead contains a distinct peak at gz = 2.92, indicating that in this
mutant the heme remains low spin in its oxidized state but has a
significantly altered environment compared with the wild type.

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Fig. 4.
[3Fe-4S] cluster EPR spectra
(A), high spin heme spectra (B), and
low spin heme spectra (C), of membranes
enriched in SdhCDAB, SdhCH84LDAB, and
SdhCDH71QAB. Illustrated are EPR spectra of membranes
containing no SdhCDAB (i) (E. coli DW35), SdhCDAB
(ii), SdhCH84LDAB (iii), and
SdhCDH71QAB (iv). Samples were prepared in 100 mM MOPS and 5 mM EDTA (pH 7.0). Oxidation was
achieved by incubation in the presence of 0.8 mM dichlorophenolindophenol for 5 min. Spectra were
normalized to a protein concentration of 30 mg of protein per ml and
were recorded under the following conditions: temperature, 12 K;
microwave power, 20 milliwatts at 9.47 GHz; modulation amplitude, 10 Gpp. For A and B, only a single scan was
necessary; five scans were accumulated for C.
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Kinetic Properties of Isolated Enzymes--
The ratio of quinone
reductase activity to that with artificial electron acceptors such as
PES and DCIP has been shown to indicate the ability of complex II to
interact with quinones (2). Both mutants showed catalytic activity with
PES and Q1, although the SdhC H84L mutant showed a 4-fold
lower turnover number in its ability to reduce Q1 (Table
II). There was also an increase in the Km for
Q1 in the SdhC H84L mutant, whereas the Km was similar to wild-type enzyme for the SdhD H71Q mutant. Interestingly, the quinone site competitive inhibitor pentachlorophenol (PCP) (29) showed an increased Ki for the SdhC mutant, and there was no change of the
Ki for the SdhD mutant.
The diheme cytochrome b of the B. subtilis SQR
complex has its absorption spectrum perturbed by the addition of the
quinone site inhibitor 2-n-heptyl
4-hydroxyquinoline-N-oxide (HQNO) (39). Although HQNO is a
potent inhibitor of B. subtilis SQR and E. coli
QFR, it does not inhibit E. coli SQR (29). The effects of
quinone site inhibitors on the absorption spectra of E. coli SQR cytochrome b have not been reported, so it was of
interest to determine if an inhibitor like PCP affected the absorption spectrum. As shown in Fig. 5, PCP does
affect the absorption spectrum of the Soret and
absorptions of wild
type E. coli SQR. PCP induced a shift in the difference
spectrum of wild type SQR so that the Soret absorption shifted with
max = 421 nm,
min = 432 nm, and 
max-min of about 10.2 mM
1
cm
1. Similar effects on difference spectra of
the Soret region have been seen after treatment of B. subtilis SQR (39) and mitochondrial cytochrome
bc1 complex with HQNO (40). The change in the
Soret region induced by PCP was accompanied by shifts in
and
absorptions. There was a decreased absorption in the
absorption
with a broad minimum at 530-540 nm. The
absorption was
blue-shifted with a
max = 556 nm and a
min = 564 nm and a significant increase in the
extinction (
max-min ~3.8
mM
1
cm
1). The Kd for PCP was
determined based on the absorbance changes (564-556 nm) induced upon
the addition of the inhibitor. The value obtained of 30 µM for the wild-type enzyme corresponds well to the
KiPCP for reduced SQR (29). The SdhCD
cytochrome b domain can be isolated independently from the
soluble SdhAB domain (36). When incubated with PCP, the isolated
wild-type SdhCD subunits show difference spectra identical to that seen
in the intact complex (Fig. 5). The similar spectral changes in both
the SQR complex and the isolated SdhCD subunits caused by PCP suggest
that the inhibitor binds near to the cytochrome b in SQR.
The effect of PCP on the mutant forms of SQR was also examined. The
dithionite-reduced difference spectrum of the SdhD H71Q enzyme
incubated with PCP is similar to that seen for the wild-type enzyme.
The Soret absorption, however, shows no detectable maximum, and
min = 425 nm, maximum (
= 8.7 mM
1
cm
1). There was only a very slight
absorption, but the
absorption was blue-shifted like wild type with
a
min = 562 nm (
= 1.9 mM
1
cm
1). It was not possible to reliably
determine the KdPCP for the SdhD H71Q
enzyme due to the instability of the mutant enzymes in the reduced
state (see below). By contrast to wild-type and SdhD H71Q SQR, PCP had
very little effect on the difference spectrum of the SdhC H84L enzyme
(Fig. 5). There was a very broad absorbance with a minimum at 425 nm
and a minor absorbance change at 560-565 nm. These data along with the
changes in Ki and Km (Table II)
suggest that the association of PCP with the enzyme has been affected
in SdhC H84L.

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Fig. 5.
Effect of pentachlorophenol on the visible
absorption difference (dithionite-reduced minus dithionite-reduced plus
PCP) spectrum of cytochrome b in purified wild-type
and mutant E. coli SQR enzymes. Shown are
wild-type SQR (dashed line), SdhC H84L
(dotted line), and SdhD H71Q (solid
line). Conditions of the reduction are the same as for Fig.
2. After complete reduction of the enzyme with dithionite, PCP in
ethanol was added to a final concentration of 0.4 mM, and
the spectra were recorded. The control cuvette contained the same
concentration of ethanol, and the absorbance of 0.4 mM PCP
in the presence of dithionite was subtracted from the difference
spectrum.
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Redox Properties of the Heme--
The favorable redox potential of
cytochrome b556 of wild type E. coli
SQR (Em = +36 mV) allows complete reduction by
succinate under anaerobic conditions (36). By contrast, the cytochrome in the mutant SQRs investigated in this study are only partially reduced by succinate under anaerobic conditions. The SdhC H84L heme is
only reduced some 20% (compared with dithionite reduction) after 12 min (pH 7.2, 25 °C), and the SdhD H71Q mutant is reduced about 30%
using the same conditions (data not shown). Overall, these results
suggest that there is a thermodynamic or kinetic block in electron
transfer to the heme in both mutants. One possible explanation for this
is that the Em,7 of the heme is significantly lowered in these mutants.
In order to determine if the heme present in the two mutants does have
a lower midpoint potential (Em,7) than
the low spin heme of the wild-type enzyme, we subjected the two mutant and wild-type enzymes to potentiometric analysis in combination with
EPR spectroscopy. Fig. 6A
shows that the high spin heme signal of membranes containing wild-type
SdhCDAB titrates with an Em,7 of
210
mV, consistent with this signal arising from high potential pentacoordinate hemes present in either cytochrome
bo3 (37) or cytochrome bd (28). The
g = 6 signal from membranes enriched in SdhD H71Q titrates as a
single species with an Em,7 of
approximately
97 mV. Potentiometric titration of the g = 2.92 signal of the SdhC H84L mutant reveals that its
Em,7 is approximately +15 mV. Similarly,
analysis of the g = 6.0 signal of membranes enriched in this
mutant reveals that it titrates with two components, one major one at
Em,7 =
210 mV and a minor one at
Em,7 = +15 mV. However, given the low
concentration of high spin heme in membranes enriched in this mutant,
it is unlikely that the high spin Em,7 = +15 mV component contributes significantly to the analyses reported herein (cf. Figs. 4B and 6B). Overall,
the data suggest that the heme b556 is
overwhelmingly in a low spin hexacoordinate state in membranes enriched
in the SdhC H84L mutant.

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Fig. 6.
Potentiometric titrations of high and low
spin signals attributable to wild-type and mutant enzyme.
A, comparison of potentiometric titrations of the g = 6.0 high spin signals from membranes enriched in wild-type and SdhD
H71Q enzyme. , titration of the g = 6.0 (high spin heme) from
membranes enriched in the SdhD H71Q mutant
(Em,7 = 97 mV); , titration of the
g = 6.0 signal from membranes enriched in wild-type enzyme
(Em,7 +210 mV). B,
comparison of potentiometric titrations of the g = 2.92 low spin
( ; Em,7 = +15 mV) and the g = 6.0 high spin ( ; Eh = +15 mV (33%) and 210 mV
(67%)) signals of membranes enriched with the SdhC H84L mutant. Data
were obtained from EPR spectra recorded as described in the legend to
Fig. 4, except that the microwave power used was 2 milliwatts, and
three scans were accumulated for titration of the g = 2.92 signal.
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In order to investigate the possibility that changes in the
coordination of heme b556 have any effect on the
properties of the [3Fe-4S] cluster located in SdhB, we also
determined the Em,7 of the [3Fe-4S]
cluster in membranes enriched in wild-type, SdhC H84L, and SdhD H71Q
SQR. The Em,7 values for the [3Fe-4S] cluster were determined to be +75, +65, and +83 mV for the wild type
and SdhC H84L and SdhD H71Q mutants, respectively (data not shown).
These values are in reasonable agreement with those previously reported
for the SdhB [3Fe-4S] cluster (Em = +65 mV
(41)).
Stability of the Mutant Enzymes--
Both the SdhC H84L and SdhD
H71Q mutant enzymes in the oxidized state and neutral pH remain
catalytically active for several days at 4 °C; however, incubation
at 30 °C and pH 7.8 results in inactivation of both mutants (Fig.
7A). As seen in Fig. 7, the
quinone reductase activity is lost at about twice the rate of the
succinate oxidase activity. The succinate oxidase activity measured
with PES/DCIP decreased with higher pH and temperature. Incubation of
the SdhD H71Q mutant enzyme with 10 mM succinate increased
the rate of inactivation of the quinone reductase activity some
10-fold; however, succinate oxidase activity was affected to a lesser
extent (Fig. 7B). The inset in Fig. 7B
shows the amplitude of the dithionite-reduced signal attributable to
the heme during incubation of the SdhD H71Q enzyme with succinate. The
decrease of the signal of the reduced heme b556,
but not its spectral nature, indicates the release of protoheme IX from
apocytochrome. The rate of release of heme b is more rapid
in aerobic than anaerobically incubated enzyme, and the loss of heme
directly correlates with the decrease of succinate-Q1
reductase activity. The correlation of the rapid loss of quinone
reductase activity with the decrease in the spectral signal for
cytochrome b556 suggests that the dissociation of SdhAB caused the loss of protoheme IX from SdhCD.

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Fig. 7.
Kinetics of inactivation of SdhC H84L and
SdhD H71Q mutants and wild type SQR during incubation at 30 °C at pH
7.8. Purified wild-type and mutant SQRs (at a concentration of 50 µM enzyme based on cytochrome b content) were
activated with 10 mM malonate at pH 7.0 as described (29)
and then diluted with 50 mM potassium phosphate (pH 7.8) to
2 µM concentration of b556 and
incubated at 30 °C in the absence (Fig. 7A) or presence
(Fig. 7B) of 10 mM succinate. A,
succinate-Q1 reductase activity ( and ) and
succinate-PES reductase activity ( and ) of purified mutant SdhC
H84L (open symbols) and SdhD H71Q
(closed symbols) enzymes.
Succinate-Q1 reductase activity for control wild-type SQR
purified enzyme ( ) is also shown in A. B, the
affect of anaerobic versus aerobic incubation on the SdhD
H71Q mutant purified enzyme. SdhD H71Q was incubated with 10 mM succinate aerobically (filled
symbols) and anaerobically after saturation of the enzyme
incubation mixture with argon (open symbols).
Succinate-Q1 reductase activity for SdhD H71Q ( and )
and succinate-PES reductase activity ( ) are shown. Inset,
the effect of 10 mM succinate on the dissociation of the
heme b from SdhD H71Q during incubation at 30 °C under
aerobic ( ) and anaerobic ( ) conditions. The amplitude of the
difference of the dithionite-reduced spectra at 559-575 nm shows the
loss of the heme from the enzyme.
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Effect of Growth Conditions on Heme Assembly--
The data
reported above indicates that pentacoordinated heme is assembled in the
SdhC H84L and SdhD H71Q mutants when grown anaerobically in strain DW35
or GV141. Previous studies using these same mutants expressed in GV141
had suggested that heme was not assembled in the mutants, although SQR
assembled in the membrane and was functionally active (27). In these
studies, SQR was expressed aerobically, conditions that are different
from those in the current paper. Therefore, wild-type SQR and the SdhC H84L and SdhD H71Q mutant enzymes were expressed aerobically in minimal
medium with succinate in strain GV141 or DW35. The content of
cytochrome b in membranes isolated from the aerobically
grown cells was 1.1 nmol/mg of protein for the SdhC H84L mutant and 1.5 nmol/mg of protein for the SdhD H71Q mutant for strain GV141 (data not
shown). This is less than half the amount found in anaerobically grown
GV141 used in the current studies (see Table I). Even higher levels of
heme were found in the membranes from aerobically grown DW35 containing
the mutant plasmids, consistent with previous results showing higher
expression levels for SQR in this strain (1). The enzyme isolated from
membranes of aerobically grown cells was found to have identical
catalytic properties, protoheme IX content, and spectral and redox
characteristics as to the enzyme from anaerobically grown cells (data
not shown).
Previous studies with E. coli SQR had shown that assembly of
the enzyme was perturbed when the sdhCDAB genes were
introduced into a heme synthesis mutant (26), suggesting the importance of heme in the assembly of complex II. These results were consistent with those found for B. subtilis complex II (25). The data
suggesting that a heme did not assemble in E. coli SQR in
the SdhC H84L and SdhD H71Q mutants but that a functional enzyme
complex was formed (27) were thus not consistent with the results using
heme-deficient mutants. To reinvestigate this question in light of the
above results, a heme-deficient strain of E. coli (SASX41B)
(42) was transformed with the SdhC H84L- and SdhD H71Q-encoding
plasmids. The strain is unable to grow aerobically on LB medium unless
supplemented with ALA or with a fermentable substrate such as glucose.
In agreement with the results reported by Nakamura et al.
(26), it was found that SQR could not assemble in the membrane in
aerobically grown cells (data not shown). This was found for either
wild-type or mutant forms of SQR unless the medium was supplemented
with ALA. Anaerobic growth of SQR using the PFRD promoter
results in a higher yield of membrane bound SQR (1), so the effect of
anaerobic growth in the heme-deficient strain was investigated. The
same results were found; i.e. unless E. coli
SASX41B grown anaerobically with glucose was supplemented with ALA, no
assembled SQR was found in the membranes of the cell based on catalytic
activity using either wild-type or mutant SQR plasmids for expression
of the enzyme. (Anaerobically, fumarate reductase is expressed from the chromosomal copy of the frd operon in E. coli
SASX41B. The fumarate reductase activity in the membrane can be
discriminated from the succinate quinone reductase activity of SQR
based on the sensitivity of fumarate reductase to HQNO, an inhibitor to
which SQR is insensitive (29).) These results are consistent with the
requirement of heme for assembly of SQR and based on the data
presented in Tables I and II and Figs. 2, 3, and 5 indicating
that heme is assembled in the SdhC H84L and SdhD H71Q mutants.
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DISCUSSION |
The role of the heme in SQR has yet to be established. It has been
suggested that the heme has a structural and/or catalytic role in the
function of Complex II (4, 21). The structural role for the heme in
B. subtilis SQR has been well documented (4, 25), and the
majority of available evidence has also suggested such a structural
role for E. coli SQR (26). The recent crystal structure of
the diheme W. succinogenes QFR shows that heme
bH (equivalent to the single heme of
E. coli SQR) has amino acid side chains from four of the
five
-helices in the membrane domain that aid in binding the heme
(12). This suggests the importance of the heme in the assembly and
structure of the complex. The results obtained with mutants of the
histidyl ligands of the heme in E. coli SQR that suggested
that the enzyme assembled and functioned in the membrane, in the
absence of heme, were therefore not entirely consistent with the other
available data (27). The results reported in this study show that heme
is indeed assembled in the SdhC H84L and SdhD H71Q enzymes. In the case
of the SdhC H84L mutant, the heme appears to be retained in a
hexacoordinate low spin form, with a gz = 2.92, whereas in
the SdhD H71Q mutant, the heme appears to become pentacoordinate and
high spin with a gxy at 6.0. For the latter mutant, the
Em,7 is also significantly lowered to
approximately
97 mV. In the case of the SdhD H71Q mutant, it is not
surprising that the high spin heme is able to bind CO. However, CO
binding also occurs in the SdhC H84L mutant, indicating that it is able
to displace the ligand that presumably replaces the imidazole nitrogen
of His84. The results reported here are in agreement with
those of others that show that the heme (26), when present, is
important for proper assembly of complex II.
The retention of low spin heme in the SdhC H84L mutant suggests that
His84 might not be the heme ligand or that another residue
within SQR can serve as an axial ligand for heme
b556. Given the results of sequence alignments
of complex II from many sources (4, 20) and the high resolution
structure of the heme containing W. succinogenes QFR (12),
it is unlikely, however, that an SdhC residue other than SdhC
His84 provides one of the ligands to heme
b556 in the wild-type enzyme. This conclusion is
also supported by the site-directed mutagenesis studies of E. coli SQR, where substitution of histidyl residues in the membrane
binding domain suggests that SdhC His84 and SdhD
His71 are the axial ligands to the heme (16, 27). An
example of the swapping of the heme axial ligands has been observed in
both beef (43) and E. coli SQR (26), when the small membrane
anchor subunit QPs3 or SdhD respectively, is expressed alone. In these examples, it appears that the histidyl ligands come from two different molecules of the respective small subunit (26, 43). In this study, the
observation of a gz at 2.92 (Fig. 4) is also typical for a
heme with bis-histidine ligation with a small interplanal angle between
the planes of the ligating imidazoles (44, 45). This suggests that the
replacement ligand is another residue, possibly His91 or
His30 from SdhC or alternatively His14 of SdhD.
Future site-directed mutagenesis experiments will address this
question. Such ligand displacement has also been observed in the
CO-sensing CooA protein from Rhodospirilium rubrum (46).
The reason for the discrepancy between the results reported here and
the previous results (27) with the SdhC His84 and SdhD
His71 mutants is not entirely clear. In the case of the
SdhD H71Q mutant, the succinate-reduced
TMPD/ascorbate-reduced
difference spectrum would not be expected to show the presence of the
heme because, as the present studies document, the heme is of much
lower potential. In the case of the SdhC H84L mutant, there may be a
blockage in electron transfer from the [3Fe-4S] cluster to the heme,
possibly as a result of disruption of the quinone binding site. More
difficult to explain is the reported inability to detect heme extracted from purified SdhC H84L and SdhD H71Q mutant SQR (27). As shown in this
paper (Fig. 7) and as noted in the previous study (27) the mutant SQR
complexes are less stable than wild type and are particularly sensitive
to temperature and aeration. Therefore, it is conceivable that in the
previous study the heme could have been lost during the purification
procedure, resulting in the inability to detect it in the final samples
obtained from the chromatographic column. Nevertheless, as shown in the
current study, heme is present in the purified mutant enzymes whether it is produced from anaerobically or aerobically grown E. coli.
The results in Table II and Fig. 5 show that PCP affects the ability of
SQR to interact with quinones and perturbs the heme environment. In
Table II, it can also be seen that the SdhC H84L enzyme is much more
severely affected in its ability to interact with Q1 than
is the SdhD H71Q mutant. Also, the inhibitor PCP shows a 5-fold
increase in its Ki in the SdhC H84L mutant, whereas
the SdhD H71Q enzyme shows no change in inhibition. These results are
consistent with SdhC His84 being part of the quinone binding site, in
addition to being a ligand of the b heme of SQR. Alternatively, the apparent ligand displacement observed might result
in the disruption of the quinone binding site being a secondary effect;
i.e. whichever residue replaces SdhC H84L may in fact be
essential for ubiquinone binding and oxidation. Azidoquinones have been
used to label the SdhC subunit of SQR and have implicated Ser27 and Arg31 of SdhC as being part of a
quinone binding site in the enzyme (16). Although the primary sequence
of the membrane anchor subunits of complex IIs are not highly
conserved, the available structures and models (11, 12, 20, 21) all
suggest a very similar transmembrane topology. In two subunit membrane
anchors, like E. coli SQR, this would place SdhC
His84 in helix II on the cytoplasmic membrane face and on
the opposite side of a pocket from SdhC Ser27 and SdhC
Arg31 as previously suggested (16). The structure of QFR
from E. coli shows two quinone binding pockets on the
opposite side of the membrane (11), and SdhC His84 would be
localized near the Qp (quinone-proximal) binding site. PCP
perturbs the heme environment in E. coli SQR similar to that seen with HQNO in B. subtilis SQR, and it has been suggested
for B. subtilis SQR that the cytochrome participates in
binding and stabilization of the semiquinone generated during electron
transfer in the enzyme (39). The semiquinone radical attributed to the Qp site demonstrates rapid relaxation behavior during EPR
analysis, and this has been attributed to a relaxation pathway
involving the heme and the [3Fe-4S] center of the enzyme, suggesting
that they are near to the Qp site (47). The need for proper
assembly of the heme to maintain the integrity of the quinone binding
sites is also supported by the data in Fig. 7 showing that alteration of the histidyl ligands affects the stability of the enzyme. The more
rapid loss of quinone reductase activity, as compared with succinate-PES reductase activity, suggests that if the heme is lost
upon incubation of the mutant enzymes then the catalytic SdhAB subunits
dissociate from the enzyme. Taken together, these data are consistent
with Qp being located near the [3Fe-4S] cluster in the
SdhB subunit and near the SdhC His84 histidyl ligand of
cytochrome b of SQR.
These studies do not directly address the role of the heme in electron
transfer in complex II. QFR from E. coli catalyzes with
similar efficiency the same reactions as SQR and does so in the absence
of heme. This suggests that heme is not essential for electron
transfer, but the results presented demonstrate that heme is present in
SdhC H84L and SdhD H71Q mutants and that the mutations also have an
effect on quinone reductase activity. This leaves open the possibility
that heme is directly involved in reaction with quinone/quinol. A
mutation in the human SdhD gene equivalent to the E. coli
SdhD His71 residue results in hereditary paraganglioma
(48). As shown in Fig. 6, such a mutation may drop the redox potential
of the heme b by more than 100 mV. If SQR heme acts as a
sensor for the redox state of the quinone pool or as an oxygen sensor,
the hypoxic phenotype observed (48) would be consistent with a lowered
potential of human heme b. Structural information for SQR
and further characterization of the cytochrome b should help
resolve these issues.