©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Ubiquinone-binding Domain in QPs1 of Succinate-Ubiquinone Reductase (*)

(Received for publication, December 6, 1994; and in revised form, January 6, 1995)

Gyesoon Yoon Lee Da-Yan He Linda Yu Chang-An Yu

From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An azidoubiquinone derivative, 3-azido-2-methyl5-methoxy[^3H]-6-decyl-1,4-benzoquinone ([^3H]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 [^3H]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, [^3H]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 [^3H]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, [^3H]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.


INTRODUCTION

Bovine heart mitochondrial succinate-ubiquinone reductase, which catalyzes electron transfer from succinate to ubiquinone (Q), (^1)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/F1bullet and Q/Qbullet 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 [^3H]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.


EXPERIMENTAL PROCEDURES

Materials

Bovine serum albumin, dichlorophenolindophenol (DCPIP), Triton X-100, sodium cholate, TTFA, and potassium deoxycholate were obtained from Sigma. SDS, molecular weight standards, and urea were from Bio-Rad. n-Dodecyl-beta-D-maltoside was from Anatrace. Silica gel 1B-F (2.5 times 7.5 cm) was from J. T. Baker. Insta-gel liquid scintillation mixture was from Packard Instrument Co. Other chemicals were of the highest purity commercially available.

The ubiquinone derivatives, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q(0)C), 3-azido-2-methoxy- and 3-azido-2-methoxy[^3H]-5-methyl-6-decyl-1,4-benzoquinones (3-azido-Q(0)C and [^3H]3-azido-Q(0)C), 5-azido-2,3-dimethoxy- and 5-azido-2,3-dimethoxy[^3H]-6-decyl-1,4-benzoquinones (5-azido-Q(0)C and [^3H]-5-azido-Q(0)C), and 3-azido-2-methyl-5-methoxy- and 3-azido-2-methyl-5-methoxy[^3H]-6-decyl-1,4-benzoquinones (azido-Q and [^3H]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.

Enzyme Preparations and Assays

Bovine heart succinate-Q reductase was prepared from succinate-cytochrome c reductase by the method reported previously (16) except that succinate was omitted from all buffers used in purification procedure. Succinate-Q reductase containing fractions obtained from the second calcium phosphate-cellulose column were concentrated by 43% ammonium sulfate saturation and centrifugation at 48,000 times g for 20 min. The precipitate obtained was dissolved in 50 mM potassium/sodium phosphate, pH 7.8, containing 0.2% sodium cholate and 10% glycerol.

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 Q(0)C, and 0.01% n-dodecyl-beta-maltoside in the total volume of 1 ml. The reduction of Q(0)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(0)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.

Isolation of [^3H]Azido-Q-labeled QPs1

10 ml of succinate-Q reductase, 1.5 mg/ml, in 50 mM sodium/potassium phosphate buffer, pH 7.8, containing 10% glycerol and 0.2% sodium cholate were incubated with 125 µl of [^3H]azido-Q (9.6 mM in 95% ethanol) at 0 °C for 10 min in the dark. The specific radioactivity of azido-Q used was 1.6 times 10^4 in 95% ethanol and 6.6 times 10^3 cpm/nmol in 50 mM potassium/sodium phosphate buffer, pH 7.8, containing 10% glycerol and 0.2% sodium cholate in the presence of succinate-Q reductase. This mixture was transferred to an illumination apparatus made from two quartz glasses sandwiched by a Teflon ring. The apparatus was immersed in cold water in a Petri dish surrounded with salted ice to maintain the temperature of the water at 0 °C. The sample was illuminated with long wavelength UV light for 7 min at a distance of 5 cm from the light source.

The illuminated [^3H]azido-Q-treated succinate-Q reductase was precipitated by 43% ammonium sulfate saturation and centrifuged at 48,000 times 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(2)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 beta-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-beta-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.

Reductive Carboxymethylation of Azido-Q-labeled QPs1

The QPs1 protein solution was subjected to reductive carboxymethylation by a modification of the method reported previously(21) . The electrophoretically eluted QPs1 protein solution was concentrated by membrane filtration, using Centricon-10, to a protein concentration of about 3 mg/ml and precipitated with 50% cold acetone (-20 °C). The protein precipitate was suspended in 1 ml of cold water and centrifuged at 100,000 times g for 1 h to remove the trace acetone remaining in the protein. The precipitate thus obtained was homogenized in 0.1 M Tris, 6 M guanidine HCl, 1 mM EDTA, pH 8.3. Dithiothreitol was added to the suspension to a final concentration of 2 mM. The solution was made anaerobic by passing argon through it and then incubated at 37 °C for 1 h. Iodoacetic acid, neutralized with NaOH, was added to give a concentration of 5 mM. Argon was flushed over the surface of the reaction mixture, the vessel was sealed, and alkylation was allowed to go on in the dark at 37 °C for 1 h. Complete alkylation was achieved by an additional treatment with 1 mM dithiothreitol and incubation with 2 mM iodoacetate under the same conditions. The alkylated protein solution was treated with beta-mercaptoethanol to a final concentration of 1% and dialyzed against 50 mM ammonium bicarbonate, pH 8.5, overnight, at 4 °C, with one change of buffer.

Trypsin Digestion of the Carboxymethylated Azido-Q-labeled QPs1

The carboxymethylated QPs1 protein was collected from the dialyzed sample by centrifugation at 100,000 times g for 30 min. The collected precipitates were suspended in 0.1 M ammonium bicarbonate, 0.1 mM CaCl(2) to a final protein concentration of 1 mg/ml and digested with trypsin at 37 °C for 2 h using a trypsin:QPs1 ratio of 1:50 (w/w). After a 2-h incubation, a second addition of trypsin was made (1:100) and the incubation continued at 37 °C for 22 h.

Isolation of Ubiquinone-binding Peptides

One-hundred-microliter aliquots of the trypsin-digested QPs1 were separated by high performance liquid chromatography (HPLC) on a Synchropak RP-8 column (0.46 times 25 cm) using a gradient formed from 0.1% trifluoroacetic acid and 90% acetonitrile containing 0.1% trifluoroacetic acid with a flow rate of 1 ml/min. One-milliliter fractions were collected. The absorbance at 214 nm and the radioactivity of each fraction were measured.

Electroblotting of Succinate-Q Reductase Protein to Polyvinylidene Difluoride Membrane for Microsequencing of QPs1

Succinate-Q reductase was digested with 1% beta-mercaptoethanol and 1% SDS for 2 h at 37 °C. 20-µl aliquots of the digested samples were applied to wells of an SDS-PAGE gel which had been pre-electrophoresed for overnight at 45 V with an anode buffer containing 0.2 M Tris-Cl, pH 8.9, and 0.1 mM sodium thioglycolate and a cathode buffer containing 0.1 M Tris, 0.1 M Tricine, 0.1% SDS, and 0.1 mM sodium thioglycolate, pH 8.2. The proteins in SDS-PAGE gel were electroblotted to a polyvinylidene difluoride membrane according to the procedure of LeGendre et al.(22) with CAPS transfer buffer (10 mM CAPS, 10% methanol, pH 11) for 15 min. After the transfer was complete, the membrane was rinsed several times with HPLC-grade H(2)O before being stained with Coomassie Blue R-250 (0.1% in 50% methanol) for 2 min to visualize the proteins bound to the membrane. The blue band corresponding to QPs1 protein was cut out and subjected to NH(2)-terminal sequence analysis.

Amino Acid Sequence Determination

Amino acid sequence 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.


RESULTS AND DISCUSSION

Preparation of Succinate-Q Reductase

Succinate, the substrate for succinate dehydrogenase, has been routinely included in the preparation of succinate-Q reductase to stabilize the enzyme complex. However, the succinate-containing succinate-Q reductase preparation is not suitable for studying Q:protein interactions using the azido-Q derivative because in the presence of succinate, the azido-Q derivative is slowly reduced by succinate-Q reductase to aminoquinol, thus losing its photoaffinity labeling ability. Although the rate of conversion of azido-Q to aminoquinol by succinate-Q reductase in the presence of succinate is very slow, the reaction is completed within 20 min at 0 °C in the dark when the molar ratio of azido-Q to succinate-Q reductase in the system is less than 50. The conversion of azido-Q to aminoquinol was evident from the change of absorption characteristics and of mobility on TLC. Upon reduction, a purple spot with the R(F) value of 0.57 (aminoquinol) was observed, with concurrent disappearance of a yellow spot with the R(F) value of 0.90 (azido-Q), on a TLC plate developed with a hexane:ether (2.5:1) mixture. When a succinate-free succinate-Q reductase was incubated with 10-fold molar excess of azido-Q, no purple spot was observed on the TLC plate after 20-min incubation at 0 °C in the dark.

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(1) 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(0)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.

Properties of Azido-Q Derivatives

Three azido-Q derivatives, 3-azido-2-methoxy-5-methyl-6-decyl-1,4-benzoquinone (3-azido-Q(0)C), 5-azido-2,3-dimethoxy-6-decyl-1,4-benzoquinone (5-azido-Q(0)C), and 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone (azido-Q), were synthesized and tested for their suitability for identifying Q-binding sites in succinate-Q reductase. All these derivatives exhibit partial electron acceptor activity for succinate-Q reductase. The maximal activity was 35, 23, and 8% for 5-azido-Q(0)C, 3-azido-Q(0)C, and azido-Q, respectively, compared with 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q(0)C).

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[^3H]-5-methyl-6-decyl-1,4-benzoquinone ([^3H]3-azido-Q(0)C) 5-azido-2,3-dimethoxy[^3H]-6-decyl-1,4-benzoquinone ([^3H]5-azido-Q(0)C), and 3-azido-2-methyl-5-methoxy[^3H]-6-decyl-1,4-benzoquinone ([^3H]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[^3H](3)I before being subjected to neucleophilic substitution with NaN(3). These [^3H]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 [^3H]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[^3H]-6-decyl-1,4-benzoquinone-treated sample showed inactivation and radioactivity uptake by protein (Table 1), indicating that this [^3H]azido-Q derivative is suitable for studying the Q:protein interaction in succinate-Q reductase. The failure to detect 3-azido-Q(0)C and 5-azido-Q(0)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(1) complexes from several sources(19, 23, 24) .



Effect of Azido-Q Concentration on Succinate-Q Reductase Activity after Illumination

When succinate-Q reductase was incubated with various concentrations of 3-azido-2-methyl-5-methoxy[^3H]-6-decyl-1,4-benzoquinone ([^3H]azido-Q) and illuminated, the activity decreased as the concentration of azido-Q increased. Maximum inactivation of approximately 35% was obtained when 40 mol of azido-Q/mol of FAD was used (Fig. 1). Inactivation was not due to inhibition of succinate-Q reductase by photolyzed products of azido-Q, because when azido-Q was photolyzed in the absence of reductase and then added to succinate-Q reductase, no inhibition was observed. The maximum inactivation of 35%, which is in close agreement with the 40-50% deficiency of Q in the preparation, suggests that the affinity of endogenous Q for its binding site is much stronger than that of the azido-Q derivative. Since succinate-Q reductase activity is assayed in the presence of excess Q(0)C (25 µM), the extent of inactivation of the azido-Q-treated succinate-Q reductase, after illumination, is a measure of the fraction of Q-binding sites covalently linked to azido-Q.


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 (bullet) and after (circle) 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 Q(0)C. The failure to observe a decrease in activity prior to illumination is due to the fact that the concentration of Q(0)C in the assay mixture is several orders of magnitude higher than that of azido-Q. Thus Q(0)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(0)C and inhibition occurs.

Correlation between Azido-Q Incorporation and Inactivation of Succinate-Q Reductase

When azido-Q-treated succinate-Q reductase was illuminated at 0 °C for various lengths of time, activity decreased as illumination time was increased; maximum inactivation (35%) was reached in 7 min. The amount of azido-Q uptake by protein paralleled the extent of inactivation until the maximum was reached, indicating that inactivation results from binding of azido-Q to the ubiquinone binding site. Although illuminating for longer than 7 min caused no further decrease in activity, azido-Q uptake continued, but at a much slower rate, suggesting that this slower incorporation is due to nonspecific binding of azido-Q to proteins. The photoinactivation rate of succinate-Q reductase was affected by the protein, alcohol, and detergent concentrations in the system. Interaction with azido-Q derivative was most effective (i.e. showed the greatest degree of inactivation after illumination) when the reaction system contained succinate-Q reductase, 1-1.5 mg/ml, sodium cholate, 0.2%, and alcohol, <3%.

Radioactivity Distribution of Azido-Q among Subunits of Succinate-Q Reductase

Since the uptake of azido-Q derivative by succinate-Q reductase during illumination correlated with the enzymatic inactivation, it is reasonable to assume that the azido-Q derivative is bound specifically to the Q-binding site(s). Thus, the distribution of the covalently bound azido-Q among the subunits of succinate-Q reductase after SDS-PAGE should indicate the specific Q-binding protein in this enzyme complex. Fig. 2shows the ^3H radioactivity distribution among subunits of succinate-Q reductase. Prior to SDS-PAGE the reductase was extracted with organic solvent to remove most of the non-protein-bound azido-Q, such as free azido-Q or detergent-azido-Q or lipid-azido-Q adducts. Radioactivity was found predominately in QPs1, suggesting that this protein provides the Q-binding site in this segment of the electron transfer chain. Some radioactivity was found in QPs2 and QPs3; about 45 and 50% of that observed in QPs1. No radioactivity was found in the subunits of succinate dehydrogenase. Since the amount of radioactivity labeling in QPs1 was proportional to the extent of inactivation of the reductase, participation of QPs1 in Q-binding is established.


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 [^3H]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% beta-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.

Preparation and Properties of Reductive Carboxymethylated Azido-Q-labeled QPs1

In order to facilitate the identification of the Q-binding domain in QPs1 through isolation and sequencing of an azido-Q-linked peptide, it is absolutely necessary to have isolated azido-Q-labeled QPs1 free from contamination with unbound azido-Q and completely susceptible to proteolytic enzyme digestion. The [^3H]azido-Qlabeled QPs1 was isolated from illuminated, [^3H]azido-Q-treated succinate-Q reductase by a procedure involving ammonium sulfate fractionation, dialysis, organic solvent extraction, lyophilization, preparative SDS-PAGE, electrophoretic elution, and cold acetone precipitation. Three steps in the isolation procedure, organic solvent extraction, SDS-PAGE, and 50% cold acetone precipitation, are used to remove non-protein-bound azido-Q, some of which occurs as adducts with detergent or phospholipid. The electrophoretically eluted QPs1 shows only one band in SDS-PAGE (see lane 1 of Fig. 3). The SDS, free azido-Q, and detergent-azido-Q adducts present in eluted QPs1 preparation was removed by the acetone precipitation step. About 35% of the QPs1 protein present in succinate-Q reductase was recovered in the final purification step, assuming that the molecular mass of succinate-Q reductase is 136 kDa and that it contains 1 mol of QPs1/mol of reductase. This relatively low yield of QPs1 is probably due to a very thin slicing of the QPs1 band from SDS-PAGE gel to avoid contamination with QPs2 protein.


Figure 3: SDS-PAGE of purified [^3H]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.

Isolation and Characterization of Ubiquinone-binding Peptides of QPs1

Fig. 4shows radioactivity distribution among the tryptic peptides of QPs1 separated by HPLC. When carboxymethylated [^3H]azido-Q-labeled QPs1 protein was digested with trypsin followed by HPLC separation using a Synchropak RP-8 column eluted with a gradient formed from 0.1% trifluoroacetic acid and 90% acetonitrile in 0.1% trifluoroacetic acid, the majority of the radioactivity was found in a fraction with retention time of 66.9 min (P-67). Although the presence of 0.1% trifluoroacetic acid in the buffer system greatly increased the peptide resolution of QPs1 on HPLC, these acid conditions cause a partial release of radioactivity during chromatography(21) . Radioactivity recovery from the HPLC separation of trypsin-digested QPs1 is about 62%.


Figure 4: ^3H radioactivity distribution on HPLC chromatogram of trypsin-digested, [^3H]azido-Q-labeled QPs1 protein. The [^3H]azido-Q-labeled QPs1 (1 mg/ml, 7.8 times 10^4 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(2)-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(2) 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(2) terminus were added upon determination of the partial NH(2)-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(2) 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(2)-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(2)-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM30721. This publication is approved by the director of the Agricultural Experiment Station, Oklahoma State University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: Q, ubiquinone; DCPIP, dichlorophenolindophenol; Q(0)C, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; 3-azido-Q(0)C, 3-azido-2-methoxy-5-methyl-6-decyl-1,4-benzoquinone; 5-azido-Q(0)C, 5-azido-2,3-dimethoxy-6-decyl-1,4-benzoquinone; azidoQ, 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone; [^3H]azidoQ, 3-azido-2-methyl-5-methoxy[^3H]-6-decyl-1,4-benzoquinone; PAGE, polyacrylamide gel electrophoresis; TTFA, 2-thenoyltrifluoroacetone; HPLC, high performance liquid chromatography; CAPS, 3-(cyclohexylamino)propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Dr. R. A. Capaldi for calling our attention to the revised coding sequence for QPs1 in the QPs1 cDNA clone and Dr. Roger Koeppe for critical review of this manuscript.


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