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INTRODUCTION |
Bovine heart mitochondrial succinate:ubiquinone
(Q)1 reductase, also known as
complex II, which catalyzes electron transfer from succinate to
ubiquinone, has been purified and characterized (1-3). Purified
reductase shows five protein bands (4) in a high resolution sodium
dodecylsulfate-polyacrylamide gel electrophoretic (SDS-PAGE) system,
with apparent molecular masses of 70, 27, 15, 13, and 11 kDa. The
reductase contains five prosthetic groups: one covalently linked FAD,
three iron-sulfur clusters (2Fe-2S, 4Fe-4S, and 3Fe-4S), and cytochrome
b560. The larger two subunits (Fp & Ip) are
succinate dehydrogenase and house the FAD and the three-iron sulfur
clusters, respectively. The smaller three subunits (QPs1, QPs2, and
QPs3) are membrane-anchoring proteins. It is still unknown which of
these three membrane-anchoring subunits house cytochrome
b560 heme.
Bovine heart mitochondrial succinate:Q reductase has been resolved into
two reconstitutively active fractions: soluble succinate dehydrogenase
(5) and the membrane-anchoring fraction (QPs) (6). Purified succinate
dehydrogenase can catalyze electron transfer from succinate to
artificial electron acceptors such as phenazine methosulfate but not to
its physiological electron acceptor, Q. Addition of QPs to succinate
dehydrogenase reconstitutes membrane-bound succinate:Q reductase, which
catalyzes TTFA-sensitive electron transfer from succinate to Q,
indicating that QPs provide membrane docking for succinate
dehydrogenase and Q-binding for the reductase.
The involvement of QPs in the Q-binding of succinate:Q reductase is
further supported by the detection of ubisemiquinone radicals in intact
or reconstituted succinate:Q reductase formed from QPs and succinate
dehydrogenase but not in succinate dehydrogenase alone (7).
Furthermore, when succinate:Q reductase is photoaffinity-labeled with
an azido-[3H]Q derivative, radioactivity is found in the
QPs subunits but not in the succinate dehydrogenase subunits (4). The
radioactivity distribution is 45, 22, and 25% in QPs1, QPs2, and QPs3,
respectively (4).
The Q-binding domains in QPs1 and QPs3 have been identified as residues
113-140 and 29-37, respectively, by matching the sequences of
azido-Q-linked peptides to their respective protein sequences. The
amino acid sequences of QPs1 and QPs3 are obtained from cloning and
nucleotide sequencing of the cDNAs (4, 8, 9) encoding these two
proteins. The Q-binding domain in the proposed model of QPs1 is located
at the connecting loop between transmembrane helices II and III toward
the matrix side (4). The Q-binding domain in the proposed model of QPs3
is located at the end of transmembrane helix I toward the C side of the
mitochondrial inner membrane (9). Location of Q-binding domains of QPs1
and QPs3 on opposite sides of the membrane is in line with a
two-Q-binding site hypothesis formulated from inhibitor binding studies
of this enzyme complex (10).
Isolated QPs contains 27 nmol of cytochrome
b560/mg of protein. The role of this cytochrome
in beef succinate:Q reductase is controversial. Because cytochrome
b560 in succinate:Q reductase is not reduced by
succinate and because it is present in a substoichiometric amount with
respect to FAD, its direct involvement in succinate:Q reductase
catalysis has been ruled out by some investigators (6). On the other
hand, it has been proposed (11) that cytochrome b560 functions as a mediator between low
potential Fl/Fl· and Q/Q· couples in a dual pathway model
of electron flow through cardiac succinate:Q reductase. Despite its
rather unclear catalytic role, the involvement of cytochrome
b560 in the binding of succinate dehydrogenase
to QPs is clearly indicated by restoration of the absorption
properties, redox potential, and EPR characteristics of cytochrome
b560 in QPs during formation of TTFA-sensitive
succinate:ubiquinone reductase from isolated QPs and succinate
dehydrogenase (6).
The ligand for cytochrome b in succinate:Q reductase from
beef heart mitochondria (b560) and
Escherichia coli (b556) has been identified as bishistidine (12, 13). Both the membrane-anchoring subunits (SdhC and SdhD) in the E. coli enzyme are involved
in heme ligation of cytochrome b556
(14). His-84 of the SdhC and His-71 of the SdhD were
identified as ligands for cytochrome b556 (15).
However, information about amino acid residues involved in the
bishistidine ligand of bovine cytochrome b560 is lacking.
A better understanding of the structure-function relationship of
succinate:Q reductase, especially of the amino acid residues involved
in Q-binding, heme b560 ligation, and succinate
dehydrogenase docking, requires functionally active QPs subunits. There
are two ways to obtain purified QPs subunits: one is by biochemical resolution of QPs into their individual subunits; the other is by gene
expression to generate recombinant QPs proteins. The availability of
the cDNA for QPs3 (9) in our laboratory together with our past
experience in overexpressing the small molecular weight Q-binding proteins of mitochondria (16, 17) and Rhodobacter
sphaeroides (18) in E. coli encouraged us to obtain
purified QPs3 by the gene expression approach. The pGEX expression
system was used because it allows one-step purification of recombinant
fusion protein with glutathione-agarose gel. Herein we report the
construction of the expression vector, pGEX/QPs3, growth conditions for
overexpression of the active soluble form of GST-QPs3 fusion protein in
E. coli JM109 and properties of recombinant QPs3. The
Q-binding function of recombinant QPs3 is established by its ability to
cause a spectral blue shift of ubiquinone. The heme
b560 ligating property of recombinant QPs3 is
established by its ability to restore the spectral properties of
cytochrome b560 upon addition of hemin chloride.
The amino acid residues of QPs3 involved in Q-binding and heme ligation were identified by site-directed mutagenesis.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes, T4 polynucleotide kinase, T4
DNA polymerase, and T4 DNA ligase were obtained either from Promega or
Life Technologies Inc. Plasmid and fragment isolation kits were
obtained from Qiagen. Nitrocellulose membranes were from Schleicher & Schuell. Bovine serum albumin,
isopropyl-
-D-thiogalactopyranoside (IPTG), D-glucose, ampicillin, tetracycline,
-aminolevulinic
acid, ferrous sulfate, gelatin, hemin chloride, sorbitol, betaine,
glutathione (reduced form), glutathione-agarose beads, thrombin,
leupeptin, phenylmethylsulfonyl fluoride, and 2,6-dichlorophenol
indophenol were from Sigma. Agarose, acrylamide, bisacrylamide,
horseradish peroxidase color development reagent, protein A horseradish
peroxidase conjugate, and Bradford reagent were obtained from Bio-Rad.
LB agar, LB broth base, SELECT peptone 140, and SELECT yeast extract were from Life Technologies Inc. Dodecyl maltoside was from Anatrace. Antibodies against the QPs3-connecting peptide and GST-QPs3 were raised
in rabbits and purified as previously reported (9). Oligonucleotides
were synthesized in the Recombinant DNA/protein resource facility at
Oklahoma State University.
Bacterial Strains and Plasmids--
E. coli
strain INV
F' was used as host for pCR2.1 vector (Invitrogen);
E. coli JM109 or DH5
was used as the host for pSelect (Promega) and pGEX2T (Amersham Pharmacia Biotech).
DNA Manipulation and DNA Sequencing--
General molecular
genetic techniques were performed according to procedures described in
Sambrook et al. (19). DNA sequencing was performed with an
Applied Biosystems model 373 automatic DNA sequencer at the recombinant
DNA/protein resource facility at Oklahoma State University.
Construction of E. coli Strains Expressing Wild-type and Mutant
QPs3--
The 331-base pair BamHI-EcoRI cDNA
fragment encoding mature QPs3 was amplified from a bovine heart
cDNA library by polymerase chain reaction using two
synthetic primers, 5'-GGATCCTCTGGTTCCAAG-3' (the sense
primer) and 5'-GAATTCTAAAAGGTCAGAGC3' (the antisense primer). This fragment was cloned into pCR2.1 vector and confirmed by
sequence analysis before being subcloned into the BamHI and EcoRI site of pGEX2T vector to generate pGEX/QPs3. E. coli transformants producing the GST-QPs3 fusion protein were
identified by immunological screening of colonies with antipeptide QPs3
antibodies (9). Both E. coli strains JM109 and DH5
were
found to be suitable hosts for pGEX/QPs3.
QPs3 DNA mutations were generated by site-directed mutagenesis using
the Altered SitesTM Mutagenesis system from Promega. A
342-base pair EcoRI fragment was excised from pCR2.1/QPs3
plasmid (9) and cloned into the EcoRI site of pSELECT-1
vector to generate pSELECT/QPs3. The single-stranded pSELECT/QPs3 was
used as the template in the mutagenesis reactions. The mutagenic
oligonucleotides used were as follows: H9N, 5'-GCATCTCTCAACTGGACTGGT; H9Y, 5'-GCATCTCTCTACTGGACTGGT; H9D, 5'-GCATCTCTCGACTGGACTGGT; H46N,
5'-CTCACTCTTAACAGTCACTGG; H46Y, 5'-CTCACTCTTTACAGTCACTGG; H46D,
5'-CTCACTCTTGACAGTCACTGG; H48N, 5'-CTTCACAGTAACTGGGGCATT; H48Y,
5'-CTTCACAGTTACTGGGGCATT; H48D, 5'-TTCACAGTGACTGGGGCATT; H60N,
5'-GACTATGTTAATGGAGATGCA; H60Y, 5'-GACTATGTTTATGGAGATGCA; H60D,
5'-GACTATGTTGATGGAGATGCA; H89N, 5'-TTCAACTATAATGACGTGGGC; H89Y,
5'-TTCAACTATTATGACGTGGGC; H89D- 5'-TTCAACTATGATGACGTGGGC; S33A,
5'-AATCCGTGTGCTGCGATGGAC; D36A, 5'-TGTTCTGCGATGGCCTACTCT; Y37A,
5'-TCTGCGATGGACGCCTCTCGTGCG.
Each of these oligonucleotides was used in combination with an
ampicillin repair oligonucleotide and annealed to the single-stranded pSELECT/QPs3. The double mutant, H46N,H60N, was constructed by annealing the H46N-mutated oligonucleotide to the single-stranded pSELECT/QPs3(H60N).
A 331-base pair BamHI-EcoRI fragment
containing mutated QPs3 was excised from pSELECT/QPs3m and
cloned into BamHI and EcoRI sites of pGEX2T
vector to generate pGEX2T/QPs3m, which was then transformed
into JM109 cells. Mutations were confirmed by DNA sequencing of both
pSELECT/QPs3m and pGEX2T/QPs3m. Transformants expressing the GST-QPs3m protein were identified by
immunological screening of colonies with antibodies against the
QPs3-connecting peptide (9). Except for H60Y and H89D, all the 18 mutational constructions produced recombinant mutant QPs3-GST fusion
proteins in E. coli JM109 cells. Twelve of these yielded
purified recombinant mutant QPs3. They are the S33A, D36A, Y37A, H9D,
H9N, H9Y, H46N, H46Y, H48N, H48Y, H60N, and H89N substitutions. The
failure to obtain QPs3 mutants H46D, H48D, H60D, and H89Y was because
of their production as inclusion body precipitates in E. coli cells, which were insoluble after treatment with 6 M urea and subsequent dialysis.
Isolation of Recombinant GST-QPs3 Fusion Protein--
400 ml of
an overnight culture of E. coli JM109/pGEX/QPs3 was used to
inoculate 12 liters of SOC medium (2.0% tryptone, 0.5% yeast extract,
10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl2, 20 mM glucose) containing 440 mM sorbitol, 2.5 mM betaine, and 60 mg/liter
ampicillin. The culture was grown in a fermentor chamber at 37 °C
with aeration until the A660 nm reached 0.9 (about 3.5 h) and cooled to 23 °C, and IPTG was added to a
final concentration of 0.6 mM. Growth was continued for
3.5 h at 23 °C before the cells were harvested by
centrifugation at 8,000 × g for 15 min. The cell paste
(48 g) was suspended in 144 ml of PBS buffer (20 mM
sodium/potassium phosphate, pH 7.3, containing 150 mM NaCl)
and sonicated at 30 milliwatts at 0 °C with four 20-s pulses at 3 to
4 min intervals. During sonication, protease inhibitor,
phenylmethylsulfonyl fluoride, was added to a final concentration of 1 mM. Triton X-100 was added to the broken cell suspension to
a final concentration of 1% (w/v). This mixture was stirred gently on
ice for 1 h before being centrifuged at 30,000 × g. The supernatant was mixed with an equal volume of
glutathione-agarose gel equilibrated with PBS. The mixture was gently
shaken on a Varimix (Thermolyne) at 4 °C for 1 h and packed
into a column. The column was washed extensively with the equilibrating
buffer and then eluted with 50 mM Tris-Cl, pH 8.0, containing 5 mM reduced glutathione and 0.25 M
sucrose. Fractions containing the fusion protein were pooled and
dialyzed against 50 mM Tris-Cl, pH 8.0, containing 0.25 M sucrose for 8 h with 2 changes of buffer to remove
glutathione. The dialyzed sample was concentrated with a Centriprep-30
(Amicon) to a protein concentration of 10 mg/ml, mixed with glycerol to
a final concentration of 10%, and frozen at
80 °C until use. QPs3
protein was released from GST-QPs3 fusion protein by thrombin digestion
(1 µg/500 µg of protein) and recovered by gel filtration using a
fast protein liquid chromatography Superose-12 column. Recombinant QPs3
mutants were obtained in the same manner as the wild type.
Enzyme Preparations and General Biochemical
Techniques--
Succinate:Q reductase (1) and QPs (6) were prepared as
reported previously. Absorption spectra and enzyme assays were performed at room temperature in a Shimadzu UV-2101PC. Protein concentration was determined by the Lowry method (20) or by Bradford
assay (21) using a kit from Bio-Rad. The heme content was determined
from the pyridine hemochromogen spectra using a millimolar extinction
coefficient of 34.6 for the absorbance at 557 nm minus that at 600 nm
(22). SDS-PAGE was done according to Laemmli (23) or for high
resolution, according to Schägger et al. (24). The EPR
measurements were made with a Bruker ER-200D equipped with an Air
Product Heli-Tran System. EPR instrument settings are given in the
figure legends.
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RESULTS AND DISCUSSION |
Effect of Induction Growth Conditions on Production of Recombinant
GST-QPs3 Fusion Protein--
The successful overexpression of
functionally active subunit IV of the R. sphaeroides
cytochrome bc1 complex (18), the QPc-9.5 kDa of
beef ubiquinol-cytochrome c reductase (16), and QPs1 of beef
succinate:Q reductase (17) in E. coli, using the pGEX system, encouraged us to use this system to obtain recombinant QPs3.
Production of recombinant GST-QPs3 fusion protein depends on IPTG
concentration, induction growth time, and induction growth medium. The
yield increases as the IPTG concentration and induction growth time are
increased, reaching a maximum at 0.4 mM IPTG and 3.5 h
post-induction growth (data not shown). When IPTG concentration is
increased to 1 mM, no further increase in expression yield is observed. When cells are grown for more than 3.5 h, the total yield decreases and degradative products increase, as determined by
Western blotting using anti-QPs3 peptide antibodies.
The yield of recombinant GST-QPs3 fusion protein in E. coli
is increased by using an induction growth medium containing magnesium. When LB or peptone phosphate-enriched medium was used, production of
GST-QPs3 fusion protein in E. coli accounts for less than
1% total cellular protein. However, including 5 mM
magnesium in the enriched medium or using SOC medium increases the
yield of GST-QPs3 fusion protein to about 15% total cellular protein
(see Table I). Although the yield of
fusion protein in E. coli is slightly higher with enriched
medium supplemented with magnesium than with the SOC medium, the latter
is preferred because severe degradation of the fusion protein is
observed with the former.
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Table I
Effect of induction growth medium on the yield of soluble recombinant
GST-QPs3 by E. coli JM109/pGEX-QPs3 cells
The % yield of recombinant GST-QPs3 fusion protein was estimated by
comparing the color intensity of the 37-kDa GST-QPs3 fusion protein
with that of the total cellular protein bands in SDS-PAGE.
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Although the expression level for GST-QPs3 fusion protein in E. coli is high using SOC medium at 37 °C, about 90% expressed protein is in the inclusion body precipitate. Purification of fusion
protein from inclusion bodies is not practical, at least in our hands,
because the recovery of soluble, active GST-QPs3 from dialyzed,
urea-solubilized inclusion body is very low (less than 1%). Because it
has been reported that including betaine and sorbitol in the induction
growth medium and lowering the induction growth temperature greatly
increases the soluble yield of GST fusion proteins (16, 17, 25), these
induction conditions were adopted. About 40% of the expressed GST-QPs3
fusion protein in E. coli is in the soluble form when IPTG
induction growth is in SOC medium, containing 0.44 M
sorbitol and 2.5 mM betaine at 23-25 °C for 3.5 h.
About 4 mg of purified fusion protein is obtained from a liter of cell
culture. When the purified fusion protein is subjected to SDS-PAGE, a
protein band with apparent molecular mass of 37 kDa is obtained. This
protein band is confirmed to be GST-QPs3 fusion protein by Western
blotting with antibodies against QPs3 and GST-QPs3.
Ubiquinone-binding Property of Recombinant QPs3--
Purified
recombinant QPs3 disperses in 0.01% dodecylmaltoside with an apparent
molecular mass of more than 1 million. This is expected because QPs is
a very hydrophobic protein containing three transmembrane helices. Upon
SDS-PAGE, purified recombinant QPs3 shows only one band, corresponding
to the fifth subunit in succinate:Q reductase (Fig.
1, panel A, lane
5).

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Fig. 1.
Identification of recombinant QPs3 by Western
blot. A, SDS-PAGE of succinate:ubiquinone reductase
(lane 2), recombinant GST-QPs3 fusion protein (lane
3), thrombin-treated fusion protein (lane 4), purified
recombinant QPs3 (lane 5). The molecular mass standard
containing phosphorylase B (108 kDa), bovine serum albumin (84 kDa),
ovalbumin (53 kDa), carbonic anhydrase (35 kDa), soybean trypsin
inhibitor (28 kDa), and lysozyme (20 kDa) is in the lane 1.
The proteins in A were electrophoretically transferred to
nitrocellulose membrane and reacted with anti-QPs3 peptide antibodies
(B) and anti-GST-QPs3 antibodies (C). Protein A
horseradish peroxidase conjugate was used as a second antibody.
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Because QPs3 has been identified as one of the Q-binding proteins in
succinate:Q reductase (4), it is important to know whether or not
recombinant QPs3 binds Q. The Q-binding function of recombinant QPs3 is
indicated by its ability to cause the blue spectral shift of Q. This
method has been used to establish the Q-binding function of recombinant
QPc-9.5 kDa (a small molecular mass Q-binding protein in
ubiquinol-cytochrome c reductase) (16). When
Q0C10 is added to recombinant QPs3, a blue
spectral shift of Q is observed (Fig. 2,
the curve with solid circles). This spectral blue
shift of Q is not observed with Pronase-treated recombinant QPs3 or
with recombinant GST (Fig. 2, the curve with open
circles). Titration of recombinant QPs3 with
Q0C10 shows saturation at around 1.1 mol of
Q/mol of protein (see Fig. 2, the curve with solid
circles), suggesting that the binding is specific and that the
recombinant QPs3 is in the functionally active form for Q-binding.

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Fig. 2.
Effect of recombinant wild-type and mutant
QPs3s on the absorption maximum of Q. To 1.8-ml aliquots of 20 mM sodium phosphate buffer, pH 7.3, containing 150 mM NaCl, 2.5 mM CaCl2, 10%
ethanol, and 0.01% dodecyl maltoside was added 100 µl of 50 mM Tris-Cl buffer, pH 8.0, containing recombinant GST
( ), recombinant wild-type QPs3 ( ), S33A ( ), D36A ( ), or
Y37A ( ). The final protein concentration was 300 µg/ml. The
mixtures were placed equally in a pair of identical cuvettes (1-cm
light path). Q0C10Br in 95% alcohol was added
to one sample cuvette in 1-µl increments to obtain the indicated
concentrations. At the same time, alcohol (95%) was added to the
second sample (reference) cuvette in an identical manner. A difference
spectra between the Q0C10Br-added and
alcohol-added samples was recorded from 320 to 250 nm after 5 min
incubation.
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Identification of Amino Acid Residues Involved in Q-binding of
QPs3--
Previous photoaffinity labeling studies indicate that the
Q-binding domain in QPs3 (residues 29-37) is at the end of
transmembrane helix 1 toward the C side of the mitochondrial membrane
(9). Once the Q-binding property of recombinant QPs3 was established, site-directed mutagenesis coupled with Q-binding spectral analysis was
used to identify the amino acid residues in the putative Q-binding domain responsible for Q-binding. Serine 33 and tyrosine 37 were selected for mutagenesis because they can form hydrogen bonds with the
carbonyl group of the benzoquinone ring of Q similar to those found in
the photosynthetic bacterial reaction center (26). Aspartic acid 36 was
selected because it is a conserved residue in this region of QPs3
proteins from bovine mitochondria, Ascaris suum (adult) and
yeast (9). Replacing Ser-33 or Tyr-37 with alanine results in
recombinant mutant QPs3 (S33A or Y37A) unable to bind Q, as no spectral
blue shift of Q0C10 is observed upon its
addition (see Fig. 2, the curve with open
triangles or with solid diamonds), indicating that
these two amino acid residues are involved in Q-binding. Replacing
Asp-36 with alanine results in recombinant mutant QPs3 (D36A) having
the same Q-binding activity as the recombinant wild-type protein; added
Q0C10 shows a spectral blue shift (see Fig. 2,
the curve with open squares), indicating that
Asp-36 is not involved in Q-binding.
Reconstitution of Cytochrome b560 from Recombinant
QPs3 and Hemin Chloride--
Isolated QPs contains three
protein subunits (QPs1, QPs2, and QPs3) with a heme
b560 content of 27 nmol/mg of protein (6). The
ligand for this cytochrome has been identified as bishistidine (12).
However, unknown is which QPs subunit is involved in heme b560 ligation, and whether the bishistidine
ligands are provided by a single subunit or by two different subunits,
as reported for cytochrome b556 of E. coli succinate:Q reductase (14, 15). Because recombinant QPs3
contains little cytochrome b560 heme, the
involvement of QPs3 in heme ligation was investigated by testing the
ability of recombinant QPs3 to reconstitute in vitro with hemin chloride to form cytochrome b560.
Reconstitution was first attempted with the fusion protein because it
is soluble in aqueous solution.
When hemin chloride in Me2SO was added to GST-QPs3, the
maximum absorption peak (Soret band) of the oxidized form of heme progressively shifts from 398 to 411 nm, with increasing absorption intensity during the incubation. It takes 1 h to complete the spectral red shift and to reach maximum absorption. When sample is
reduced with dithionite, it shows symmetrical
-absorption at 560 nm,
a broad
-absorption between 526 and 528 nm, and Soret absorption at
424 nm (see Fig. 3A). These
spectral characteristics are identical to those of cytochrome
b560 in an isolated, reconstitutively active QPs
preparation (6), indicating that b560 is
restored in GST-QPs3 fusion protein by heme addition. Because no
cytochrome b560 spectral properties are observed
with heme-treated GST (see Fig. 3B), the cytochrome
b560 restored in GST-QPs3 is in the QPs3 moiety
of the fusion protein.

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Fig. 3.
Absorption spectra of reconstituted
cytochrome b560 in recombinant GST-QPs3
fusion protein. Three-µl aliquots of hemin chloride (6 mM) in Me2SO were added to 1 ml of purified
recombinant GST-QPs3 (1.5 mg) (A) and 1 ml of recombinant
GST (1.5 mg) (B) in Tris-Cl buffer, pH 8.0, containing 0.25 M sucrose. The mixtures were incubated at room temperature,
and absorption spectra were recorded from time to time during the
incubation period. When the oxidized Soret absorption peak no longer
changed (solid line), a small amount of dithionite was added
and spectra recorded (dashed line). The inset on
A displays the difference spectra of the dithionite-reduced
versus oxidized form in the and absorption
regions.
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Reconstituted cytochrome b560 in GST-QPs3 shows
an EPR peak at g = 2.92 (see Fig. 5). This signal
differs with the EPR characteristics of heme-treated GST
(g = 3.50 and g = 3.86) and of free
heme (a broad peak with g = 3.80) (17). It resembles
the one in the isolated QPs (g = 2.92 and
g = 3.07), which do not respond to interaction with
succinate dehydrogenase to form succinate:Q reductase (6). The EPR
signal of cytochrome b560 in intact succinate:Q reductase is at g = 3.46. When succinate dehydrogenase
is removed from the reductase, the EPR signals of cytochrome
b560 in the resulting QPs preparation are at
g = 3.07 and 2.92. The g = 3.07 signal
converts to g = 3.46, whereas the g = 2.92 signal remains unchanged upon reconstitution with succinate
dehydrogenase to form succinate:Q reductase. The inability of the
b560 with g = 2.92 to convert to
g = 3.46 upon reconstitution with succinate dehydrogenase suggests that this cytochrome b560
has been somewhat modified during the isolation of QPs.
The observation that absorption and EPR spectral properties of
reconstituted cytochrome b560 in GST-QPs3 fusion
protein remain unchanged upon thrombin digestion together with the fact
that heme-treated GST does not have the absorption or EPR spectral properties of cytochrome b560 indicates that the
ligands of reconstituted b560 heme are from
QPs3. Moreover, the spectral properties of cytochrome
b560 are associated with the recombinant
QPs3-containing fractions when thrombin-treated, heme-reconstituted
fusion protein is subjected to gel filtration chromatography (Superose
12, Amersham Pharmacia Biotech) to separate GST from QPs3. The
QPs3-containing fraction has a heme b560 to
protein ratio of 0.75. This stoichiometry may result from part of the
recombinant protein not being in the right orientation for ligation,
from part of reconstituted heme b560 being
released during gel filtration, or from dimerization of recombinant
protein. Perhaps part of the observed b560
spectral properties result from heme-ligated with two molecules of QPs3.
Because recombinant QPs1 has also been reported to restore cytochrome
b560 spectral properties upon addition of hemin
chloride (17), it is of interest to see whether or not restoration of cytochrome b560 by recombinant QPs3 is affected
by the presence of recombinant QPs1. When hemin chloride was added to a
1:1 mixture of recombinant QPs1 and QPs3, the amount of cytochrome
b560 restoration equals the sum of cytochrome
b560 restored by the individual recombinant proteins, suggesting that each of these proteins can provide
bishistidine ligands for cytochrome b560 in
bovine heart mitochondrial succinate:Q reductase. This differs from the
report that cytochrome b556 in E. coli succinate:Q reductase is ligated to two histidine residues located, respectively, at SdhC and SdhD (14).
Identification of Amino Acid Residues of QPs3 Involved in Ligation
of Heme b560--
QPs3 contains histidine residues at
positions 9, 46, 48, 60, and 89. To locate the heme-ligating residues,
we altered each of these residues to asparagine, tyrosine, or aspartate
by site-directed mutagenesis followed by spectral (absorption and EPR)
characterizations of heme-reconstituted recombinant QPs3 mutants.
Fig. 4 shows absorption spectra of
heme-reconstituted recombinant QPs3 mutants. The absorption spectra of
heme-reconstituted wild-type QPs3 and GST are included for comparison.
The addition of hemin chloride to the H9D, H9N, H9Y, H48N, H48Y, or
H89N mutant yields absorption spectra similar to those of reconstituted
wild type, indicating that H9, H48, and H89 of QPs3 are not involved in
heme b560 ligation. The Soret absorption peaks
of heme-reconstituted mutants H46Y (Fig. 4F) and H60N (Fig.
4I) are very different, with a 14-nm red shift of the peak maximum and
a drastic decrease in absorbance. Thus His-46 and His-60 of QPs3 are
involved in heme b560 ligation. The involvement
of His-46 is further supported by the diminishing of the
-absorption
peak in the heme-reconstituted H46N mutant QPs3 (see Fig.
4E). As expected, when a double mutant, H46N,H60N was
reconstituted with hemin chloride, no cytochrome b560 spectral characteristics were obtained (see
Fig. 4K).

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Fig. 4.
The dithionite-reduced minus -oxidized
spectra of thrombin-treated, heme-reconstituted mutant QPs3 fusion
proteins. The panels are as follows: A, wild
type; B, H9D; C, H9N; D, H9Y;
E, H46N; F, H46Y; G, H48N;
H, H48Y; I, H60N; J, H89N;
K, H46N,H60N; and L, GST.
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Fig. 5 compares the EPR characteristics
of heme-reconstituted wild-type and mutants QPs3. The
heme-reconstituted H9D, H9N, H9Y, H48N, and H89N mutants of QPs3 have
EPR spectra similar to reconstituted wild type, indicating that
histidines at positions 9, 48, and 89 of QPs3 are not involved in heme
b560 ligation. Mutants H46N, H46Y, H60N, and
H46N,H60N did not produce a g = 2.92 signal upon
treatment with hemin chloride, indicating that cytochrome
b560 is not formed in these mutants and
consistent with the lack of cytochrome b560
absorption characteristics when these mutants are treated with hemin
chloride. Therefore, histidines 46 and 60 provide ligands for
reconstituted cytochrome b560 in recombinant
QPs3.

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Fig. 5.
EPR spectra of heme-reconstituted wild-type
and QPs3 mutants. 15 µl of hemin chloride in Me2SO
was added to 0.6 ml of Tris-Cl buffer, pH 8.0, containing the indicated
fusion proteins (10 mg/ml). The mixtures were incubated at room
temperature for 1 h, treated with thrombin (0.01 unit/µg of
protein), and then subjected to EPR measurements. The EPR instrument
settings were: modulation frequency, 100 KHz; modulation amplitude, 20 G; time constant, 0.5 s; microwave frequency, 9.42 GHz; microwave
power, 20 mW; scan rate, 200 s; temperature 10 K.
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Sequence alignments of the smallest membrane-anchoring subunit of
succinate:Q reductases (9) reveal that His-46 of mitochondrial QPs3
corresponds to histidine 71 of the E. coli SdhD subunit, which has been identified as one of the bishistidine ligands for cytochrome (15). The involvement of this histidine residue in cytochrome b560 heme-ligating is also supported
by the observation that yeast succinate:Q reductase and E. coli fumarate-Q reductase contain no cytochrome
b560, and the residues corresponding to His-46
of beef QPs3 in the yeast Sdh4 and FrdD subunits are tyrosine and
valine, respectively. His-60 of mitochondrial QPs3 is tissue-specific (9).
Because His-60 of mitochondrial QPs3 is not conserved in the Sdh4
subunit of cytochrome b556-containing bacterial succinate:Q reductases, the heme-ligating function of this histidine residue merits
discussion. Because according to the proposed structure of QPs3, His-46
is located in the transmembrane helix, it would be difficult for
His-60, in the same molecule, to serve as the second ligand for
cytochrome b560. Probably cytochrome
b560 heme in recombinant QPs3, as shown in this
investigation, has two histidine ligands, His-60 and His-46, from two
different molecules of QPs3. The heme-ligating property of His-60 is
observed only in isolated QPs3. In intact beef succinate:Q reductase
His-60 of QPs3 may be shielded by other protein subunits and not
involved in cytochrome b560 heme ligation. The
second histidine ligand for cytochrome b560 in
intact reductase is from QPs1. When QPs3 is detached from the
reductase, the His-60 in QPs3 is unshielded and may replace the
histidine ligand from QPs1. The fact that the EPR signal of reconstituted cytochrome b560 in recombinant
QPs3 (g = 2.92) resembles the one in isolated QPs
(g = 2.92 and g = 3.07), which does not respond to interaction with succinate dehydrogenase to form succinate:Q reductase (6), supports the idea that the cytochrome
b560 heme in this reconstituted system is
somewhat different. It is unclear whether or not QPs2 plays a role in
the proper ligation of b560 heme. We failed to
generate cytochrome b560 in a mixture of
recombinant QPs1 and QPs3, which has an EPR signal at g = 3.07 that converts to g = 3.46 upon reconstituting
with succinate dehydrogenase to form succinate:Q reductase. However,
this failure can be explained by polymerization of the recombinant
proteins or by the lack of QPs2. Further investigations on QPs2 and on
the three-dimensional structure of succinate:Q reductase should yield
information concerning the structure of this cytochrome b
and its role in this enzyme complex.