ARCHIVES
OF BIOCHEMISTRY
Vol. 283, No. 1, November
AND
BIOPHYSICS
15, pp. 102-106,199O
Fluorescence Studies with Malate Dehydrogenase Bradyrhizobium japonicum 311B-l 43 Bacteroids: A Two-Tryptophan Containing Protein
from
Camillo
A. Ghiron,* Maurice R. Eftink,?,’ James K. Waters,* and David W. Emerich* *Department of Biochemistr.y, University of Missouri, Columbia, Missouri 65211; and TDepartment of Chemistry, Untversity
of ikssissippi,
University,
Mksippi
38677
Received April 24,1990, and in revised form July 23,199O
A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Brudyrhizobium japonicum 3IlB-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tar0 1990 Academic PW+, 1~. trOniC acid or D-malate.
A number of fluorescence studies, of both tryptophanyl (trp)2 residues and bound reduced nicotinamide adenine dinucleotide (NADH), have been reported for procine malate dehydrogenase (MDH) (l-3). The alterations in the fluorescence properties of NADH usually involve an increase in the quantum yield and a blueshift in the emission maximum upon binding to the coenzyme site. The extent of these changes is usually different for binary and ternary complexes (2). The large number (6) of trp residues complicates the interpretation of studies of the intrinsic fluorescence of this enzyme (4). To circumvent this we have performed i To whom correspondence should be addressed. ’ Abbreviations used: trp, tryptophanyl; MDH, malate dehydrogenase; b-MDH, bacteroid malate dehydrogenase; PRS, phase-resolved spectra.
studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3IlB-143 bacteroids. The bacteroid malate dehydrogenase (b-MDH) is a tetramer with a subunit molecular weight of 36,000 Da. The pH optimum for conversion of oxaloacetate to Lmalate is 8.0-8.5, while the pH optimum in the reverse direction is 8.6-9.0. NADH, but not oxaloacetate, provides almost complete protection against heat inactivation. Kinetics analysis of b-MDH indicates an ordered bi-bi mechanism with NAD+ adding first and NADH leaving last. The Michaelis constants for b-MDH are similar to those of other MDH’s (5). Here we report steady-state and frequency domain fluorescence studies with b-MDH. Our primary intent is to provide background information for the possible future exploitation of the intrinsic fluorescence at the two trp residues of b-MDH as reporter groups for studies of the conformation of the protein. Secondarily, we studied the binding of NADH to the enzyme. The coenzyme dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in the fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate. EXPERIMENTAL
SECTION
Materials. The b-MDH was purified as described elsewhere (5). The protein was usually maintained in a pH 7 to 8 phosphate buffer (5 to 50 mM) in which sodium chloride (0.1-0.2 M) was sometimes added. Porcine heart mitochondrial MDH was purchased from Sigma Chemical Co. Acrylamide was recrystallized from ethyl acetate before use; KI was freshly prepared to avoid I; formation. NADH, tartronic acid, and Dmalate were obtained from Sigma Chemical Co. Methods. Steady-state fluorescence spectra were obtained on a Perkin-Elmer MPF 44A. Unless specified, measurements were made at room temperature. Quenching of the fluorescence of b-MDH by acrylamide was performed by adding aliquots of an 8 M stock solution of the quencher to 2.0 ml of protein solution in a 1 X l-cm fluorescence
102 All
0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
MALATE
DEHYDROGENASE
FROM
Bradyrhizobium
japonicum
103
cuvette. Fluorescence was measured with an excitation wavelength of 295 nm, an emission wavelength of 340 nm, and using a 5-nm bandpasses. Correction was made for absorptive screening by the added acrylamide (6). Quenching data were analyzed using the Stern-Volmer equation
111
t v, z r Z
where F and FO are the fluorescence intensities in the absence and presence of quencher, Q, K,; and V; are the dynamic and static quenching constants for component i, and f, is the fractional steadystate intensity associated with component i. We fitted this equation, for n = 1 and 2, to the data using a grid search nonlinear least squares program. Multifrequency fluorescence lifetime measurements and phase resolved spectral measurements were performed on an SLM 4800, which was updated to operate up to 200 MHz with equipment from ISS, Inc. The procedures for these measurements are given in previous papers (7-9). Sample excitation was achieved by using either a 290-nm filtered xenon light or the 300-nm line of an Innova 200 argon ion laser. The multifrequency phase and modulation data were analyzed, via a nonlinear least squares program, using the following impulse-response function,
WAVELENGTH,
nm
FIG. 1. Emission spectra of b-MDH and 295 nm (- - -). Conditions: 25°C.
with excitation at 280 nm (-) pH 7.6 in 50 mM KIHPOl, temperature
dure) yielded, as expected, 3.93 + 0.35 Trp residues per molecule (15). Steady-state fluorescence studies. In Fig. 1 are shown uncorrected fluorescence spectra of b-MDH with excitaz(t) = zo c a,exp(-tlri), PI tion at 295 and 280 nm. The wavelength maxima (X max) i=1 are 335 and 336 nm, respectively. The additional emiswhere ri and oi are the fluorescence decay time and preexponential sion seen in the 300- to 350-nm region upon 280-nm exterm for component i, and I0 and Z(t) are the intensities at time zero citation is from the tyrosinyl residues of the protein (i.e., and t (10). the difference spectrum shows a maximum below 310 In the dynamic polarization measurements the sample is illuminm, as expected for emission from tyrosine). nated by light polarized parallel to the vertical laboratory axis with The excitation anisotropy spectrum of b-MDH was intensity modulated at variable frequencies. The differential phase delay and modulation ratio for the perpendicular and parallel polarizadetermined. The spectrum obtained is typical of that for tion components of the emission were determined as a function of tryptophan fluorescence in proteins. A maximum value modulation frequency. The following anisotropy decay function was of 0.29 is found upon 305-nm excitation. A value of this fitted to these data as described elsewhere (16). magnitude indicates that the emitting Trp residues are relatively immobilized. r(t) = r. i g,exp(-t/&). 131 The acrylamide quenching of the Trp fluorescence of i=l b-MDH, monitored at 335 nm with excitation at 295 nm, yielded a slightly upward curving Stern-Volmer plot. In this equation & and gi are the rotational correlation time and fracComputer analysis of these data gave a single compotional degree of depolarization for process i, r,, is the limiting anisotropy in the absence of motion, and r(t) is the anisotropy at time t. A nent fit with a dynamic quenching constant, K,, of 3.6 nonassociated analysis, with respect to the intensity decay, was perM-l and a static quenching constant, V, of 0.14 M-‘. Informed. clusion of more terms did not lead to a significant reducPhase-resolved spectra (PRS) were obtained using the computation in the x2 for the fit. tional procedures of Gratton and Jameson (11,12). Incremental addition of urea to a b-MDH solution (0.1 The tryptophan content of b-MDH was determined chemically by the reaction withp-dimethylaminobenzaldehyde as described by Spies M NaCl, pH 7.0) decreased the fluorescence yield slightly and Chambers (13). Standard curves were prepared from free trypto(excitation X = 290 nm, emission X = 320 nm). The fluophan and from trypsin. rescence X,,, remained unchanged at 335 nm even when Dissociation constants were obtained by titration of enzyme solua total of 4 M urea was added. Incremental reduction of tions with coenzyme NADH in the presence or absence of saturating amounts of a second ligand. The decrease in protein fluorescence at the pH of a b-MDH solution from 7.0 to 3.25 caused a 340 nm, upon 295nm excitation, was used to monitor the complexprogressive blue shift in the emission X,,, from 335 to ation. 328 nm (excitation X = 295 nm). This shift was accompanied by a large reduction in the fluorescence yield. HowRESULTS ever, the magnitude of the fluorescence anisotropy of this sample (excitation X = 295 nm, emission X = 335 Trp content of b-MDH. Three independent determinations utilizing p-dimethylaminobenzaldehyde gave nm) was found to be unaffected by this change in pH. NADH binding studies. The addition of NADH to b1.9 t 0.14 Trp residues per b-MDH monomer unit. ConMDH causes a quenching of the tryptophanyl fluorestrol experiments with trypsin (using the same proce-
104
GHIRON
[ NADH]
, M
FIG. 2. Binding isotherm of NADH to b-MDH, determined by the quenching of the intrinsic protein fluorescence as NADH is added. Closed circles are data obtained in the presence of 50 mM succinate. The solid line is a fit to a simple 1:l binding model with & = 2.3 X 10-a M and AF,,. = -50.9 (relative units). Similar studies with other preparations of b-MDH yielded Kd values of 2.5 X 1Om6M and 2.3 X 10m6M in the absence and presence of 7.4 mM tartronic acid, and (in a study at 0.5 M ionic strength) Kd values of 5.1 X 10e6 M and 6.2 X lo-” M in the absence and presence of 3.5 mM D-malate. In all cases the data were well described by a hyperbolic binding function. An attempt to fit the data with a function having two types of sites (i.e., different &, and AFmsl,J did not significantly improve the reduced x square of the fit. These observations suggest that the binding sites are equivalent and noninteracting and that the signal change is linear in coverage of the sites.
cence of the protein. This quenching most likely occurs due to resonance energy transfer to bound NADH. As shown in Fig. 2, the fluorescence change can be fitted to a binding isotherm with dissociation constant, Kd, equal to 2.5 X 10e6 M and AF,,, = -58% (for emission at 335 nm). This Kd value is similar to that for NADH binding to other MDH’s under similar experimental conditions (3). Surprisingly, inclusion of tartronic acid, D-malate, or succinate does not appreciably change the Kd for NADH. A control experiment with porcine heart mitochondrial MDH yielded a lo-fold decrease in the dissociation constant for NADH upon the addition of 7.5 mM tartronic acid. Also, a large enhancement in the fluorescence intensity of bound NADH is seen in the ternary complex formed with porcine heart mitochondrial MDH and either D-malate or tartronic acid (l-3). Again, in contrast, no enhancement is seen for the fluorescence of NADH bound to b-MDH when in the presence of tartronic acid or D-malate. Time-resolved fluorescelzce studies. Various frequency domain fluorescence measurements were made with b-MDH at 20°C to try to distinguish, if possible, contributions from the two trp residues. Our early measurements were made with a xenon light source. Later measurements were made with an argon ion laser light source. In both cases, the emission was observed through a bandpass filter (Corning 7-60) centered at 350 nm. Multifrequency phase and modulation measurements were made with a xenon lamp (290 nm interference filter) and the data obtained show that the fluorescence decay is nonexponential. An adequate fit was obtained to a biexponential with 71 = 6.5 ns, TV = 1.2 ns, fi = 0.82 and xg = 3.7. (A wg 320 filter was also added to assure
ET AL.
that the short-lived component was real and not due to scattered light.) See Table I for these and other results. Subsequent phase and modulation measurements made with the 300-nm line of an argon ion laser also showed the decay to be nonexponential, as shown in Fig. 3. The two-component fit of these data of 71 = 6.6 ns, r2 = 1.1 ns, fi = 0.9 and xk = 8.6 are similar to that for the less precise data with the xenon lamp. The quality of the data obtained with the laser enabled us to perform a triexponential fit, with the fitting parameters given in Table I and Fig. 3. A lo-fold reduction in & is achieved in going from the biexponential to the triexponential fit. Using the biexponential fluorescence lifetimes, we measured the phase lag across the emission spectrum, to calculate the spectrum associated with each decay time. The details of this procedure, usually referred to as PRS, TABLE
I
Fluorescence Lifetime Data for b-MDH” Xenon lamp excitation* Temperature (“C)
71 bs)
72 W
011
Xi,
5
6.71
10
6.71
1.49 1.49
0.389 0.416
18.7
3.6
20 20 30
6.41 6.51 6.57
0.96 1.22 1.06
0.410 0.423 0.403
4.6 3.7 10.5
011
Xtle
0.552 0.550 0.551 0.527 0.444
8.6 7.2 5.3 3.0 3.4
Laser excitationd Biexponential 71
WI (M)
fits 72
bs) 6.56 5.03 4.27 3.87 3.60
0
0.12 0.22 0.32 0.51
1.11
0.875 0.826 0.806 0.916
Laser excitation Triexponential 72 73
0.12 0.22 0.32 0.51
a1
e?
Xft’
0.297 0.372
0.297 0.372
0.81
0.395
0.395
0.375 0.443
0.356 0.255
0.378 0.287 0.267 0.318 0.435
t-1
[KU (M) 0
fits
8.25 5.56 4.49 4.32 4.26
0.698 1.913 1.398
1.772 1.743
1.9
3.5 0.57 0.66
a Conditions: pH 7.6,35 mM KzHPOl, 0.1 M NaCl, broad band emission. * Global analysis with E.i = 1.00 kcal/mole, E,, = 2.02 kcal/mol, preexponential factors Al = 0.872 and A2 = 27.76, (Ye = 0.419, and global xi = 9.1. ’ xz calculated using (us= 0.5” and cM = 0.005. d Global analysis gives 7, = 6.53 ns, TV = 1.13 ns, k,, = 0.33 X 10’ M-k’, kqs = 1.25 X 10’ M-k’, 01~= 0.539, and x& = 8.1. e xi calculated using np = 0.2” and (TV = 0.002.
MALATE
DEHYDROGENASE
MHz
FIG. 3. Multifrequency phase and modulation data for b-MDH in the absence (0) and presence (0) of 50 X 10m6M NADH. Conditions: pH 7.6, 35 mM K*HPO,, 0.1 M NaCl, excitation at 300 nm with argon laser, magic angle emission polarizer, broad band emission observed. Biexponential fits are shown. For b-MDH alone, ri = 6.51 ns, r2 = 1.22 ns, fi = 0.879. In the presence of NADH, ri = 4.79 ns, r2 = 0.97 ns, and fi = 0.853.
has been described previously in detail (11, 12, 14). The 1.2-ns component is found to have a blue spectrum (X,,, = 328 nm). The 6.5ns component is more intense and fluoresces to the red (X,,, = 346 nm). Phase and modulation measurements with 290-nm xenon lamp excitation were performed on b-MDH over the temperature range 5 to 30°C. The two component fits are given in Table I. As can be seen, the magnitudes of the two lifetimes are relatively insensitive to temperature. The temperature-dependence data sets were also analyzed in a global manner (18). This involved linking the data sets by the Arrhenius function (i.e., for l/7) and by requiring (Y~to be the same at each temperature. The global fit was obtained with activation energies of 1.00 kcal/mol and 2.02 kcal/mol for the long (6.25 ns at 25°C) and short (1.09 ns at 25°C) components, respectively, and (Ye= 0.419 (long component). The & for this fiveparameter global fit is virtually the same as the average & for the individual biexponential fits (for which there are 15 fitting parameters). As described earlier, the addition of saturating amounts of NADH to b-MDH reduces the latter’s fluorescence yield. The consequences of this effect can be readily seen in Fig. 3, where the phase and modulation lifetime data for b-MDH, in the presence of 50 PM NADH, are also shown. The two- and three-component fits for these data are: 71 = 4.79 ns, fi = 0.85, r2 = 0.97 ns, xk = 4.5 and pi = 6.26 ns, fi = 0.46, 72 = 3.2 ns, fi = 0.45, ‘TV= 0.73 ns, f3 = 0.09, xi = 1.8. In terms of the biexponential analysis, both the long- and short-lived components are partially quenched by the binding of (and energy transfer to) NADH. The quenching action of iodide ion was also studied by time-resolved fluorescence spectroscopy. The two- and three-component fits for increasing amounts of added potassium iodide are shown in Table I. Again, in terms of the biexponential analysis, iodide ion appears to quench both components, with the longer-lived component being quenched to a greater degree. Global analysis of the
FROM
Bradyrhizobium
juponicum
105
data sets, with linkage by the Stern-Volmer equation (l/ 7qi = l/~i + k+[Q], where kqi is the quenching rate constant for component i and with the requirement that (Y; is the same for all [Q], yielded a biexponential fit with 7i = 6.53 ns, 72 = 1.13 ns, k,, = 0.33 X 10gM-‘s-l, ke2 = 1.25 X 10’ M-%-l, (Y~= 0.539, and & = 8.12. This global fit is not very satisfactory, considering the quality of the data. A global analysis in terms of a triexponential decay was also unsatisfactory (xi = 20), in comparison with the individual fits in Table I. Possible explanations for these poor global fits are that (i) linear Stern-Volmer plots do not describe the quenching of the individual components (which is possible since a constant ionic strength was not maintained), or (ii) that the CZ~ are not constant at all [Q] (which may be a consequence of a static quenching component). Differential polarized phase and modulation data for the tryptophan emission of b-MDH are shown in Fig. 4. The dynamic polarization results were best fit with two rotational correlation times, r$, of 80.5 and 0.238 ns, with limiting anisotropy contributions of 0.240 and 0.051, respectively. DISCUSSION
The fluorescence of b-MDH comes predominantly from the tryptophan residues of this protein. A relatively small tyrosine contribution centered around 305 nm is seen upon 280-nm excitation. Since we have shown, by chemical analysis, that there are two tryptophanyl residues in each monomeric unit, it is not surprising to find that the tryptophanyl fluorescence of b-MDH is heterogeneous. The simplest interpretation of the lifetime and quenching data can be made by assuming that these trp residues are emitting independently. If a biexponential decay is assumed, phase-resolved emission spectra show that one of the trp components has a r = 6.5 ns and X,,, = 346 nm, while the other is shorter lived with a T = 1.2 decay law does ns and X,, = 328 nm. A biexponential not appear to be adequate, but this phase-resolved spectral measurement shows the shorter decay time(s) to contribute to the blue side of the emission. It is possible
FIG. 4. Differential polarized phase and modulation anisotropy decay measurements with b-MDH. Conditions as in Fig. 3. Fit is with $i = 80.5 ns, & = 0.238 ns, g,r,, = 0.240 and g2roz = 0.051. This fit is a nonassociated fit with respect to the individual intensity decay times.
106
GHIRON
that excited state energy transfer occurs between the two types of tryptophan residues and that this is a basis for the multiexponential decay. However, we have used an excitation wavelength of 295-303 nm in all studies and trp + trp energy transfer is known to be minimized at the red edge of its absorption spectrum (20). Steady state anisotropy and dynamic polarization measurements indicate that both these residues are relatively immobile. The small activation energies obtained for thermal quenching of both lifetime components (for an assumed biexponential decay) support this contention. A small degree of rapid, restricted motion of the trp residues is detected by the 0.238-ns rotational correlation time. This rapid motion can be modeled in terms of the wobbling of the fluorophore in a cone of semiangle 13” (19). The anisotropy data also permit us to obtain an estimate of b-MDH’s molecular weight. The molecular weight of a spherical protein should be in direct proportion to its rotational correlation times, 4, by the equation Cp= uM (E + h)/RT, where u (= 0.89 CP at 25°C) is the bulk viscosity, M is the molecular weight of the macromolecule, U= 0.73 ml/g is the partial specific volume of the protein, and h is the hydration of the protein (which we take to be 0.3 ml/g). From the experimental value of Q,= 80.5 ns for global rotation of the protein, we calculate that b-MDH has an effective molecular weight of 213 kDa. This result is larger than the molecular weight of 139 kDa that has been estimated for b-MDH by an electrophoretic method (5). The discrepancy (i.e., longer than expected $Jfor a spherical protein of 139 kDa) suggests that the tetrameric protein has a nonspherical shape and/or that there is a large degree of hydration. The oligomeric structure of b-MDH is not easily dissociated and/or unfolded. The trp fluorescence X,,, was not red-shifted to 350 nm when the pH of the protein solution was decreased from 7.0 to 3.25 or when urea was added up to a concentration of 4 M. These findings indicate that the trp residues were not exposed to the aqueous solvent. Since the steady state anisotropy of b-MDH remained unchanged as the pH was decreased, we conclude that the decrease in this enzyme’s activity at low pH cannot be caused by tetramer dissociation and/or denaturation (5). The binding of NADH dynamically quenches both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in the fluorescence of bound NADH upon addition of either tartronic acid or D-malate. Also the association constant for NADH does not change appreciably by the addition of tartronic acid or D-malate. These results suggest that these ligands do not bind to b-MDH. This proposition is supported by the observation that D-malate does not inhibit b-MDH, but is contradicted by the preliminary observation that tartronic acid is a competitive inhibitor of b-MDH with an inhibition constant approximately
ET AL.
equal to 1 mM. More studies will be needed to resolve the later discrepancy. We have interpreted our fluorescence data in terms of contributions from two types of tryptophanyl residues. The emission from each of the individual trp residues may be heterogeneous as well. The fluorescence lifetime of individual tryptophanyl residues in proteins is rarely a single exponential (17). The more precise data obtained with 300-nm laser excitation suggests that each b-MDH trp residue may have a multiexponential decay. Thus, our description may be an oversimplification. However, such complexity is very difficult to model from an experimental and interpretative point of view. The simplified interpretation presented here should be adequate for the intended structural studies. ACKNOWLEDGMENTS This research was supported by NSF Grant DMB 88-06113 to M.R.E. andby USDA Competitive Grant 85CRCR-1-1734 to D.W.E. This is contribution No. 11,168 of the Agricultural Experiment Station of the University of Missouri.
REFERENCES 1. Jameson, D. M., Thomas, V., and Zhoa, D. M. (1989) Biochim. Biophys. Actu 994,187-190. 2. Baumgarten, B., and Joachim Hones. (1988) Photo&em. Photobiol. 47, 201-205. 3. Holbrook, J. J., and Wolf, R. G. (1972) Biochemistry 11, 24992502. 4. Banaszak, L. J., and Bradshaw, R. A. (1975) The Enzymes, Vol. 11, p. 369, Academic Press, New York. 5. Waters, J. K., Karr, D. B., and Emerich, D. W. (1985) Biochemistry 24,6479-6486. 6. Ghiron, C. A., Eftink, M. R., Porter, M. A., and Hartman, F. C. (1988) Arch. Biochem. Biophys. 260,267-272. 7. Jameson, D. M., Gratton, E., and Hall, R. D. (1984) Appl. Spectrosc. Rev. 20,55-85. 8. Wasylewski, Z., and Eftink, M. R. (1987) Eur. J. Biochem. 167, 513-518. 9. Eftink, M. R., and Ghiron, C. A. (1987) Biophys. J. 52,467-473. 10. Lakowicz, J. R., Laczko, G., Cherek, H., Gratton, E., and Limkeman, M. (1984) Biophys. J. 46,463-477. 11. Gratton, E., and Jameson, D. M. (1985) Anal. Chem. 57, 16941697. 12. Eftink, M. R., Ghiron, C. A., and Wasylewski, Z. (1987) Biochemistry 26,8338-8346. 13. Spies, J. R., and Chambers, D. C. (1948) Anal. Chem. 20,30-39. 14. Lakowicz, J. R., and Cherek, H. (1981) J. Biol. Chem. 256,63486353. 15. Dayhoff, M. D. (1972) Atlas of Proteins Sequence and Structure, Vol. 5, p. D-105, N.B.R. Foundation Press, Washington DC. 16. Gratton, E., Jameson, D. M., and Hail, R. D. (1984) Annu. Rev. Biophys. Bioeng. 13,105-124. 17. Beechem, J. M., and Brand, L. (1985) Annu. Rev. Biochem. 54, 43-71. 18. Beechem, J. M., and Gratton, E. (1988) SPZE Proc. 909.70-81. 19. Lipari, G., and Szabo, A. (1980) Biophys. J. 30,489~506. 20. Weber, G., and Shinitzky, M. (1970) Proc. Natl. Acad. Sci. USA 65,823-830.
OF BIOCHEMISTRY
Vol. 283, No. 1, November
AND
BIOPHYSICS
15, pp. 102-106,199O
Fluorescence Studies with Malate Dehydrogenase Bradyrhizobium japonicum 311B-l 43 Bacteroids: A Two-Tryptophan Containing Protein
from
Camillo
A. Ghiron,* Maurice R. Eftink,?,’ James K. Waters,* and David W. Emerich* *Department of Biochemistr.y, University of Missouri, Columbia, Missouri 65211; and TDepartment of Chemistry, Untversity
of ikssissippi,
University,
Mksippi
38677
Received April 24,1990, and in revised form July 23,199O
A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Brudyrhizobium japonicum 3IlB-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tar0 1990 Academic PW+, 1~. trOniC acid or D-malate.
A number of fluorescence studies, of both tryptophanyl (trp)2 residues and bound reduced nicotinamide adenine dinucleotide (NADH), have been reported for procine malate dehydrogenase (MDH) (l-3). The alterations in the fluorescence properties of NADH usually involve an increase in the quantum yield and a blueshift in the emission maximum upon binding to the coenzyme site. The extent of these changes is usually different for binary and ternary complexes (2). The large number (6) of trp residues complicates the interpretation of studies of the intrinsic fluorescence of this enzyme (4). To circumvent this we have performed i To whom correspondence should be addressed. ’ Abbreviations used: trp, tryptophanyl; MDH, malate dehydrogenase; b-MDH, bacteroid malate dehydrogenase; PRS, phase-resolved spectra.
studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3IlB-143 bacteroids. The bacteroid malate dehydrogenase (b-MDH) is a tetramer with a subunit molecular weight of 36,000 Da. The pH optimum for conversion of oxaloacetate to Lmalate is 8.0-8.5, while the pH optimum in the reverse direction is 8.6-9.0. NADH, but not oxaloacetate, provides almost complete protection against heat inactivation. Kinetics analysis of b-MDH indicates an ordered bi-bi mechanism with NAD+ adding first and NADH leaving last. The Michaelis constants for b-MDH are similar to those of other MDH’s (5). Here we report steady-state and frequency domain fluorescence studies with b-MDH. Our primary intent is to provide background information for the possible future exploitation of the intrinsic fluorescence at the two trp residues of b-MDH as reporter groups for studies of the conformation of the protein. Secondarily, we studied the binding of NADH to the enzyme. The coenzyme dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in the fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate. EXPERIMENTAL
SECTION
Materials. The b-MDH was purified as described elsewhere (5). The protein was usually maintained in a pH 7 to 8 phosphate buffer (5 to 50 mM) in which sodium chloride (0.1-0.2 M) was sometimes added. Porcine heart mitochondrial MDH was purchased from Sigma Chemical Co. Acrylamide was recrystallized from ethyl acetate before use; KI was freshly prepared to avoid I; formation. NADH, tartronic acid, and Dmalate were obtained from Sigma Chemical Co. Methods. Steady-state fluorescence spectra were obtained on a Perkin-Elmer MPF 44A. Unless specified, measurements were made at room temperature. Quenching of the fluorescence of b-MDH by acrylamide was performed by adding aliquots of an 8 M stock solution of the quencher to 2.0 ml of protein solution in a 1 X l-cm fluorescence
102 All
0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
MALATE
DEHYDROGENASE
FROM
Bradyrhizobium
japonicum
103
cuvette. Fluorescence was measured with an excitation wavelength of 295 nm, an emission wavelength of 340 nm, and using a 5-nm bandpasses. Correction was made for absorptive screening by the added acrylamide (6). Quenching data were analyzed using the Stern-Volmer equation
111
t v, z r Z
where F and FO are the fluorescence intensities in the absence and presence of quencher, Q, K,; and V; are the dynamic and static quenching constants for component i, and f, is the fractional steadystate intensity associated with component i. We fitted this equation, for n = 1 and 2, to the data using a grid search nonlinear least squares program. Multifrequency fluorescence lifetime measurements and phase resolved spectral measurements were performed on an SLM 4800, which was updated to operate up to 200 MHz with equipment from ISS, Inc. The procedures for these measurements are given in previous papers (7-9). Sample excitation was achieved by using either a 290-nm filtered xenon light or the 300-nm line of an Innova 200 argon ion laser. The multifrequency phase and modulation data were analyzed, via a nonlinear least squares program, using the following impulse-response function,
WAVELENGTH,
nm
FIG. 1. Emission spectra of b-MDH and 295 nm (- - -). Conditions: 25°C.
with excitation at 280 nm (-) pH 7.6 in 50 mM KIHPOl, temperature
dure) yielded, as expected, 3.93 + 0.35 Trp residues per molecule (15). Steady-state fluorescence studies. In Fig. 1 are shown uncorrected fluorescence spectra of b-MDH with excitaz(t) = zo c a,exp(-tlri), PI tion at 295 and 280 nm. The wavelength maxima (X max) i=1 are 335 and 336 nm, respectively. The additional emiswhere ri and oi are the fluorescence decay time and preexponential sion seen in the 300- to 350-nm region upon 280-nm exterm for component i, and I0 and Z(t) are the intensities at time zero citation is from the tyrosinyl residues of the protein (i.e., and t (10). the difference spectrum shows a maximum below 310 In the dynamic polarization measurements the sample is illuminm, as expected for emission from tyrosine). nated by light polarized parallel to the vertical laboratory axis with The excitation anisotropy spectrum of b-MDH was intensity modulated at variable frequencies. The differential phase delay and modulation ratio for the perpendicular and parallel polarizadetermined. The spectrum obtained is typical of that for tion components of the emission were determined as a function of tryptophan fluorescence in proteins. A maximum value modulation frequency. The following anisotropy decay function was of 0.29 is found upon 305-nm excitation. A value of this fitted to these data as described elsewhere (16). magnitude indicates that the emitting Trp residues are relatively immobilized. r(t) = r. i g,exp(-t/&). 131 The acrylamide quenching of the Trp fluorescence of i=l b-MDH, monitored at 335 nm with excitation at 295 nm, yielded a slightly upward curving Stern-Volmer plot. In this equation & and gi are the rotational correlation time and fracComputer analysis of these data gave a single compotional degree of depolarization for process i, r,, is the limiting anisotropy in the absence of motion, and r(t) is the anisotropy at time t. A nent fit with a dynamic quenching constant, K,, of 3.6 nonassociated analysis, with respect to the intensity decay, was perM-l and a static quenching constant, V, of 0.14 M-‘. Informed. clusion of more terms did not lead to a significant reducPhase-resolved spectra (PRS) were obtained using the computation in the x2 for the fit. tional procedures of Gratton and Jameson (11,12). Incremental addition of urea to a b-MDH solution (0.1 The tryptophan content of b-MDH was determined chemically by the reaction withp-dimethylaminobenzaldehyde as described by Spies M NaCl, pH 7.0) decreased the fluorescence yield slightly and Chambers (13). Standard curves were prepared from free trypto(excitation X = 290 nm, emission X = 320 nm). The fluophan and from trypsin. rescence X,,, remained unchanged at 335 nm even when Dissociation constants were obtained by titration of enzyme solua total of 4 M urea was added. Incremental reduction of tions with coenzyme NADH in the presence or absence of saturating amounts of a second ligand. The decrease in protein fluorescence at the pH of a b-MDH solution from 7.0 to 3.25 caused a 340 nm, upon 295nm excitation, was used to monitor the complexprogressive blue shift in the emission X,,, from 335 to ation. 328 nm (excitation X = 295 nm). This shift was accompanied by a large reduction in the fluorescence yield. HowRESULTS ever, the magnitude of the fluorescence anisotropy of this sample (excitation X = 295 nm, emission X = 335 Trp content of b-MDH. Three independent determinations utilizing p-dimethylaminobenzaldehyde gave nm) was found to be unaffected by this change in pH. NADH binding studies. The addition of NADH to b1.9 t 0.14 Trp residues per b-MDH monomer unit. ConMDH causes a quenching of the tryptophanyl fluorestrol experiments with trypsin (using the same proce-
104
GHIRON
[ NADH]
, M
FIG. 2. Binding isotherm of NADH to b-MDH, determined by the quenching of the intrinsic protein fluorescence as NADH is added. Closed circles are data obtained in the presence of 50 mM succinate. The solid line is a fit to a simple 1:l binding model with & = 2.3 X 10-a M and AF,,. = -50.9 (relative units). Similar studies with other preparations of b-MDH yielded Kd values of 2.5 X 1Om6M and 2.3 X 10m6M in the absence and presence of 7.4 mM tartronic acid, and (in a study at 0.5 M ionic strength) Kd values of 5.1 X 10e6 M and 6.2 X lo-” M in the absence and presence of 3.5 mM D-malate. In all cases the data were well described by a hyperbolic binding function. An attempt to fit the data with a function having two types of sites (i.e., different &, and AFmsl,J did not significantly improve the reduced x square of the fit. These observations suggest that the binding sites are equivalent and noninteracting and that the signal change is linear in coverage of the sites.
cence of the protein. This quenching most likely occurs due to resonance energy transfer to bound NADH. As shown in Fig. 2, the fluorescence change can be fitted to a binding isotherm with dissociation constant, Kd, equal to 2.5 X 10e6 M and AF,,, = -58% (for emission at 335 nm). This Kd value is similar to that for NADH binding to other MDH’s under similar experimental conditions (3). Surprisingly, inclusion of tartronic acid, D-malate, or succinate does not appreciably change the Kd for NADH. A control experiment with porcine heart mitochondrial MDH yielded a lo-fold decrease in the dissociation constant for NADH upon the addition of 7.5 mM tartronic acid. Also, a large enhancement in the fluorescence intensity of bound NADH is seen in the ternary complex formed with porcine heart mitochondrial MDH and either D-malate or tartronic acid (l-3). Again, in contrast, no enhancement is seen for the fluorescence of NADH bound to b-MDH when in the presence of tartronic acid or D-malate. Time-resolved fluorescelzce studies. Various frequency domain fluorescence measurements were made with b-MDH at 20°C to try to distinguish, if possible, contributions from the two trp residues. Our early measurements were made with a xenon light source. Later measurements were made with an argon ion laser light source. In both cases, the emission was observed through a bandpass filter (Corning 7-60) centered at 350 nm. Multifrequency phase and modulation measurements were made with a xenon lamp (290 nm interference filter) and the data obtained show that the fluorescence decay is nonexponential. An adequate fit was obtained to a biexponential with 71 = 6.5 ns, TV = 1.2 ns, fi = 0.82 and xg = 3.7. (A wg 320 filter was also added to assure
ET AL.
that the short-lived component was real and not due to scattered light.) See Table I for these and other results. Subsequent phase and modulation measurements made with the 300-nm line of an argon ion laser also showed the decay to be nonexponential, as shown in Fig. 3. The two-component fit of these data of 71 = 6.6 ns, r2 = 1.1 ns, fi = 0.9 and xk = 8.6 are similar to that for the less precise data with the xenon lamp. The quality of the data obtained with the laser enabled us to perform a triexponential fit, with the fitting parameters given in Table I and Fig. 3. A lo-fold reduction in & is achieved in going from the biexponential to the triexponential fit. Using the biexponential fluorescence lifetimes, we measured the phase lag across the emission spectrum, to calculate the spectrum associated with each decay time. The details of this procedure, usually referred to as PRS, TABLE
I
Fluorescence Lifetime Data for b-MDH” Xenon lamp excitation* Temperature (“C)
71 bs)
72 W
011
Xi,
5
6.71
10
6.71
1.49 1.49
0.389 0.416
18.7
3.6
20 20 30
6.41 6.51 6.57
0.96 1.22 1.06
0.410 0.423 0.403
4.6 3.7 10.5
011
Xtle
0.552 0.550 0.551 0.527 0.444
8.6 7.2 5.3 3.0 3.4
Laser excitationd Biexponential 71
WI (M)
fits 72
bs) 6.56 5.03 4.27 3.87 3.60
0
0.12 0.22 0.32 0.51
1.11
0.875 0.826 0.806 0.916
Laser excitation Triexponential 72 73
0.12 0.22 0.32 0.51
a1
e?
Xft’
0.297 0.372
0.297 0.372
0.81
0.395
0.395
0.375 0.443
0.356 0.255
0.378 0.287 0.267 0.318 0.435
t-1
[KU (M) 0
fits
8.25 5.56 4.49 4.32 4.26
0.698 1.913 1.398
1.772 1.743
1.9
3.5 0.57 0.66
a Conditions: pH 7.6,35 mM KzHPOl, 0.1 M NaCl, broad band emission. * Global analysis with E.i = 1.00 kcal/mole, E,, = 2.02 kcal/mol, preexponential factors Al = 0.872 and A2 = 27.76, (Ye = 0.419, and global xi = 9.1. ’ xz calculated using (us= 0.5” and cM = 0.005. d Global analysis gives 7, = 6.53 ns, TV = 1.13 ns, k,, = 0.33 X 10’ M-k’, kqs = 1.25 X 10’ M-k’, 01~= 0.539, and x& = 8.1. e xi calculated using np = 0.2” and (TV = 0.002.
MALATE
DEHYDROGENASE
MHz
FIG. 3. Multifrequency phase and modulation data for b-MDH in the absence (0) and presence (0) of 50 X 10m6M NADH. Conditions: pH 7.6, 35 mM K*HPO,, 0.1 M NaCl, excitation at 300 nm with argon laser, magic angle emission polarizer, broad band emission observed. Biexponential fits are shown. For b-MDH alone, ri = 6.51 ns, r2 = 1.22 ns, fi = 0.879. In the presence of NADH, ri = 4.79 ns, r2 = 0.97 ns, and fi = 0.853.
has been described previously in detail (11, 12, 14). The 1.2-ns component is found to have a blue spectrum (X,,, = 328 nm). The 6.5ns component is more intense and fluoresces to the red (X,,, = 346 nm). Phase and modulation measurements with 290-nm xenon lamp excitation were performed on b-MDH over the temperature range 5 to 30°C. The two component fits are given in Table I. As can be seen, the magnitudes of the two lifetimes are relatively insensitive to temperature. The temperature-dependence data sets were also analyzed in a global manner (18). This involved linking the data sets by the Arrhenius function (i.e., for l/7) and by requiring (Y~to be the same at each temperature. The global fit was obtained with activation energies of 1.00 kcal/mol and 2.02 kcal/mol for the long (6.25 ns at 25°C) and short (1.09 ns at 25°C) components, respectively, and (Ye= 0.419 (long component). The & for this fiveparameter global fit is virtually the same as the average & for the individual biexponential fits (for which there are 15 fitting parameters). As described earlier, the addition of saturating amounts of NADH to b-MDH reduces the latter’s fluorescence yield. The consequences of this effect can be readily seen in Fig. 3, where the phase and modulation lifetime data for b-MDH, in the presence of 50 PM NADH, are also shown. The two- and three-component fits for these data are: 71 = 4.79 ns, fi = 0.85, r2 = 0.97 ns, xk = 4.5 and pi = 6.26 ns, fi = 0.46, 72 = 3.2 ns, fi = 0.45, ‘TV= 0.73 ns, f3 = 0.09, xi = 1.8. In terms of the biexponential analysis, both the long- and short-lived components are partially quenched by the binding of (and energy transfer to) NADH. The quenching action of iodide ion was also studied by time-resolved fluorescence spectroscopy. The two- and three-component fits for increasing amounts of added potassium iodide are shown in Table I. Again, in terms of the biexponential analysis, iodide ion appears to quench both components, with the longer-lived component being quenched to a greater degree. Global analysis of the
FROM
Bradyrhizobium
juponicum
105
data sets, with linkage by the Stern-Volmer equation (l/ 7qi = l/~i + k+[Q], where kqi is the quenching rate constant for component i and with the requirement that (Y; is the same for all [Q], yielded a biexponential fit with 7i = 6.53 ns, 72 = 1.13 ns, k,, = 0.33 X 10gM-‘s-l, ke2 = 1.25 X 10’ M-%-l, (Y~= 0.539, and & = 8.12. This global fit is not very satisfactory, considering the quality of the data. A global analysis in terms of a triexponential decay was also unsatisfactory (xi = 20), in comparison with the individual fits in Table I. Possible explanations for these poor global fits are that (i) linear Stern-Volmer plots do not describe the quenching of the individual components (which is possible since a constant ionic strength was not maintained), or (ii) that the CZ~ are not constant at all [Q] (which may be a consequence of a static quenching component). Differential polarized phase and modulation data for the tryptophan emission of b-MDH are shown in Fig. 4. The dynamic polarization results were best fit with two rotational correlation times, r$, of 80.5 and 0.238 ns, with limiting anisotropy contributions of 0.240 and 0.051, respectively. DISCUSSION
The fluorescence of b-MDH comes predominantly from the tryptophan residues of this protein. A relatively small tyrosine contribution centered around 305 nm is seen upon 280-nm excitation. Since we have shown, by chemical analysis, that there are two tryptophanyl residues in each monomeric unit, it is not surprising to find that the tryptophanyl fluorescence of b-MDH is heterogeneous. The simplest interpretation of the lifetime and quenching data can be made by assuming that these trp residues are emitting independently. If a biexponential decay is assumed, phase-resolved emission spectra show that one of the trp components has a r = 6.5 ns and X,,, = 346 nm, while the other is shorter lived with a T = 1.2 decay law does ns and X,, = 328 nm. A biexponential not appear to be adequate, but this phase-resolved spectral measurement shows the shorter decay time(s) to contribute to the blue side of the emission. It is possible
FIG. 4. Differential polarized phase and modulation anisotropy decay measurements with b-MDH. Conditions as in Fig. 3. Fit is with $i = 80.5 ns, & = 0.238 ns, g,r,, = 0.240 and g2roz = 0.051. This fit is a nonassociated fit with respect to the individual intensity decay times.
106
GHIRON
that excited state energy transfer occurs between the two types of tryptophan residues and that this is a basis for the multiexponential decay. However, we have used an excitation wavelength of 295-303 nm in all studies and trp + trp energy transfer is known to be minimized at the red edge of its absorption spectrum (20). Steady state anisotropy and dynamic polarization measurements indicate that both these residues are relatively immobile. The small activation energies obtained for thermal quenching of both lifetime components (for an assumed biexponential decay) support this contention. A small degree of rapid, restricted motion of the trp residues is detected by the 0.238-ns rotational correlation time. This rapid motion can be modeled in terms of the wobbling of the fluorophore in a cone of semiangle 13” (19). The anisotropy data also permit us to obtain an estimate of b-MDH’s molecular weight. The molecular weight of a spherical protein should be in direct proportion to its rotational correlation times, 4, by the equation Cp= uM (E + h)/RT, where u (= 0.89 CP at 25°C) is the bulk viscosity, M is the molecular weight of the macromolecule, U= 0.73 ml/g is the partial specific volume of the protein, and h is the hydration of the protein (which we take to be 0.3 ml/g). From the experimental value of Q,= 80.5 ns for global rotation of the protein, we calculate that b-MDH has an effective molecular weight of 213 kDa. This result is larger than the molecular weight of 139 kDa that has been estimated for b-MDH by an electrophoretic method (5). The discrepancy (i.e., longer than expected $Jfor a spherical protein of 139 kDa) suggests that the tetrameric protein has a nonspherical shape and/or that there is a large degree of hydration. The oligomeric structure of b-MDH is not easily dissociated and/or unfolded. The trp fluorescence X,,, was not red-shifted to 350 nm when the pH of the protein solution was decreased from 7.0 to 3.25 or when urea was added up to a concentration of 4 M. These findings indicate that the trp residues were not exposed to the aqueous solvent. Since the steady state anisotropy of b-MDH remained unchanged as the pH was decreased, we conclude that the decrease in this enzyme’s activity at low pH cannot be caused by tetramer dissociation and/or denaturation (5). The binding of NADH dynamically quenches both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in the fluorescence of bound NADH upon addition of either tartronic acid or D-malate. Also the association constant for NADH does not change appreciably by the addition of tartronic acid or D-malate. These results suggest that these ligands do not bind to b-MDH. This proposition is supported by the observation that D-malate does not inhibit b-MDH, but is contradicted by the preliminary observation that tartronic acid is a competitive inhibitor of b-MDH with an inhibition constant approximately
ET AL.
equal to 1 mM. More studies will be needed to resolve the later discrepancy. We have interpreted our fluorescence data in terms of contributions from two types of tryptophanyl residues. The emission from each of the individual trp residues may be heterogeneous as well. The fluorescence lifetime of individual tryptophanyl residues in proteins is rarely a single exponential (17). The more precise data obtained with 300-nm laser excitation suggests that each b-MDH trp residue may have a multiexponential decay. Thus, our description may be an oversimplification. However, such complexity is very difficult to model from an experimental and interpretative point of view. The simplified interpretation presented here should be adequate for the intended structural studies. ACKNOWLEDGMENTS This research was supported by NSF Grant DMB 88-06113 to M.R.E. andby USDA Competitive Grant 85CRCR-1-1734 to D.W.E. This is contribution No. 11,168 of the Agricultural Experiment Station of the University of Missouri.
REFERENCES 1. Jameson, D. M., Thomas, V., and Zhoa, D. M. (1989) Biochim. Biophys. Actu 994,187-190. 2. Baumgarten, B., and Joachim Hones. (1988) Photo&em. Photobiol. 47, 201-205. 3. Holbrook, J. J., and Wolf, R. G. (1972) Biochemistry 11, 24992502. 4. Banaszak, L. J., and Bradshaw, R. A. (1975) The Enzymes, Vol. 11, p. 369, Academic Press, New York. 5. Waters, J. K., Karr, D. B., and Emerich, D. W. (1985) Biochemistry 24,6479-6486. 6. Ghiron, C. A., Eftink, M. R., Porter, M. A., and Hartman, F. C. (1988) Arch. Biochem. Biophys. 260,267-272. 7. Jameson, D. M., Gratton, E., and Hall, R. D. (1984) Appl. Spectrosc. Rev. 20,55-85. 8. Wasylewski, Z., and Eftink, M. R. (1987) Eur. J. Biochem. 167, 513-518. 9. Eftink, M. R., and Ghiron, C. A. (1987) Biophys. J. 52,467-473. 10. Lakowicz, J. R., Laczko, G., Cherek, H., Gratton, E., and Limkeman, M. (1984) Biophys. J. 46,463-477. 11. Gratton, E., and Jameson, D. M. (1985) Anal. Chem. 57, 16941697. 12. Eftink, M. R., Ghiron, C. A., and Wasylewski, Z. (1987) Biochemistry 26,8338-8346. 13. Spies, J. R., and Chambers, D. C. (1948) Anal. Chem. 20,30-39. 14. Lakowicz, J. R., and Cherek, H. (1981) J. Biol. Chem. 256,63486353. 15. Dayhoff, M. D. (1972) Atlas of Proteins Sequence and Structure, Vol. 5, p. D-105, N.B.R. Foundation Press, Washington DC. 16. Gratton, E., Jameson, D. M., and Hail, R. D. (1984) Annu. Rev. Biophys. Bioeng. 13,105-124. 17. Beechem, J. M., and Brand, L. (1985) Annu. Rev. Biochem. 54, 43-71. 18. Beechem, J. M., and Gratton, E. (1988) SPZE Proc. 909.70-81. 19. Lipari, G., and Szabo, A. (1980) Biophys. J. 30,489~506. 20. Weber, G., and Shinitzky, M. (1970) Proc. Natl. Acad. Sci. USA 65,823-830.
