6298
Biochemistry 1981, 20, 6298-6305
Further Characterization of Nucleotide Binding Sites on Chloroplast Coupling Factor One+ Michael F. Bruistt and Gordon G. Hammes*
ABSTRACT: The solubilized coupling factor from spinach chloroplasts (CF,) contains one nondissociable ADP/CF, which exchanges slowly with medium ADP in the presence of Ca2+,Mg2+,or EDTA; medium ATP also exchanges in the presence of Ca2+or EDTA, but it is hydrolyzed, and only ADP is found bound to CF,. The rate of ATP exchange with heat-activated CF1 is approximately 1000 times slower than the rate of ATP hydrolysis. In the presence of Mg2+, both latent CFI and heat-activated CFl bind one ATP/CFI, in addition to the ADP. This MgATP is not removed by dialysis, by gel filtration, or by the substrate CaATP during catalytic turnover; however, it is released when the enzyme is stored several days as an ammonium sulfate precipitate. The photoaffinity label 3'-0-[ 3-[N-(4-azido-2-nitrophenyl)amino]propionyl]-ATP binds to the MgATP site, and photolysis results in labeling of the 0subunit of CF1. Equilibrium binding
measurements indicate that CFI has two identical binding sites for ADP with a dissociation constant of 3.9 pM (in addition to the nondissociable ADP site). When MgATP is bound to CFI, one ADP binding site with a dissociation constant of 2.9 pM is found. One ATP binding site is found in addition to the MgATP site with a dissociation constant of 2.9 pM. Reaction of CF1 with the photoaffinity label 3'-0-[3-[N-(4azido-2-nitrophenyl)amino]propionyl]-ADP indicates that the ADP binding site which is not blocked by MgATP is located near the interface of CY and 0subunits. No additional binding sites with dissociation constants less than 200 pM are observed for MgATP with latent CF1 and for CaADP with heat-activated CFI. Thus, three distinct nucleotide binding sites can be identified on CFI, and the tightly bound ADP and MgATP are not at the catalytic site. The active site is either the third ADP and ATP binding site or a site not yet detected.
C o u p l i n g factor 1 (CF,)' is a soluble portion of the ATP synthesizing complex from chloroplasts and contains the catalytic site. The synthesis of ATP requires a pH gradient or potential across the membrane, and Mg2+ is the normal cofactor. While isolated CF1 cannot synthesize ATP, it hydrolyzes ATP when activated by various methods (Vambutas & Racker, 1965). In contrast to the synthetic reaction, Ca2+ is a much more effective cofactor than Mg2+for this ATPase activity. Five different types of subunits are found in CFI, with a probable stoichiometry of (Binder et al., 1978; Baird & Hammes, 1976). Only the CY and 0 subunits are required for ATPase activity (Deters et al., 1975), but the y subunit is also involved in photophosphorylation (Nelson et al., 1972). The structural relationships of the subunits have been studied extensively (cf. Baird & Hammes, 1979). In this work, the interaction of CF1 with ADP and ATP has been studied. Previous studies have shown that CFl has several nucleotide binding sites. These sites are responsible for catalysis, for ADP and ATP inhibition of the ATPase activity (Cantley & Hammes, 1975), for modulation of the proton permeability of CF, on the membrane, and for control of susceptibility to inhibitors and uncouplers (McCarty, 1979). A tightly bound ADP of unknown function also has been observed (Harris & Slater, 1975; Magnusson & McCarty, 1976; Carlier & Hammes, 1979). Earlier work from this laboratory demonstrated that CF, reversibly binds two ADP molecules (Cantley & Hammes, 1975) and that the additional tightly bound ADP is nondissociable but exchanges with solution ADP and ATP (Carlier & Hammes, 1979). This study describes a site which binds MgATP very tightly and is distinct from the site which binds ADP tightly. In all, three types of nucleotide binding sites are characterized and can be designated as follows: the nondissociable ADP site contains the
tightly bound ADP which can be exchanged but not removed without denaturing CFI, the MgATP site binds ATP essentially irreversibly in the presence of Mg2+,and the dissociable nucleotide sites bind ADP reversibly in the presence or absence of Mg2+or Ca2+. The relationship of these sites to the catalytic site has been investigated. Materials and Methods Chemicals. The ADP and ATP (vanadium free) were from Sigma Chemical Co. The 3H-labeled ADP and ATP were from New England Nuclear, and the 14C- and 32P-labeled nucleotides from Amersham Corp. The radioactive ADP was purified by paper chromatography using isobutyric acid-1 N ammonia (100:60 v/v). Both radioactive and nonradioactive ATP were purified by column chromatography on Bio-Rad AG-1-X4 C1- (0.4 X 4 cm). The ATP on the column was washed with 50 mM NaCl-10 mM HCl and eluted with 500 mM NaCl-20 mM HCl. The eluant was neutralized with 1 M Tris base, and the ATP was concentrated and desalted by precipitating with 0.2 mL of 2 M barium acetate per 1 mL of solution, washing the precipitate with water, and solubilizing the ATP by the addition of Bio-Rad AG-50W-X8 Lif cation-exchange resin. The 4-fluoro-3-nitroaniline was from Aldrich Chemical Co., and the chemicals for electrophoresis were from Bio-Rad Laboratories. All other chemicals were analytical grade, and all solutions were made with deionized distilled water. Unless indicated, experiments were performed at room temperature, 19 f 2 "C. Enzyme. The CFI was prepared from fresh market spinach (Lien & Racker, 1971; Binder et al., 1978). Enzyme having a 305:340 nm fluorescence ratio (excitation at 280 nm) greater than 1.5 was collected and stored as an ammonium sulfate precipitate in 2 M ammonium sulfate, 10 mM Tris-HCl (pH
'From the Department of Chemistry, Cornel1 University, Ithaca, New 14, 1981. This work was supported by a grant from the National Institutes of Health (GM 13292). *National Institutes of Health Predoctoral Trainee (GM 07273).
Abbreviations used: CF,, chloroplast coupling factor 1; EDTA, ethylenediaminetetraacetic acid; arylazido-ADPor -ATP, 3'-0- [3- [N(4-azido-2-nitrophenyl)amino]propionyl]-ADPor -ATP; Tris, tris(hydroxymethyl)aminomethane.
York 14853. Received April
0006-296018 110420-6298$01.25/0 0 1981 American Chemical Society
NUCLEOTIDE BINDING SITES ON C F I
7.2), 1 mM EDTA, and 0.5 mM ATP. Its purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF1 was activated by heating at 60 OC for 4 rnin in 40 mM ATP, 30 mM Tris-HC1 (pH 8.0), 7 mM dithiothreitol, and 2 mM EDTA (Farron & Racker, 1970), and the ATPase activity was measured on a Radiometer pH stat (Cantley & Hammes, 1975) or by determination of [32P]Piliberated from [ Y ~ ~ P ] A T(Avron, P 1960). The specific activity immediately after activation was 8-12 pmol of Pi/(mg.min) in 50 mM NaC1,6 mM CaC12, 1 mM EDTA, and 0.5 mM ATP at pH 8.0, 20 OC. The extinction coefficient of CFI was determined by dry weight measurements (Kupke & Dorrier, 1978). A value of 0.483 cm2/mg at 277 nm in 50 mM NaCl, 10 mM Tris-HC1 (pH KO), and 1 mM EDTA was obtained and used for determining concentrations. Turbid solutions were clarified by centrifuging at 17OOOg for 20 rnin at room temperature before the absorbance was measured. A molecular weight of 325 000 was used for determining molar concentrations of CF1 (Farron, 1970). Dissociable nucleotides were removed from CFI by passage through a Sephadex G-25 medium column (1 X 60 cm) equilibrated with 25 mM sucrose, 10 mM Tris-HC1 (pH 8.0), and 2 mM EDTA (Cantley & Hammes, 1975). The eluted enzyme was concentrated either by dialyzing within a collodion membrane against the appropriate buffer or by precipitating the enzyme with an equal volume of column buffer saturated with ammonium sulfate. The precipitated enzyme was cooled on ice and centrifuged at 12000g for 10 min at 2 OC. The pellet was dissolved in a minimal amount of 50% glycerol and 50% column buffer (v/v) Holowka & Hammes, 1977) and then passed through a 3.5-mL centrifuge column (Penefsky, 1977) of Sephadex G-50 fine equilibrated with appropriate buffer. Analysis of Tightly Bound Nucleotides. Bound nucleotides were extracted from CFI freed of dissociable nucleotides in 10 mM Tris-HC1 (pH 8.0) by perchloric acid denaturation followed by potassium hydroxide neutralization (Rosing & Slater, 1972; Carlier & Hammes, 1979). Correction for the concentration decrease during the extraction was made by determining the change in nucleotide radioactivity per unit volume or by the change in weight when no radioactivity was present. The nucleotide-containing extract was analyzed on poly(ethylenimine)-cellulose columns (ICN Nutritional Biochemicals, 0.5 X 7 cm; Magnusson et al., 1976; Randerath & Randerath, 1967). The poly(ethylenimine)-cellulose was washed with 1.5 M LiCl and 0.1 N HCl before column preparation, and the columns were rinsed with 3 mL of 0.05 M H3P04raised to pH 3.0 with LiOH (H3P04-LiOH) before addition of the nucleotide sample (1-2 mL). Hydrolyzed protein and other interfering substances were washed off with 3 mL of H3P04-LiOH, AMP was eluted with 3 mL of 0.05 M LiCl in H3P04-LiOH, ADP was eluted with 5 mL of 0.3 M LiCl in H3P04-LiOH, and ATP was eluted with 7 mL of 1.2 M LiCl in H3P04-LiOH. The eluant was monitored at 254 nm, and l-mL fractions were collected. Each set of columns was calibrated with a standard mixture of [3H]ADP and [ T - ~ ~ P I A T PThe . amount of nucleotide in each fraction was determined spectrophotometrically by using an extinction coefficient of 15.0 X lo3 M-' cm-I at 256 nm for adenine nucleotides at pH 3.0. Measurement of Nucleotide Incorporation into CF1. The nondissociable ADP site of both latent CF1 and heat-activated CFl was labeled by incubating the enzyme with radioactive ADP or ATP as described by Carlier & Hammes (1979). The nondissociable site was prelabeled by incubation of heat-ac-
VOL. 20, NO. 22, 1981
6299
tivated CF1 with 200 pM [3H]ADP in 50 mM NaCl, 10 mM Tris-HC1 (pH 8.0), 5 mM dithiothreitol, 1 mM EDTA for 2 h to measure the rate of exchange of ATP into the nondissociable ADP site of heat-activated CF1. The CF1 again was freed of dissociable nucleotides and put into 50 mM NaCl, 10 mM Tris-HC1 (pH 8.0), 6 mM CaC12, and 1 mM EDTA. The exchange of ATP into the nondissociable ADP site of CFI in a 0.5-mL aliquot was initiated by the addition of nonradioactive ATP (final concentration 0.5 mM), and it was quenched by the addition of 50 pL of 200 mM EDTA in 20 mM Tris-HC1 (pH 8.0). The quenched solution was immediately pipetted onto a freshly prepared 3.5-mL centrifuge column equilibrated with quench buffer. The eluant then was passed through a second centrifuge column equilibrated with 20 mM Tris-HC1 (pH 8.0). The addition of 50 pL of buffer to the centrifuge columns after the preliminary centrifugation increased the amount of CFI recovered. The amount of radioactive nucleotide in the enzyme was determined by scintillation counting in 10 mL of Ready-Solv GP (Beckman). For determination of whether or not nucleotide in the MgATP site dissociated during ATP hydrolysis, the MgATP site of heat-activated CF1 was labeled with [ T - ~ ~ P , ~ H ] A T P by incubation of 5 pM enzyme with 50 pM [ T - ~ ~ P , ~ H ] A T P in 50 mM NaC1, 10 mM Tris-HC1 (pH KO), 6 mM MgC12, and 1 mM EDTA for 5 min. The dissociable nucleotides were removed, and the labeled enzyme was put into 50 mM NaCl, 10 mM Tris-HC1 (pH 8.0), and 1 mM EDTA. Nonradioactive ATP (final concentration 0.5 mM) in the presence of 6 mM CaC12 was incubated with the labeled enzyme for 2 min, and the reaction was quenched by the addition of EDTA (final concentration 10 mM). The solution then was pipetted onto a 5-mL centrifuge column equilibrated with 40 mM Tris-HC1 (pH 8.0) and 2 mM EDTA. The eluant was put through a second 5-mL centrifuge column equilibrated with the same buffer. The amount of 3H and 32Pbound to the enzyme was determined by scintillation counting. Equilibrium Binding Measurements. The binding of ADP and ATP to CF1 was studied by the forced dialysis technique (Cantley & Hammes, 1973). Ligand was incubated with 1-35 pM CF1 (freed of dissociable nucleotides) for 30 min in 0.3-0.5 mL before the forced dialysis was started. When the binding of high concentrations of [3H]ADP to heat-activated CF1 was studied, the nondissociable ADP site was prelabeled with [3H]ADPof the same specific radioactivity as that used in the forced dialysis experiment. Photoaffinity Labeling and Gel Electrophoresis. Arylazidoadenine nucleotides were synthesized with nonradioactive and IT-labeled ATP and ADP (Jeng & Guillory, 1975). High pressure liquid chromatography on a Whatman Partisil PXS 10/25 SAX anion-exchange column with 0.8 M potassium phosphate (pH 3.4) indicated the products were better than 95% pure. Prior to irradiation, the arylazido nucleotides were handled with only a 25-W red safe light for illumination. Samples were photolyzed by irradiating them in 50 mM NaCl and 0.5 mM Tris-HC1 (pH 8.0) for 15 min in a l-cm water-jacketed cuvette at 20 OC with a focused 200-W Hanovia Xe-Hg arc lamp 10 cm away from the sample. U1traviolet radiation below 350 nm was eliminated with a Corning CSO-5 1 filter. No heating of the sample occurred. The extent of covalent labeling was determined by denaturing an aliquot of the photolyzed CF1-arylazido nucleotide complex by the addition of perchloric acid to 4% (w/v) and filtering the precipitated protein on a Whatman glass-fiber C filter. The filters were washed sequentially with 5 mL of 4% perchloric acid, 5 mL of water, and 5 mL of 95% ethanol. The
6300 B I o c H E M I ST R Y
BRUIST A N D HAMMES
Table I: Incorporation of Nucleotides into CF, 3
nucleotide &M)
addition
['HIADP (74)b [ 3H]ATP (170) [y3'P,'H] ATP (49)b [y'?,'H]ATP (59)c [y-32P,'H]ATP [ 3H]ADP(43)e [Y-~~P,'H]ATP (49)f ['HIADP (61)g [y3'P,'H]ATP (50)e
2 mM EDTA 2 mM EDTA 5 mM CaCl, 6 mM CaCl, 6 mM CaCl, 2 mM EDTA 5 mM MgC1, 6 mM MgCI, 6 mM MgCl,
~
13 2 ~ 1
CF, CF, ADPICF," 0.70 0.64 0.88 0.31 0.60 1.26 1.16 0.10 1.08
0.90 (0.75) 0.93 (0.68) 0.33 1.43 (0.41) 0.15 1.21 (0.13) 0.03 0.97 (0.59)
t
ATP/ CF,
