(Received for publication, March 20, 1997, and in revised form, May 10, 1997)
From the Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK 74078
The cDNA encoding the smallest
membrane-anchoring subunit (QPs3) of bovine heart mitochondrial
succinate-ubiquinone reductase was cloned and sequenced. This cDNA
is 1330 base pairs long with an open reading frame of 474 base pairs
that encodes the 103 amino acid residues of mature QPs3 and a 55-amino
acid residue presequence. The cDNA insert has an 820-base pair long
3-untranslated region, including a poly(A) tail. The molecular mass of
QPs3, deduced from the nucleotide sequence, is 10,989 Da. QPs3 is a
very hydrophobic protein; the hydropathy plot of the amino acid
sequence reveals three transmembrane helices. Previous photoaffinity
labeling studies of succinate-ubiquinone reductase, using
3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone
([3H]azido-Q), identified QPs3 as one of the
putative Q-binding proteins in this reductase. An azido-Q-linked
peptide with a retention time of 66 min is obtained by high performance
liquid chromatography of the chymotrypsin digest of carboxymethylated
and succinylated [3H]azido-Q-labeled QPs3 purified from
labeled succinate-ubiquinone reductase by a procedure involving
phenyl-Sepharose 4B column chromatography, preparative
SDS-polyacrylamide gel electrophoresis, and acetone precipitation. The
amino acid sequence of this peptide is
NH2-L-N-P-C-S-A-M-D-Y-COOH, corresponding to residues
29-37. The structure of QPs3 in the inner mitochondrial membrane is
proposed based on the hydropathy profile of the amino acid sequence, on the predicted tendencies to form
-helices and
-sheets, and on immunobinding of Fab
fragmenthorseradish peroxidase conjugates prepared from antibodies against two synthetic peptides,
corresponding to the NH2 terminus region and the loop
connecting helices 2 and 3 of QPs3, in mitoplasts and submitochondrial
particles. The ubiquinone-binding domain in the proposed model of QPs3
is probably located at the end of transmembrane helix 1 toward the
C-side of the mitochondrial inner membrane.
Bovine heart mitochondrial succinate-ubiquinone (Q)1 reductase, also known as Complex II (1), catalyzes electron transfer from succinate to Q. Succinate-Q reductase is composed of two parts, soluble succinate dehydrogenase and a membrane anchoring protein fraction (QPs). Succinate dehydrogenase contains two protein subunits, a 70-kDa flavoprotein (FP) with a covalently linked flavin adenine dinucleotide and a 27-kDa iron-sulfur protein (IP) with three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S) (2, 3). The amino acid sequences of FP and IP have been determined by peptide (4) and nucleotide (5) sequencing.
Reconstitutively active QPs has been isolated and characterized (6-8). Purified QPs shows two protein bands (6-8) in the SDS-PAGE system of Weber and Osborn (9) and three (10) in the high resolution SDS-PAGE system of Schägger et al. (11). These three subunits, with apparent molecular masses of 14, 11, and 9 kDa, are named QPs1, QPs2, and QPs3, respectively. The function of QPs is to provide membrane docking for succinate dehydrogenase and the Q-binding sites for succinate-Q reductase. Soluble succinate dehydrogenase catalyzes electron transfer from succinate to redox dyes, such as phenazine methosulfate and ferricyanide (12), but it cannnot catalyze the 2-thenoyltrifluoroacetone (TTFA)-sensitive electron transfer from succinate to Q. Addition of QPs to soluble succinate dehydrogenase converts succinate dehydrogenase to the membrane-bound, TTFA-sensitive succinate-Q reductase (13) with detectable ubisemiquinone radicals (14).
Participation of QPs in the Q-binding of succinate-Q reductase was further established by photoaffinity labeling studies with [3H]azido-Q derivatives (10). When a succinate-free, partially Q-deficient succinate-Q reductase is treated with [3H]azido-Q derivatives in the dark followed by illumination with a long wavelength UV light, about 50% of bound Q is located on QPs1 and the other 50% is equally distributed between QPs2 and QPs3 (10). The Q-binding domain in the proposed structure of QPs1, based on the deduced amino acid sequence (15, 16), is located in a loop connecting transmembrane helices 2 and 3, which protrude from the surface of the M side of the inner membrane (10).
Although lesser amounts of azido-Q were found in QPs2 and QPs3, involvement of these two subunits in the Q-binding site cannot be ruled out. The low azido-Q uptake by QPs2 and QPs3 may result from (i) only part of a Q-binding site being formed by these two subunits, (ii) preferential binding of these two subunits by endogenous Q present in the partially Q-deficient succinate-Q reductase used in these photoaffinity labeling studies, or (iii) QPs2 and QPs3 being the same peptide, i.e. either QPs2 is incompletely processed QPs3 or QPs3 is a COOH-terminal truncate of QPs2. If the latter is true, the amount of azido-Q uptake by QPs2 or QPs3 equals that by QPs1. Isolation of an azido-Q-linked peptide from azido-Q-labeled QPs2 or QPs3, obtained from azido-Q-labeled succinate-Q reductase, will help determine the Q-binding role of QPs2 or QPs3. However, identification of the Q-binding domains in QPs2 or QPs3 requires knowledge of the amino acid sequences of these two subunits. Herein, we report cloning and nucleotide sequencing the cDNA encoding QPs3, the immunological determination of the topology of QPs3 in the inner mitochondrial membrane using monospecific polyclonal antibodies against two synthetic peptides corresponding to residues 1-14 and 55-66, isolating and sequencing an azido-Q-linked peptide from labeled QPs3, and the localization of the Q-binding domain in the proposed model of QPs3. The identity of QPs2 is also discussed.
Bovine serum albumin, dichlorophenolindophenol (DCPIP), Triton X-100, sodium cholate, TTFA, and phenyl-Sepharose CL-4B, were obtained from Sigma. Protein A-horseradish peroxidase conjugate, protein molecular weight standards, SDS, acrylamide, urea, and DEAE Affi-Gel blue were from Bio-Rad. Taq polymerase was from Promega. TA cloning kit was from Invitrogen. The bovine heart cDNA library constructed in the Uni-ZAP XR vector was from Stratagene. Insta-gel liquid scintillation mixture was from Packard Instrument Co. Oligonucleotides and peptides were synthesized by the DNA/Protein Core Facility at Oklahoma State University. Nitrocellulose membranes were from Schleicher & Schuell. All other chemicals were of the highest purity commercially available.
The ubiquinone derivatives, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q0C10) and 3-azido-2-methyl-5-methoxy- and 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone (azido-Q and [3H]azido-Q, respectively) were synthesized according to previously reported methods (17). Calcium phosphate was prepared according to Jenner (18) and mixed at a 3:1 ratio with cellulose powder prior to use in column chromatography.
Enzyme Preparation and AssaysIntact bovine heart
mitochondria (19), mitoplasts (20), submitochondrial particles (21),
and succinate-Q reductase (22) were prepared and assayed as previously
reported. Succinate-Q reductase was assayed, at room temperature, for
its ability to catalyze TTFA-sensitive Q-stimulated DCPIP reduction by
succinate using a Shimadzu UV-2101PC. The reaction mixture (1 ml)
contains 40 µmol of DCPIP, 100 µmol of sodium potassium phosphate
buffer, pH 7.4, 20 µmol of succinate, 10 nmol of EDTA, 25 nmol of
Q0C10, and 0.01% of Triton X-100. The
reduction of DCPIP was followed by measuring the absorption decrease at
600 nm, using a millimolar extinction coefficient of 21 mmol1 cm
1. The concentration of TTFA used
was 10
4 M.
PCR amplification was performed in a minicycler from M. J. Research. The thermal cycle was set-up as follows: step 1, 95 °C for 3 min; step 2, 94 °C for 1 min for denaturation; step 3, 37 °C for 2 min for annealing; and step 4, 70 °C for 90 s for extension. A total of 30 cycles were performed with a final extension step of 7 min.
DNA SequencingThis was done at the Core Facility of Oklahoma State University with an automatic DNA sequencer from Applied Biosystems, model 373 A.
Purification and Partial NH2-terminal Amino Acid Sequencing of QPs3Isolated succinate-Q reductase was diluted to 2 mg/ml with 50 mM Tris-Cl, pH 7.4, containing 0.2% sodium cholate. The solution was stirred at room temperature for 30 min and applied to a phenyl-Sepharose CL-4B column equilibrated with 50 mM Tris-Cl, pH 7.4, containing 0.2% sodium cholate. The column was, in sequence, washed with 50 mM Tris-Cl, pH 7.4, containing 0.2% sodium cholate; 50 mM Tris-Cl, pH 7.4, containing 2% sodium cholate and 4 M urea; and 50 mM Tris-Cl, pH 7.4, containing 2% sodium cholate. QPs was eluted from the column with 50 mM Tris-Cl, pH 7.4, containing 0.15% SDS.
Pure QPs3 was obtained from QPs by preparative SDS-PAGE essentially
according to the previously reported method (10) except that the gels
were pre-run for 10 h at 45 V with an anode buffer containing 0.1 M Tris-Cl, pH 8.9, 0.1 mM sodium thioglycolate and a cathode buffer containing 2% M Tris, 0.1 M Tricine, 0.1% SDS, and 0.1 mM sodium
thioglycolate. The gel-eluted QPs3 protein was concentrated by membrane
filtration, using centricon-10, to a protein concentration of 2 mg/ml
and precipitated with cold acetone (20 °C). These precipitates
were washed with 50% acetone, dried under argon, and subjected to
NH2-terminal sequence analysis. These analyses were done at
the Molecular Biology Resource Facility, Saint Francis Hospital of
Tulsa Medical Research Institute, University of Oklahoma Health
Sciences Center under the supervision of Dr. Ken Jackson.
Purified succinate-Q reductase was photoaffinity labeled with [3H]azido-Q derivative as reported previously (10). The azido-Q-labeled reductase was precipitated by 43% ammonium sulfate saturation, separated by centrifugation at 48,000 × g for 20 min, dissolved in 20 mM Tris-Cl, pH 7.8, and dialyzed against double-distilled water, overnight, with one change of water. [3H]azido-Q-labeled QPs3 was purified to homogeneity from this dialyzed, labeled succinate-Q reductase as described above for isolation of pure QPs3.
The acetone precipitate of electrophoretically eluted [3H]azido-Q-labeled QPs3 was dissolved in 0.1 M Tris, 6 M guanidine HCl, 1 mM EDTA, pH 8.3, and subjected to reductive carboxymethylation and succinylation as described previously (23).
Proteolytic Enzyme Digestion of QPs3The reductive carboxymethylated and succinylated QPs3, 1 mg/ml, suspended in 50 mM ammonium bicarbonate buffer, containing 1 M urea was treated with chymotrypsin at 37 °C for 2 h, using a chymotrypsin to QPs3 ratio of 1:50 (w/w). After the 2-h incubation, a second addition of chymotrypsin was made (1:100), and the digestion was continued at 37 °C for 24 h.
Isolation of [3H]Azido-Q-linked Peptides100 µl aliquots of the chymotrypsin-digested, reductive carboxymethylated, succinylated, [3H]azido-Q-labeled QPs3 were separated by reverse phase high performance liquid chromatography (HPLC) on a Synchropak RP-8 column (0.46 × 25 cm) using a gradient formed from 90% acetonitrile in 0.1% trifluoroacetic acid and 0.1% trifluoroacetic acid with a flow rate of 0.8 ml/min. 0.8-ml fractions were collected and monitored for radioactivity and absorbance at 214 nm. Fractions with radioactivity were collected, dried, and sequenced. Those containing no radioactivity, with retention times of 18.96 and 31.15 were also collected, dried, and sequenced.
Production and Purification of Antibodies against QPs3 and Its NH2-terminal and Connecting PeptidesPure QPs3 (see
above) was used as antigen to raise antibodies in rabbits (15). Two
polypeptides, one containing 14 amino acid residues
(NH2-S-G-S-K-A-A-S-L-H-W-T-G-E-R-COOH) corresponding to
residues 1-14 of QPs3 (the NH2-terminal peptide, see Fig.
4) and another containing 11 amino acid residues
(NH2-T-D-Y-V-H-G-D-A-V-Q-K-COOH corresponding to residues
56-66 (the connecting peptide, the loop between helices 2 and 3, see
Fig. 4) were synthesized, purified, and used as antigens, after
conjugation with ovalbumin, to raise antibodies in rabbits (24).
Boosters were given weekly for 5 weeks, and sera were collected by
cardiac puncture.
Purification of antibodies and preparation of the antibody Fab
fragment-horseradish peroxidase conjugates were as previously reported
(15). Horseradish peroxidase activity of the purified conjugate was
assayed using a TMB peroxidase substrate kit (Bio-Rad) according to the
manufacturer instructions.
Since the amino acid
sequences of the membrane-anchoring subunits in succinate-Q reductase
from different species show little conservation (25), we did not use
the homology probing strategy to obtain the cDNA for QPs3. The
availability of anti-QPs3 antibodies in our laboratory together with
our previous success in immunological screening of a beef heart
cDNA expression library in gt11 to obtain cDNAs for the
Rieske iron sulfur protein (26), the QPc 9.5 kDa (27) of
ubiquinol-cytochrome c reductase, and the QPs1 (15) of
succinate-Q reductase encouraged us to use the immunological screening
method to isolate the cDNA for QPs3. However, no positive result
was obtained. This failure to obtain cDNA for QPs3 by the immunological screening method is probably due to the low titer of
antibodies against QPs3 rather than the lack of QPs3 cDNA in the
cDNA library used because we also failed to obtain a positive clone
from other beef heart cDNA libraries, such as
ZAP (from Stratagene). In an effort to obtain high titer anti-QPs3 antibodies, we
added several more booster injections to rabbits and tried to raise
antibodies in chickens. However, both attempts failed.
It was reported (28) that specific cDNA inserts are obtained from
the cDNA libraries constructed in gt11 by PCR amplification using synthetic guessmers. The design of synthetic guessmers requires knowledge of a partial amino acid sequence of the target protein. When
purified QPs3 (Fig. 1A, lane 6)
was subjected to protein sequencing, 43 residues from the
NH2 terminus were obtained,
NH2SGSKAASLHWTGERVVSVLLLGLIPAAYLNPCSAMDYSLAATL-. This
enabled us to use the PCR cloning method to isolate the QPs3 cDNA
from a beef heart cDNA library in
ZAP. A 110-bp cDNA
fragment was amplified from a beef heart cDNA library in
ZAP
(4 × 106 plaque-forming unit) by PCR using two
synthetic guessmers, 5
-GCTGCCTCCCTGCACTGGAC-3
(the sense primer)
and 5
-GCAGCCAGGGAGTAGTCCAT-3
(the antisense primer). The sense
primer represents the guessed sequence for residues 5-11
(A-A-S-L-H-W-T) with the degenerate third base of the codon of
T11 being omitted. The antisense primer represents the
guessed sequence for residues 41-35 (A-A-L-S-Y-D-M). Since W and M
have no degeneracy in their genetic codes, the presence of
W10 and M35 in the QPs3 partial sequence
enables us to design these two PCR guessmers with specificity at the 3
end, five specific bases in the 3
end of the sense guessmer, and three
in the antisense guessmer.
The PCR reaction consists of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl2, 30 mM KCl, 0.05% Tween 20, 100 µg of autoclaved gelatin, 100 µM dNTP, 2 units of Taq polymerase, and 200 pmol of guessmers. The resulting 110-bp PCR product was cloned into a PCR vector (TA cloning kit) and sequenced. The DNA sequence of this 110-bp PCR product translated to match the amino acid residues between Trp10 and Met35 of the chemically determined partial NH2-terminal sequence of QPs3.
Based on the nucleotide sequence for residues 10-35 of QPs3, two gene
specific primers, 5-TTGCTCCTGGGCCTAATTCC-3
, corresponding to residues
21-27 (sense primer), and 5
-AGGAGCAAAACACTGAACAAC-3
, corresponding
to residues 22-17 (antisense primer), were synthesized and used
with the primers of vector ZAP, T7 and T3, respectively, in the
subsequent PCR reactions to yield 3
- and 5
-RACE products. These two
PCR products were cloned into the PCR II vector and sequenced. The
3
-RACE product is confirmed by matching the deduced amino acid
residues 21-42 of QPs3 with chemically determined partial NH2-terminal amino acid sequence, and residues 59-66 and
44-49 with chymotryptic peptides of QPs3 with retention times of 18.96 and 31.15 min, respectively, on an HPLC chromatogram. Although the 5
-
and 3
-RACE products can be joined together by PCR using a marathon
cDNA amplification method (29), no effort was made to obtain a
combined RACE product in this investigation. However, for future
structure-function studies of QPs3, we have obtained a 331-bp
BamHI-EcoRI cDNA fragment encoding mature
QPs3 protein by PCR amplification from the beef heart cDNA library
in
ZAP using two primers, GGATCCTCTGGTTCCAAG (sense primer) and
GAATTCTAAAAGGTCAGAGC (antisense), and cloned into a PCR vector.
Fig. 2 shows the
nucleotide sequence and the deduced amino acid sequence of QPs3. The
QPs3 cDNA is 1330 base pairs long with an open reading frame of 474 base pairs that encodes 158 amino acid residues, of which 103, starting
with serine, belong to mature QPs3 and 55, starting with methionine,
constitute an NH2-terminal presequence. In addition, the
cDNA has 820 nucleotides of 3 non-coding sequence and contains a
poly(A) tail.
The presequence of QPs3 is rich in the basic amino acid arginine
and contains the hydroxy amino acid serine. This is characteristic of
the cleavable amino acid terminal presequences that are essential for
the import of mitochondrial proteins encoded by nuclear DNA (30). Since
the QPs3 presequence lacks Arg at the 2 position relative to the
mature amino terminus, QPs3 may be matured by a two-step cleavage
process (31). Mitochondrial proteins with leader peptides containing
the R-X-(F)-X-X-(S) motif, where
r = arginine at the
10 position, X = other amino acid at
9; (F) = hydrophobic residues at
8; and (S) = serine, threonine, or glycine at
5, are thought to cleave first by a
matrix processing protease between residues at
9 and
8 since
arginine at position
10 is at position
2 relative to the cleaved
bond. The remaining octapeptide is subsequently removed by an
intermediate-specific protease. Although the QPs3 presequence lacks the
arginine
10 in the common motif of this two-step cleavage,
the maturation of QPs3 may still follow the same process because
mutation of arginine
10, in the human ornithine
transcarbamylase precursor or rat malate dehydrogenase precursor, to
alanine (32, 33) does not alter the two-step maturation of these two
proteins.
The molecular mass of mature QPs3, determined from the deduced amino
acid sequence, is 10,989 daltons, which is fairly close to the 9 kDa
estimated from SDS-PAGE. Fig. 3 compares the amino acid
sequence of bovine QPs3 with those of Saccharomyces
cerevisiae Sdh4 (34), adult Ascaris suum cytbS (35),
and Escherichia coli SdhD (36) and FrdD (37). Bovine QPs3
shares little sequence homology with gene products of S. cerevisiae sdh4, E. coli sdhD, and frdD but
has 50% sequence identity with the A. suum (adult) cytochrome b small subunit of fumarate reductase (35). It is noteworthy that the similarity between bovine QPs3 and A. suum cybS in the region from Leu-23 to His-60 of QPs3 (68%) is
higher than that of the entire peptide. This region contains a
conserved histidine residue that matches His-71 of E. coli
SdhD, which provides the heme b ligand (38). His-81 of
E. coli FrdD that was reported to be involved in Q-binding
is also in this region (39). It should be mentioned that the QPs3
cDNA was found to have 85% sequence identity to the DNA sequence
of a Homo Sapiens cDNA clone (R91018) according to a BLAST search
of the Expressed Sequence Tag (EST) data base.
The Proposed Structure of QPs3 in the Mitochondrial Inner Membrane
Fig. 4 shows the proposed structure of
QPs3 in the inner mitochondrial membrane. This structural model,
constructed from hydropathy plots of the amino acid sequence of QPs3
(40), predicted tendencies to form -helices and
-sheets and the
binding of Fab
fragment-horseradish peroxidase conjugates, prepared
from antibodies against synthetic peptides corresponding to residues
1-14 and 55-66 of QPs3, in mitoplasts, submitochondrial particles
(SMP), and alkali-treated submitochondrial particles. In this model,
QPs3 has three transmembrane helices corresponding to residues 15-34
(helix I), 37-56 (helix II), and 67-89 (helix III). The
NH2-terminal region, residues 1-14, and the loop
connecting helices II and III, residues 57-66, are extruded from the
M-side of the inner mitochondrial membrane. The loop connecting helices
I and II, residues 36-37, and the COOH-terminal region, residues
90-103, are on the C-side of the membrane.
The sidedness of the membrane in this model was determined
immunologically with Fab-horseradish peroxidase conjugates prepared from anti-QPs3, anti-NH2-terminal peptide (residues 1-14),
and anti-connecting peptide (residues 57-66) antibodies in bovine heart mitoplasts (digitonin-treated intact mitochondria), SMP (reverse
orientation), and alkaline-treated SMP (SMP devoid of succinate
dehydrogenase). The peroxidase activity assays of these three particles
are shown in Fig. 5. Since the peroxidase activity observed with preimmune Fab
-horseradish peroxidase-treated
preparations is assumed to be due to nonspecific binding, it is
subtracted from that of the anti-QPs3, anti-NH2-terminal
peptide, or anti-connecting peptide Fab
-horseradish peroxidase-treated
preparations. The intactness of mitoplasts and submitochondrial
particle preparations was established by the absence and presence of
rotenone-sensitive NADH-Q reductase activity.
When mitoplasts and SMP preparations were treated with anti-QPs3 Fab
fragment-peroxidase conjugates, peroxidase activity was detected in
both preparations. The slightly higher activity in treated SMP suggests
that QPs3 is a transmembranous protein with slightly more mass exposed
on the matrix side of the membrane. When an alkali-treated SMP
preparation is treated with anti-QPs3 Fab
fragment-horseradish
peroxidase conjugate, only a slight increase in peroxidase activity is
observed, indicating that few epitopes on the matrix side of QPs3 are
covered by succinate dehydrogenase.
When mitoplasts and SMP preparations are treated with Fab
fragment-horseradish peroxidase conjugates prepared from
anti-N-terminal and connecting peptides antibodies, peroxidase activity
is observed only on SMP, indicating that the NH2-terminal
end and the loop connecting helices II and III are exposed on the
M-side of the mitochondrial inner membrane.
It should be mentioned that during the course of immunological studies of QPs3 we observed that antibodies against QPs3, the NH2-terminal, and the connecting peptides cross-react with QPs2 (Fig. 1, B-D, lanes 3 and 5). They do not react with QPs1 or proteins in ubiquinol-cytochrome c reductase (see Fig. 1). Antibodies against QPs2 also react with QPs3 (data not shown). This immunocross-reaction of QPs2 and QPs3 is expected because the QPs3 sequence is contained in QPs2. This is evident from the following observations. (i) When an electrophoretically pure QPs2 preparation (Fig. 1A, lane 5) is subjected to protein sequencing, a major peptide with a partial NH2-terminal amino acid sequence of Ser-Pro-Ser-His-His-Ser-Gly-Ser-Lys-Ala- is obtained. This sequence contains five amino acid residues from the COOH terminus of the presequence and five amino acid residues from the NH2 terminus of mature QPs3, suggesting that QPs2 is incompletely processed QPs3. (ii) When chymotrypsin-digested QPs2 and QPs3 are subjected to HPLC separation, identical chromatograms are obtained (data not shown). (iii) When peptides with identical retention times, from the respective QPs2 and QPs3 HPLC chromatograms, are sequenced, identical sequences are obtained. (iv) The azido-Q-labeled peptide from QPs2 is identical to that from QPs3 (described in the next section). At present, we do not know whether QPs2 is the same or a different gene product than QPs3. This requires further investigation.
Isolation and Characterization of the Ubiquinone-binding Peptides of QPs3When succinate-Q reductase is photoaffinity labeled with [3H]azido-Q derivative, about 50% of bound azido-Q is in QPs1, 22% in QPs2, and 25% in QPs3 (10). The smaller azido-Q uptake by QPs2 or QPs3, compared with QPs1, questions the Q-binding role of these two subunits. One way to establish a Q binding role for QPs3 is to isolate an [3H]azido-Q-linked peptide from [3H]azido-Q-labeled QPs3 obtained from [3H]azido-Q-labeled succinate-Q reductase.
[3H]azido-Q-labeled QPs3 is isolated from [3H]azido-Q labeled succinate-Q reductase by a procedure involving phenyl-Sepharose CL-4B column chromatography, preparative SDS-PAGE, electrophoretic elution of proteins from gel slices, and acetone precipitation. The use of a hydrophobic column, phenyl-Sepharose CL-4B, and elution with different detergents results in the isolation of [3H]azido-Q-labeled QPs from [3H]azido-Q-labeled succinate-Q reductase. Since the FP and IP subunits of succinate dehydrogenase are less hydrophobic than those of QPs subunits, they are eluted with detergents having less hydrophobicity than that used for eluting QPs. This column chromatographic step also removes most of the non-protein bound [3H]azido-Q from QPs. Pure [3H]azido-Q-labeled QPs3 is isolated from the labeled QPs by preparative SDS-PAGE using high resolution gel system in the presence of 8 M urea. The use of preparative SDS-PAGE not only separates QPs3 from other QPs subunits, it also further removes non-protein bound azido-Q adducts from QPs3. QPs3 in gel slices is eluted by electrophoretic eluting. The SDS present in the eluted protein solution was removed by 50% acetone precipitation.
Although the isolated [3H]azido-Q-labeled QPs3 is pure and free of free azido-Q, it is highly aggregated and resistant to proteolytic enzyme digestion. Inclusion of 0.1% SDS and 2 M urea in the digestion mixture does not increase proteolysis. Modification of isolated azido-Q labeled QPs3 by reductive carboxymethylation followed by succinylation renders the protein susceptible to chymotrypsin digestion. Reductive carboxymethylated and succinylated QPs3 is not completely soluble in aqueous solution; the solution becomes clear only after chymotrypsin digestion. A similar situation was observed with azido-Q labeled-cytochrome b (23).
Fig. 6 shows the radioactivity distribution among the
chymotryptic peptides of QPs3 separated by HPLC. Most of the
radioactivity was found in fraction 66 (P-66). It should be mentioned
that the HPLC chromatograms and radioactivity distribution of the
chymotryptic peptides of QPs2 are identical to those of QPs3 (data not
shown).
When P-66 from QPs3 was sequenced, a partial NH2-terminal sequence of Leu-Asn-Pro-Cys-Ser-Ala-Met-Asp-Tyr, corresponding to residues 29-37 in QPs3, is obtained. An identical sequence is obtained for the radioactivity containing fraction from QPs2. Thus the Q-binding domain in the proposed structure of QPs3, is probably located at the end of transmembrane helix 1, near the C-side of the membrane. Recall that the Q-binding domain of QPs1 is on the M-side of the mitochondrial inner membrane.
The finding that the Q-binding domains in QPs3 and QPs1 of bovine succinate-Q reductase are on opposite sides of the membrane is in line with a two-Q binding site hypothesis formulated from inhibitor studies of this enzyme complex (41). The presence of two quinone-binding sites in E. coli fumarate reductase is suggested by mutational studies (39) and by inhibitor kinetics analysis of putative Q-binding site mutants (41). When these two quinone binding sites in E. coli fumarate reductase are incorporated into a proposed mechanism of Q reduction in photoreaction centers (42, 43), Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a QB-type site, and FrdD Phe-57, Glu-59, and Ser-60 in an apolar QA-type site (39). According to the proposed structure of E. coli FrdC and FrdD, the QB-type site is located at the cytoplasmic side and the QA-type site at periplasmic side. If this reasoning is applied to beef heart mitochondrial succinate-Q reductase, the Q-binding domain identified in QPs1 (10) would be the QB-type site and the domain in QPs3 is the QA-type site. More detailed information on Q-binding must await determination of the three-dimensional structure of succinate-Q reductase.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U50987.
We thank Dr. Roger Koeppe for critical review of this manuscript and Dr. Da-Yan He for synthesis of azido-Q derivatives.