Biochemistry 0 Copyright 1976 by the American Chemical Society
Volume 15, Number I
January 13, I976
Investigation of Quercetin Binding Sites on Chloroplast Coupling Factor 1 Lewis C. Cantley, Jr.,! and Gordon G . Hammes*
ABSTRACT: The quercetin binding sites on spinach chloroplast coupling factor 1 ( C F , ) have been investigated using direct and competitive binding, stopped-flow, temperaturejump, and fluorescence resonance energy transfer measurements. It was found that 8-anilino- 1-naphthalenesulfonic acid ( A N S ) competes with quercetin binding at two sites on the solubilized enzyme which are distinct from the two tight nucleotide binding sites and the 7-chloro-4-nitrobenzo-2oxa- 1,3-diazole (NBD-CI) reactive site. The bimolecular association of quercetin with C F I is too fast to measure directly and is followed by two slower conformational
changes. The distances from the tight nucleotide sites to the quercetin-ANS sites were estimated as 40-48 8, by fluorescence resonance energy transfer using 1,N6-ethenoadenosine diphosphate and 1 ,N6-ethenoadenylyl imidodiphosphate as donors and quercetin as the acceptor. The distance from the quercetin-ANS site to the NBD-CI reactive site was found to be about 30 A using A N S as a donor and NBD-C1 reacted with a tyrosine group on CFI as the energy acceptor. A model is proposed for the relative location of these sites on C F I .
P u r i f i e d preparations of coupling factor ( C F I ) solubilized ~ from spinach chloroplasts contain five different subunits (Lien et al., 1972), a , 0,7,6, and t , with respective molecular weights of 59000, 56000, 37000, 17500, and 13000. The total molecular weight is 325000 (Farron, 1970), and the solubilized C F I appears to be spherical in shape with an approximate diameter of 100 8, (Howell and Moudrianakis, 1967). The Ca2+-ATPase activity is located on the a-6 subunits, and this activity can be abolished by modification with NBD-CI of a tyrosine located on a 0subunit (Deters et al., 1975). Binding and steady-state kinetic experiments (Cantley and Hammes, 1975a) have indicated the presence of two tight binding sites on solubilized CFI which bind ADP, €ADP, AMP-PNP, and CAMP-PNP and a single active site for A T P hydrolysis on the heat-activated enzyme. The tight nucleotide sites appear to act as allosteric conformational switches of the Ca’+-ATPase activity. Fluores-
cence energy transfer measurements have shown that the tight nucleotide sites are approximately 40 8,away from the NBD-CI reactive site (Cantley and Hammes, 1975b). Quercetin has an inhibitory effect on the Ca2+-ATPase activity of C F I (Deters et al., 1975) but appears to stimulate the photophosphorylation activity of extensively dialyzed chloroplasts (Lardy et al., 1964). Quercetin also inhibits the ATPase activity of the mitochondrial coupling factor, F I , without affecting oxidative phosphorylation (Lang and Racker, 1974). In this work the quercetin binding sites on solubilized CF, are investigated using direct and competitive binding, stopped-flow, and temperaturejump measurements. A N S is found to compete with quercetin binding a t two sites on C F I . The distances from these sites to the tight nucleotide sites and to the NBD-CI reactive site were measured by the method of fluorescence resonance transfer using the donor-acceptor pairs €ADP-quercetin, CAMP-PNP-quercetin, and ANS-NBD-Tyr, and a model is proposed for the location of these sites on C F I .
From the Department of Chemistry, Cornell bniversity, Ithaca, New York 14853.Receiaed August 11, 1975. This work was supported Experimental Section by a grant from the National Institutes of Health ( G M 13292). National Science Foundation Fellow and National Institutes of Health Trainee ( G M 00834). Materials. The [3H]tADP and [3H]tAMP-PNP were Abbreviations used are: C F I , chloroplast coupling factor I : F I , miprepared as previously described (Cantley and Hammes, tochondrial coupling factor l ; NBD-CI, 7-chloro-4-nitrobenzo-2-oxa1975a). The quercetin was purchased from Eastman Chem1,3-diazole; tADP, 1 .Nh-ethenoadenosine diphosphate: AMP-PNP, adical Co., the NBD-CI from Pierce Chemical Co., the quienylyl imidodiphosphate; CAMP-PNP, I ,Nh-ethenoadenylyl imidodinine sulfate from Aldrich Chemical Co., and the A N S from phosphate; A N S , 8-anilino-I-naphthalenesulfonate: NBD-Tyr-CFI, NBD-CI reacted with the phenolic oxygen of a tyrosine moiety on C F , . Sigma Chemical Co. All other chemicals were the best
* ’
BIOCHEMISTRY, VOL.
15, NO. I , 1976
1
C.ANTLEY A N D 14AMMES
available commercial grades, and all solutions were prepared with deionized distilled water. CFI and NBD-Tyr-CFI Preparations. CFI was prepared by known procedures (Lien and Racker, 1971). An extinction coefficient of 0.476 ml/(mg cm) (Cantley and Hammes, 1975a) a t 280 nm in 0.1 M NaCI, 50 m M TrisCI, and 5 m M CaCl2 (pH 8.0), 23', was used to determine protein concentrations of C F I solutions free of exchangeable nucleotides. A molecular weight of 325000 (Farron, 1970) was used to determine the molar concentrations of enzyme. The NBD-CI modified C F , in which the N B D moiety is attached to one or two tyrosine groups per mole of enzyme (NBD-Tyr-CFI) was prepared as described elsewhere (Cantley and Hammes, 1975a), and an extinction coefficient of 10.7 X l o 3 M - ' cm-' a t 400 nm was used to determine the stoichiometry of N B D covalently bound to C F I . The enzyme was passed through two Sephadex (3-25 (medium) columns immediately before use to remove exchangeable nucleotides (Cantley and Hammes, 1975a). Difference Spectra Measurements. Rectangular quartz tandem cells (Pyrocell Manufacturing Co.) having a 0.44cm path length in each chamber were used to measure the difference spectrum resulting from the interaction of quercetin with solubilized C,Fl. The difference spectrum was recorded on a Cary 14 recording spectrophotometer equipped with a 0-0.1 -absorbance slide-wire. Difference spectrum titrations were done using a Zeiss spectrophotometer a t constant slit width (cf. Anderson et al., 1968). The quercetin was dissolved in 95% ethanol (10 m M ) and added to the enzyme in 0.1 M NaC1, 50 m M Tris-C1, 5 m M CaC12, and 1 m M dithiothreitol (pH 8.0), 23'. The dithiothreitol was present to stabilize quercetin in a reduced state. The final ethanol concentration was less than 4%. Steady-State Fluorescence Measurements. The steadystate fluorescence measurements were made with a HitachiPerkin-Elmer MPF-3 fluorescence spectrophotometer. The quantum yield of A N S bound to C F , in 0.1 M NaCI, 50 m M Tris-CI, and 5 m M CaCl2 (pH 8.0), 23', was determined by a comparative method (Parker and Rees. 1966).
Equation 1 gives the ratio of quantum yields, Q,, as a function of the area of the corrected emission spectrum, F,, and the absorbance a t the exciting wavelength, A,, for two different fluorescing compounds. The factor (1 - Th/4)-' is a correction for polarized emission (Shinitzky, 1972), and T h is the anisotropy of bound ANS. Quinine sulfate in 0.1 N H2S04 was used as a standard and was assumed to have an absolute quantum yield, Qz, of 0.70 (Scott et al., 1970) at 23'. In order to calculate the absorbance of A N S bound to C F I , A 1, it was necessary to determine the extinction coefficient and the concentration of A N S bound to C F I . The concentration of A N S bound to C F I was calculated using the dissociation constants determined as described below, and the extinction coefficient, 6.17 X lo3 M - ' cm-' a t 370 nm, was calculated from the difference spectrum of A N S with C F I . The inner filter effect due to excess free A N S which does not fluoresce was corrected for by adding A N S to the quinine sulfate solution to a final absorbance equal to the free A N S absorbance in the enzyme solution. Both A 1 and A2 were less than 0.05 a t the exciting wavelength, 370 nm, and the area of the corrected emission spectrum was determined by cutting out and weighing the recorded spectrum. I n experiments measuring A N S binding to C F I or competitive binding of quercetin or 2,4-dinitrophenol with A N S
2
BIOCHEMISTRY. VOL
15, NO
1,
1976
by fluorescence intensity, it was necessary to correct for the inner filter effect due to the absorbance of these compounds a t the exciting wavelength, 370 nm. This correction was calculated by a control experiment in which A N S was added to a quinine sulfate solution in 0.1 N H2S04. The wavelengths (370-nm excitation, 470-nm emission), slits, and cuvette were identical in the control experiment, and the percent quenching of quinine sulfate fluorescence vs. A N S absorbance was plotted. From this curve, the inner filter effect due to nonfluorescing constituents could be calculated from their absorbance a t 370 nm. The absorbance of bound A N S was always less than 0.05. Triangular ( 1 cm X 1 cm X 1.4 cm) and square micro (0.3 cm X 0.3 cm) cuvettes were used to minimize the inner filter effect. The quantum yields of cADP and CAMP-PNP i n the presence of quercetin were determined as follows. The change in fluorescence (320-nm excitation, 400-nm emission) with time of [ 3 H ] ~ A D or P [ 3 H ] ~ A M P - P N Paccompanying binding to CF1 was measured (Cantley and Hammes, 1975a). After equilibrium was reached, the solutions were titrated with 5 m M quercetin in 95% ethanol and the fluorescence intensity was recorded. The free and total nucleotide concentrations were then immediately determined (in about 10 min) using the forced dialysis technique (Cantley and Hammes, 1973). As a control experiment, a large excess of unlabeled A D P (440 &f) was equilibrated with the enzyme before adding the labeled fluorescent nucleotide. The solution was then titrated with 5 m M quercetin as before, and the free and total labeled nucleotide concentrations were determined. The A D P prevented binding of the fluorescent nucleotide so that the inner filter effect of quercetin and the effect of the ethanol (
