(Received for publication, December 6, 1994; and in revised form, January 6, 1995)
From the
An azidoubiquinone derivative,
3-azido-2-methyl5-methoxy[H]-6-decyl-1,4-benzoquinone
([
H]azido-Q), was used to study the
ubiquinone-protein interaction and to identify ubiquinone-binding
proteins in bovine heart mitochondrial succinate-ubiquinone reductase.
When the reductase was incubated with [
H]azido-Q
and illuminated with long wavelength UV light, the decrease in the
enzymatic activity correlated with the amount of azido-Q incorporated
into the protein. When the illuminated,
[
H]azido-Q-treated reductase was extracted with
organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis, radioactivity was found primarily in the QPs1
subunit. The [
H]azido-Q-labeled QPs1 was purified
from labeled reductase by a procedure involving ammonium sulfate
fractionation, dialysis, organic solvent extraction, lyophilization,
preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and cold acetone precipitation. The purified,
[
H]azido-Q-labeled QPs1 protein was subjected to
reductive carboxymethylation prior to digestion by trypsin. One
azido-Q-linked peptide, with a retention time of 66.9 min, was obtained
by high performance liquid chromatographic separation. The partial
amino-terminal sequence of this peptide is GLTISQL-, indicating that
this tryptic peptide comprises amino acid residues 113-140 of the
revised amino acid sequence of QPs1. The Q-binding domain, using the
proposed structure of QPs1, is probably located in the stretch
connecting transmembrane helices 2 and 3 that extrude from the surface
of the M side of the inner membrane.
Bovine heart mitochondrial succinate-ubiquinone reductase, which
catalyzes electron transfer from succinate to ubiquinone (Q), ()is composed of two parts: a soluble succinate
dehydrogenase and a membrane-anchoring protein
fraction(1, 2) . Reconstitutively active succinate
dehydrogenase was first isolated from a Keilin-Hartree preparation by
Keilin and King (3) in 1958. However, pure, reconstitutively
active succinate dehydrogenase was not available until
1971(4) . Succinate dehydrogenase contains two protein subunits
with apparent molecular masses of 70 and 27 kDa. The 70-kDa
flavoprotein is covalently linked to FAD, whereas the 27-kDa
iron-sulfur protein houses all the iron-sulfur clusters. The amino acid
sequences of these proteins were obtained by peptide (5) and
nucleotide sequencing(6) . Biochemical and biophysical
characterization of succinate dehydrogenase has generated a lot of
information(1, 2) .
The reconstitutively active,
membrane-anchoring protein fraction has been isolated and characterized
in several laboratories(7, 8, 9) , under
different names, such as CII-3 and -4(7) , cytochrome b fraction(8) , and QPs(9) . All
these preparations show two protein bands in the SDS-PAGE system of
Weber and Osborn(10) . The two-subunit QPs is further resolved
into three protein subunits (11) by the high resolution
SDS-PAGE system of Schägger et al. (12).
The apparent molecular masses of these three subunits are 14, 11, and 9
kDa. They are named QPs1, QPs2, and QPs3, respectively. The function of
QPs is to provide membrane docking for succinate dehydrogenase (7, 8, 9) and the Q-binding site for
succinate-Q reductase(13, 14) . Soluble succinate
dehydrogenase catalyzes electron transfer from succinate to redox dyes,
such as phenazenemethosulfate and ferricyanide(15) . It cannot
catalyze the 2-thenoyltrifluoroacetone (TTFA)-sensitive electron
transfer from succinate to ubiquinone. Addition of QPs to soluble
succinate dehydrogenase converts succinate dehydrogenase to the
membrane-bound, TTFA-sensitive succinate-Q reductase with detectable
ubisemiquinone radical formation, indicating that QPs provides the
Q-binding site for the complex. It is not clear, at present, whether
one, two, or three QPs protein subunits are required for membrane
docking and for the Q-binding functions of QPs, because attempts to
dissociate QPs into active subunits have, so far, been unsuccessful.
QPs1 is believed to house the cytochrome b heme. The role of cytochrome b
in electron
transfer within the succinate-Q reductase region has yet to be
established. The failure of succinate to reduce cytochrome b
in isolated QPs or in the succinate-Q
reductase complex (16) led some investigators to rule out a
catalytic function for cytochrome b
in
succinate-Q reductase. The presence of a substoichiometric amounts of
cytochrome b
, with respect to FAD, in isolated
succinate-Q and succinate-cytochrome c reductase (16) also raises questions as to the catalytic role of
cytochrome b
. On the other hand, it has been
proposed that cytochrome b
functions as a
mediator between low potential F1/F1
and Q/Q
couples in a
dual pathway model of electron flow through cardiac Complex
II(17) . Despite its uncertain catalytic role, cytochrome b
is involved in the binding of succinate
dehydrogenase to QPs as indicated by the restoration of absorption
properties, redox potential, and EPR characteristics of cytochrome b
, upon reconstitution of succinate-ubiquinone
reductase from isolated QPs and succinate dehydrogenase(18) .
The involvement of QPs1 in the Q-binding site for succinate-Q reductase
is established herein by photoaffinity labeling technique with
[
H]azido-Q derivative. Availability of the QPs1
amino acid sequence, deduced from nucleotide sequencing(11) ,
aids in the identification of the Q-binding domain in this protein
subunit. Herein we report the conditions for photoaffinity labeling of
Q-binding protein, in succinate-Q reductase, with the azidoQ
derivative, and the detailed isolation procedure for the Q-binding
domain in QPs1.
The ubiquinone
derivatives, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone
(QC
), 3-azido-2-methoxy- and
3-azido-2-methoxy[
H]-5-methyl-6-decyl-1,4-benzoquinones
(3-azido-Q
C
and
[
H]3-azido-Q
C
),
5-azido-2,3-dimethoxy- and
5-azido-2,3-dimethoxy[
H]-6-decyl-1,4-benzoquinones
(5-azido-Q
C
and
[
H]-5-azido-Q
C
), and
3-azido-2-methyl-5-methoxy- and
3-azido-2-methyl-5-methoxy[
H]-6-decyl-1,4-benzoquinones
(azido-Q and [
H]azido-Q) were synthesized
according to methods reported previously(19) . Calcium
phosphate was prepared according to Jenner (20) and mixed at a
3:1 ratio with cellulose powder prior to use in column chromatography.
Succinate-Q reductase activity was assayed for its
ability to catalyze Q reduction or Q-stimulated DCPIP reduction by
succinate. The assays were performed at room temperature in a Cary
spectrophotometer (model 219) or Shimadzu UV-2101PC. The reaction
mixture used for the Q reduction assay contains 100 µmol of
sodium/potassium phosphate buffer, pH 7.4, 20 µmol of succinate, 10
nmol of EDTA, 25 nmol of QC
, and 0.01% n-dodecyl-
-maltoside in the total volume of 1 ml. The
reduction of Q
C
was followed by measuring the
absorption decrease at 275 nm, using a millimolar extinction
coefficient of 12.25 mmol
cm
. The
reaction mixture used for the Q-stimulated DCPIP reduction assay
contains 38 µmol of DCPIP, 100 µmol of potassium/sodium
phosphate buffer, pH 7.4, 20 µmol of succinate, 10 nmol of EDTA, 25
nmol of Q
C
, and 0.01% Triton X-100 in the
total volume of 1 ml. The reduction of DCPIP was followed by measuring
the absorption decrease at 600 nm, using a millimolar extinction
coefficient of 21 mmol
cm
.
The illuminated
[H]azido-Q-treated succinate-Q reductase was
precipitated by 43% ammonium sulfate saturation and centrifuged at
48,000
g for 20 min. The precipitate was dissolved in
10 mM Tris-Cl buffer, pH 7.8, and dialyzed against
double-distilled water, overnight, with one change of water. The free
Q, or the phospholipid bound Q, was extracted from the protein by
organic solvent(19) . The dialyzed sample (0.8 ml) was mixed
with 2 ml of methanol and 1 ml chloroform and incubated at room
temperature for 30 min with occasional shaking. After incubation, 1 ml
of H
O and 1 ml of chloroform were added, and the mixture
was kept at room temperature for another 10 min with occasional
shaking. The chloroform layer was separated from the aqueous layer by
centrifugation and discarded. The aqueous layer was extracted once more
with 1 ml of chloroform, and the chloroform layer was removed. The
methanol in the aqueous layer was evaporated under a stream of nitrogen
gas before the solution, which contains the photolyzed protein, was
subjected to lyophilization. The lyophilized sample was suspended in 20
mM Tris-Cl, pH 8.0, containing 1% SDS. SDS (5 mg/mg protein)
and
-mercaptoethanol (1%) were added, and the solution was
incubated at 37 °C for 2 h before it was subjected to preparative
SDS-PAGE. The SDS-PAGE gel was prepared according to
Schägger et al.(12) except 7 M urea was used in the separating gel instead of 13% glycerol.
The SDS-
-mercaptoethanol-digested sample was loaded onto a gel
slab in two strips sandwiched with three reference wells (one on each
side and one at the center) loaded with the digested samples treated
with fluorescamine. The electrophoresis was run at 15 V for 2 h and
then at 35 V for another 17 h. The protein bands were visualized by
fluorescence under UV. The SDS-PAGE pattern of the
fluorescamine-treated sample was identical to that of the untreated
sample as established by Coomassie Blue staining. The QPs1 protein band
was excised from the SDS-PAGE gel, and the protein was eluted with an
electroeluter from Bio-Rad.
The succinate-free succinate-Q reductase was prepared by replacing Tris-succinate buffer with Tris-Cl buffer during the separation of succinate-Q reductase from ubiquinol-cytochrome c reductase in Triton X-100-treated succinate-cytochrome c reductase. Succinate was omitted from the subsequent buffers used in calcium phosphate column chromatography.
It has been shown that the interaction of azido-Q derivatives with
the cytochrome b-c complexes from various sources (19, 23, 24) requires prior removal of
endogenous Q from the complex. The reason for this is that the binding
affinity of azido-Q derivatives to the Q-binding proteins (sites) is
weaker than that of endogenous Q
. The succinate-Q
reductase, prepared according to the procedure described(16) ,
is 40-50% deficient in Q content. The Q deficiency in succinate-Q
reductase preparations is measured by the extent of activity increase
of Q-stimulated DCPIP reduction by succinate in the presence of
Q
C
(25 µM) in the assay mixture.
Attempts to further remove Q from succinate-Q reductase preparations by
prolonged washing of the second calcium phosphate-cellulose column or
by repeating the ammonium sulfate fractionation in the presence of 0.5%
sodium cholate, were unsuccessful. Therefore, a partially Q-deficient,
succinate-free succinate-Q reductase was used in this study.
In order to use these azido-Q
derivatives to identify the Q-binding proteins and to facilitate the
study of the Q:protein interaction, it is necessary to synthesize them
in a radioactive form.
3-Azido-2-methoxy[H]-5-methyl-6-decyl-1,4-benzoquinone
([
H]3-azido-Q
C
)
5-azido-2,3-dimethoxy[
H]-6-decyl-1,4-benzoquinone
([
H]5-azido-Q
C
), and
3-azido-2-methyl-5-methoxy[
H]-6-decyl-1,4-benzoquinone
([
H]azido-Q) were prepared by methylation of the
hydroxyl group(s) on 3-H-2-hydroxy5-methyl-,
5-H-2,3-dihydroxy-, and
3-H-2-methyl-5-hydroxy-6-decyl-1,4-benzoquinones,
respectively, with C[
H]
I before being
subjected to neucleophilic substitution with NaN
. These
[
H]azido-Q derivatives have the same electron
acceptor activity for succinate-Q reductase as their unlabeled
compounds (Table 1). However, when succinate-Q reductase was
incubated with 40 M excess of these
[
H]azido-Q derivatives for 10 min at 0 °C in
the dark prior to illumination with a long wavelength UV light for 7
min, only the
3-azido-2-methyl-5-methoxy[
H]-6-decyl-1,4-benzoquinone-treated
sample showed inactivation and radioactivity uptake by protein (Table 1), indicating that this [
H]azido-Q
derivative is suitable for studying the Q:protein interaction in
succinate-Q reductase. The failure to detect
3-azido-Q
C
and 5-azido-Q
C
uptake by succinate-Q reductase during illumination is most
likely due to intramolecular cyclization, between the generated nitrene
and its neighboring methoxy or methylene group on the Q molecule,
during illumination.
3-Azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone has been
successfully used to identify the Q-binding site in cytochrome b-c
complexes from several
sources(19, 23, 24) .
Figure 1:
Effect of azido-Q concentration on
succinate-Q reductase activity after illumination. Aliquots (0.2 ml) of
succinate-Q reductase, 1 mg/ml (5.5 µM FAD), in 50 mM phosphate buffer, pH 7.8, containing 10% glycerol and 0.2% sodium
cholate were mixed with 5 µl of an alcoholic solution of azido-Q
derivative (concentrations indicated), in the dark. After incubation at
0 °C for 10 min, the samples were illuminated for 7 min at 0
°C. Succinate-Q reductase activity was assayed before () and
after (
) illumination. 100% activity is succinate-Q reductase
without treatment with azido-Q and without
illumination.
If azido-Q can
occupy 35% of the Q-binding sites in the reductase, one would expect to
see a decrease in the activity of the azido-Q-treated sample before
illumination, because this derivative has less than 10% of the electron
transfer efficiency of QC
. The failure to
observe a decrease in activity prior to illumination is due to the fact
that the concentration of Q
C
in the assay
mixture is several orders of magnitude higher than that of azido-Q.
Thus Q
C
can easily displace azido-Q from the
binding sites, and the inferior electron transfer activity of azido-Q
is not expressed. After illumination, the covalently linked azido-Q
cannot be displaced by Q
C
and inhibition
occurs.
Figure 2:
Distribution of radioactivity among
subunits of succinate-Q reductase. 0.2 ml of succinate-Q reductase, 1.5
mg/ml, in 50 mM phosphate buffer, pH 7.8, containing 10%
glycerol and 0.2% sodium cholate were mixed with 10 µl of
[H]azido-Q (13.5 mM in 95% ethanol) in
the dark. After incubation at 0 °C for 10 min, the sample was
illuminated for 7 min at 0 °C. The illuminated sample was extracted
with a mixture of chloroform/methanol (2:1) and lyophilized. The
lyophilized sample was dissolved in 20 mM Tris-Cl buffer, pH
8.0, containing 1% SDS and 1%
-mercaptoethanol and incubated at 37
°C for 2 h. The digested sample was treated with fluorescamine and
applied to SDS-PAGE gel. The protein bands were visualized by
fluorescence under UV and sliced. The portion containing no protein was
also sliced to the same size as that of protein bands. The gel slices
were smashed and mixed with 5 ml of Insta-Gel, and the radioactivity
was determined.
Although lesser amounts of radioactivity were found in QPs2 and QPs3, involvement of these two subunits in formation of the Q-binding site of succinate-Q reductase cannot be ruled out. The smaller azido-Q uptake by QPs2 and QPs3, compared with QPs1, may result from portions of the Q-binding sites being contributed by these two subunits, or from the preferable occupation of these two subunits by the endogenous Q present in the partially Q-deficient succinate-Q reductase used in this investigation. The role of QPs2 and QPs3 in Q-binding needs further investigation. The availability of a reconstitutively active, Q-depleted succinate-Q reductase, and of the amino acid sequences of QPs2 and QPs3, would greatly help in the assessment of the involvement of QPs2 and QPs3 in Q-binding of succinate-Q reductase.
Figure 3:
SDS-PAGE of purified
[H]azido-Q-labeled QPs1. Lane 1,
electrophoretically eluted QPs1; lane 2, succinate-Q
reductase, and lane 3, molecular weight standards
(phosphorylase b, 97,400; bovine serum albumin, 66,200;
ovalbumin, 45,000; carbonic anhydrase, 31,000; soybean trypsin
inhibitor, 21,500; and lysozyme, 14,400).
That isolated azido-Q labeled QPs1 was highly aggregated and resistant to proteolytic digestion. Inclusion of 0.1% SDS and 2 M urea in the digestion mixture does not increase proteolysis efficiency. Modification of isolated azido-Q-labeled QPs1 protein by reductive carboxymethylation rendered the protein susceptible to trypsin and chymotrypsin digestion. The reductive carboxymethylation step was carried out in 50 mM Tris-Cl buffer, pH 8.3, containing 6 M guanidinium chloride. Guanidinium chloride (6 M) was found to be more effective than 50% N,N-dimethylformamide (21) in denaturation and in reduction of QPs1 with dithiothreitol.
Figure 4:
H radioactivity distribution
on HPLC chromatogram of trypsin-digested,
[
H]azido-Q-labeled QPs1 protein. The
[
H]azido-Q-labeled QPs1 (1 mg/ml, 7.8
10
cpm/mg) was digested with trypsin, and 100-µl
aliquots of digested solution were subjected to HPLC separation as
described under ``Experimental Procedures.''
One-hundred-µl aliquots were withdrawn from each tube for
radioactivity determination.
The partial NH-terminal amino acid
sequence of P-67 was determined to be GLTISQL-, which corresponds to
amino acid residues 105-111 in our previously reported sequence (11) and 113-119 in the revised amino acid sequence (Fig. 5). The only difference between the reported and revised
amino acid sequences of QPs1 is that the latter has an additional 8
amino acid residues, LGTTAKEE, on the NH
terminus. In other
words, the coding sequence for mature QPs1 starts 7 base pairs, instead
of 31 base pairs, downstream from the 5` end of our isolated QPs1 cDNA
clone(11) . The eight amino acid residues on the NH
terminus were added upon determination of the partial
NH
-terminal amino acid sequence: LGTTAKEEMER. The use of
pre-electrophoresed SDS-PAGE gel and inclusion of 0.1 mM sodium thioglycolate in the running buffer prevented blockage of
the NH
terminus during electrophoresis, thus make
sequencing possible. The molecular weight of the mature QPs1 is 15,149.
Apparently the cDNA clone for QPs1 isolated in our laboratory is a
partial clone; it contains a complete sequence for mature QPs1, but
lacks some amino acid residues of the leading peptide of QPs1. The
coding sequence for the leading peptide of QPs1 should start with ATG
and end with CCT upstream from TTC which encodes the
NH
-terminal amino acid residue of mature QPs1. Thus, the
isolated QPs1 clone contains only two amino acid residues, proline
(Pro) and valine (Val), from the COOH-terminal end of the leading
peptide. A cDNA clone for QPs1, containing the complete sequence for
the leading peptide, could be obtained by screening the bovine heart
cDNA library in
gt11 with a biotinylated DNA probe prepared by
nick translation using the obtained cDNA clone as the template. Since
for structure-function studies of QPs1, a cDNA clone containing the
presequence peptide is unnecessary, no effort was made to obtain it at
the present time.
Figure 5:
Proposed structure of QPs1. The amino acid
sequence of QPs1 was revised by the addition of 8 amino acid residues,
LGTTAKEE, to the NH-terminal end of our previously reported
sequence(11) .
Although the isolated azido-Q linked peptide
(P-67) was from a tryptic peptide containing amino acid residues
113-140 of QPs1, the Q-binding domain, using the proposed
structure of QPs1 (Fig. 5), is most likely located in the
sequence connecting transmembrane helices 2 and 3. This region of the
peptide is extruded from the surface of the M side of the inner
membrane. Threonine 115 is probably the amino acid covalently linked to
azido-Q upon illumination. This deduction is based on finding that the
intensity of the threonine peak is much less than that of the other
amino acid peaks during sequencing of P-67. Neighboring amino acid
residues, such as His, Trp
and
Asp
, probably participate in the formation of Q-binding
site. More detailed information on Q binding must await determination
of three-dimensional structure of succinate-Q reductase. The
information obtained from this study, however, can serve as a basis for
future mutagenic studies to identify the essential amino acid residues
involved in Q-binding.