Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3805-3808, August 1979 Biochemistry
Characterization of the iron-sulfur centers in succinate dehydrogenase (iron-sulfur proteins/core extrusion/core displacement/interprotein core transfer/iron-sulfur center structures)
CHRISTOPHER J. COLES*, RICHARD H. HOLMf, DONALD M. KURTZ, JR.f, WILLIAM H. ORME-JOHNSON*, JILL RAWLINGSf, THOMAS P. SINGER*, AND GEOFFREY B. WONGf *Department of Biochemistry and Biophysics, University of California, and Molecular Biology Division, Veterans Administration Hospital, San Francisco, California 94121; tDepartment of Chemistry, Stanford University, Stanford, California 94305; and tDepartment of Biochemistry, University of Wisconsin,
Madison, Wisconsin 53706 Contributed by Richard H. Holm, May 29, 1979
ABSTRACT Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.11. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magnetic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)42- (RF = PC6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers -in this enzyme.
respectively) accounted for the chemically determined nonheme iron and labile sulfide content until it was observed§ that in succinate-cytochrome c reductase, a membrane preparation, and in the most intact form of the soluble enzyme available (13) reduction with dithionite generates the same amount of EPR signal at g = 1.94 as does succinate (S. P. J. Albracht, H. Beinert, B. A. C. Ackrell, and T. P. Singer, unpublished data). These findings have led to questions concerning the existence of center S-2. § Because of uncertainties in the EPR signal quantitations and the puzzling observations of Albracht§ on center S-2, we have undertaken characterization of the Fe-S centers in succinate dehydrogenase by entirely different means. Both techniques used in this study begin with reaction 1 (14-17). apoprotein
Fe-S protein + RSH Unfolding solvent
[Fe2S2(SR)4 12-
(I)
)
and/or [ Fe4
[1]
S4(SR)4 ]2- (II)J
Although the presence of nonheme iron and of labile sulfide in succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] has been recognized for nearly 25 years (1-3), the chemical nature of the iron-sulfur centers of the enzyme has remained unsettled (4, §) despite extensive studies in many laboratories. Most investigators agree that, in relatively intact preparations, the protein contains 8 mol each of nonheme iron and acid-labile sulfur and one covalently bound flavin per mol (5-7). Two electron paramagnetic resonance (EPR)-detectable iron-sulfur centers subject to reduction by the substrate have been recognized in particulate and soluble preparations of the enzyme in amounts stoichiometric with the flavin (4,8-10). One center, S-1, in the reduced state affords an EPR signal centered near g t 1.94, which is typical of binuclear (Fe2S2) centers. The other center, S-3, when oxidized exhibits a g k 2.01 EPR signal, similar to that of the oxidized high potential protein of Chromatium (11), and has, therefore, been assumed to be a tetranuclear (Fe4S4) center. Reduction with dithionite but not with succinate elicits the appearance of an additional EPR signal at g ; 1.94 with a different line shape which has been ascribed to a third center, S-2, also assumed to be binuclear (4, 9). Contrary to earlier claims (12), the concentration of center S-2 calculated from the EPR signal generated by dithionite is less than 0.5 mol per mol of flavin, possibly because of spin coupling between centers S-1 and S-2 (4, 9). The presence of two 2-Fe and of one 4-Fe center (with Fe2S2 and Fe4S4 core structures,
In a medium capable of unfolding protein tertiary structure, Fe2S2 and Fe4S4 cores of protein centers are extruded [alternatively, displaced (16)] as their thiolate complexes I and II in the presence of excess thiol. If interfering chromophores are absent, the extruded cores may be spectrophotometrically assayed (14, 16). This procedure is not readily applicable to succinate dehydrogenase owing to the presence of a histidyl-8aFAD prosthetic group (2, 18). Two further methods of detecting and quantitating iron-sulfur cores have recently been developed which obviate the problem of interfering chromophores. One of these uses p-CF3C6H4SH (RFSH) as the extrusion reagent. Products I and II (R = p-C6H4CF3), resulting from protein 2-Fe and 4-Fe centers, respectively, exhibit contact-shifted 19F resonances that are well separated from each other and from that of excess RFSH. These species can be identified and quantitated by Fourier transform 19F nuclear magnetic resonance (NMR) spectroscopy (19, 20). In the interprotein core
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: EPR, electron paramagnetic resonance; Fd, ferredoxin; Me6phosphoramide, hexamethylphosphoramide; RF, p-C6H4CF3. § Albracht, S. P. J. (1977) Abstracts, International Symposium on Membrane Bioenergetics, Spetsai, Greece, no. 37.
3805
I2-Fe, 4-Fe apo Fd
2-Fe, 4-Fe Fdox S Folding S2042-
[2]
2-Fe, 4-Fe Fdred
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Proc. Natl. Acad. Sci. USA 76 (1979)
transfer method, cores are displaced with an appropriate thiol, 2-Fe and 4-Fe oxidized ferredoxins (Fdox) are formed by specific core transfer to acceptor apoFd proteins, and the latter are folded to normal protein configurations by dilution with aqueous buffer. Reaction sequence 2 is completed by dithionite reduction to yield Fdred proteins, which are identified and quantitated by low-temperature EPR spectroscopy. MATERIALS AND METHODS Materials. Unless otherwise mentioned, succinate dehydrogenase was isolated from complex II by the method of Ackrell et al. (13) and stored as (NH4)2SO4 pellets in liquid nitrogen. In the 19F NMR experiments (Table 1), the pellets were taken up immediately before use in a minimum volume of 50 mM Tris-HCl buffer (pH 8.5), containing 20 mM succinate, under a stream of argon. Residual (NH4)2SO4 (insoluble in the reaction medium) was removed by rapid centrifugation through a small Sephadex G-50 column (21). The specific activity of the enzyme at 38°C in the phenazine methosulfate assay at Vmax was 110 ,umol of succinate per min per mg of protein, and the histidyl flavin and nonheme iron contents were 8.6 nmol/mg and 67 nmol/mg, respectively. In one experiment, in the core transfer method (Table 2) an -70% pure sample of the enzyme, isolated by the method of Davis and Hatefi (6), was used. The specific activity was 82 ,umol/min per mg, the histidyl flavin content was 6.5 nmol/mg, and the nonheme iron content was 53 nmol/mg of protein. (Et4N)2[Fe2S2(SC6H4p-CF3)4], (Et4N)2[Fe4S4(SC6H4-p-CF3)4], p-CF3C6H4SH, CrH5CFC12, and o-xylyldithiol were prepared as described (20). N,N-Dimethylformamide was distilled from barium oxide and stored under argon. Hexamethylphosphoramide (Me6phosphoramide) was distilled under reduced pressure at -600C from barium oxide, redistilled from P205 immediately before use, and handled anaerobically. N-Methylformamide and dimethyl sulfoxide were used as supplied (Aldrich). Ferredoxins from Bacillus polymyxa (22) and adrenal cortical mitochondria (23) were converted to apoproteins by saturating 0.3 mM solutions of proteins in 0.5 M Tris-HCI (pH 8) with 2,2'-bipyridyl and adding solid dithiothreitol and urea to 25 mM and 6 M, respectively. After 1 hr at room temperature, apoprotein was recovered by passage of the solution through a Sephadex G-25 column equilibrated with 50 mM TrisIHCl, pH 8/10 mM 2-mercaptoethanol. Apoproteins were concentrated by ultrafiltration to 0.3 mM and stored under liquid nitrogen.
Chemical Determinations and Enzyme Assays. Protein was determined by the biuret method (24); histidyl flavin (25), nonheme iron (26), and succinate dehydrogenase activity (25) were determined by published procedures. Core Identification. All manipulations and measurements were carried out under strictly anaerobic conditions. 19F NMR method. The procedures for this method have been described in detail elsewhere (19, 20). Extrusions were performed in Me6phosphoramide/buffer solutions, 4:1 (vol/vol) (80%), with
FIG. 1. 19F Fourier transform NMR spectra (338 MHz) of a Me6phosphoramide/H20, 4:1 (vol/vol), solution of succinate dehydrogenase at -15°C after completion of the active site core extrusion reactions and after addition of standards. (Lower) Spectrum of succinate dehydrogenase 0.103 mM in flavin, RpSH/Fe mol ratio of 430/1. (Upper) Spectrum of 0.5 ml of the preceding solution + 5 ,l of 18.2 mM [Fe4S4(SRF)412' (t2-) + 8 Al of 24.06 mM [Fe2S2(SRF)412- (d2-) in dimethylformamide. Acquisition time of both spectra was 14 min.
RFSH as the extrusion reagent. Dithiothreitol (5 mM) was added to the protein solution prior to introduction of Me6phosphoramide. Extrusions were allowed to proceed for 45 min at 25°C.
These conditions were selected on the basis of visible spectral changes of aliquots of similar extrusion solutions monitored with a Cary model 14 spectrophotometer. The completeness of extrusion of the Fe-S centers was assayed by passing an aliquot of each extrusion mixture through a column of Sephadex G-50 equilibrated with 80%. Me6phosphoramide so as to separate the extruded clusters from the protein, and the iron content of the excluded (i.e., protein) phase was then determined. All NMR spectra were obtained in the Fourier transform mode with instruments and under the conditions described (19, 20). Extruded Fe2S2 and Fe4S4 cores were quantitated by measurement of 19F intensity ratios of spectra obtained on solutions before and after the addition of small volumes of standard solutions of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2-. Further experimental details are given in Table 1 and Fig. 1. Interprotein core transfer (EPR) method. Reagents were prepared and all reactions were carried out at room temperature in a double-septum seal apparatus (16) rendered 02 free
by repeated evacuation and flushing with purified argon. Typically, to 0.66 mg of the enzyme in 0.05 ml of 0.2 M TrisHCI (pH 8.5) was added 0.2 ml of dimethyl sulfoxide/methylformamide, 5:1 (vol/vol) containing 15 mM o-xylyldithiol. After 30 min, 0.25 ml of 150 ,uM apoferredoxin in 0.1 M TrisHC1 (pH 8.5) was added and, after an additional 60 min, 0.01 ml of an aqueous solution containing 1 M Na2S204, 1 M Tris base, and 0.25 mM methyl viologen was added to reduce the
Table 1. Core composition of succinate dehydrogenase by the 19F NMR methoda % Fe Initial conc., mM Products, mM Flavin Fe Total Fe extrudedef nd/flavin njflavin [Fe2S2(SRF)4]2- IFe4S4(SRF)4120.146 1.151b 0.112 86e 1.8 0.77 0.988 0.270 0.103 0.804c 0.080 0.786 98f 2.3 0.78 0.233 2.1 0.0516 0.402d 0.74 0.107 0.038 0.365 91f a Experimental conditions: Me6phosphoramide/H20, 4:1 (vol/vol); aqueous solution before Me6phosphoramide dilution contained enzyme, 50 mM Tris-HCl (pH 8.5), 20 mM succinate, and 5 mM dithiothreitol; extrusion reaction time was 45 min at 251C. b-d RFSH/Fe mol ratios in extrusion solutions: b 270/1, c 430/1, and d 950/1. e,f Percent initial Fe analytically determined in residual protein after extrusion: e 7% and f -0%.
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Coles et al.
3807
perimentsO with spinach Fdoxred and Clostridium pasteuri2-Fe Fdox,red under very similar experimental conditions have demonstrated, by spectrophotometric ass'ay, that both the oxidized and reduced proteins afford [Fe2S2(SRF)4]2- as the extrusion product in nearly quantitative recovery (19, 20). Similarly, extrusion of Chromatium high potential proteinx,red and C. pasteurianum 8-Fe Fd0x produces [Fe4S4(SRF)4]2- in anum
high yield (19, 20).
These observations show that Fe2S2
Fe4S4
conversions involving cores in the enzyme with those oxidation levels ([Fe2S2]2+,+, [Fe4S4]3+'2+) indicated by the presence (or absence) of EPR signals do not occur to an important extent under the present experimental conditions. Thus, the signals in Fig. 1 demonstrate unequivocally that succinate dehydrogenase contains both 2-Fe and 4-Fe centers. The results from three extrusion experiments using the 19F NMR method are compiled in Table 1. Extrusion of total iron approaches completeness, particularly in the experiment illustrated in Fig. 1. For nd = 2, the recovery of Fe2S2 cores is in each case within ±15%, the estimated error of the method, leaving no doubt that two 2-Fe centers are present per flavin. For nt = 1, the recovery of Fe4S4 cores is below the usual error limit but highly suggestive of one 4-Fe center per flavin. Our experience with this method has shown that quantitation of Fe4S4 cores is usually core
I
2.05
2.02
FIG. 2. EPR spectra of core transfer samples prepared as described in the text and Table 2. (Upper) Spectrum of reconstituted B. polymyxa ferredoxin formed in Exp. 2, representing Fe4S4 cores. (Lower) Spectrum of reconstituted adrenodoxin formed in Exp. 3, representing Fe2S2 cores. EPR conditions were as described in Table 2.
ferredoxin formed. Concentrations of ferredoxin were estimated from EPR spectra of the reduced reaction mixture, obtained at 13 K, by comparison of the heights of features near g = 2.05 (Fe4S4 cores) or 2.02 (Fe2S2 cores) to reduced ferredoxin standards. The concentrations of the latter were checked by double integration of the EPR signals (27). This method will be described in more detail elsewhere (J. R. Bale, B. A. Averill, J. Rawlings, and W. H. Orme-johnson, unpublished data). EPR measurements were performed with a Varian E-9 spectrometer operating at 9.1 GHz. By the quantitation procedures described, the number of FenSn cores of Fe-S centers extruded per histidyl flavin was calculated as follows: nd = [extruded Fe2S2]/[flavin] and nt = [extruded Fe4S4]/[flavin]. RESULTS AND DISCUSSION Shown in Fig. 1 are the 19F NMR spectra of a solution of succinate dehydrogenase 0.103 mM in flavin in 80% Me6phosphoramide after core extrusion of the Fe-S centers with RFSH and after the addition of standards. The signals at 3.7 and 6.4 ppm arise from [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2-, respectively, as shown by separate measurements of these two species under the same conditions (19, 20) and by the coincidence of these signals with those of the added standards. Control ex-
less satisfactory than for Fe2S2 cores (28). The core transfer experiments were conducted by a similar method. In Fig. 2 are shown EPR spectra corresponding to the second and third experiments described in Table 2-i.e., for cases where only one type of apoferredoxin was present in each reaction mixture. As can be seen from the data presented, the precision of the method is probably better than +10%. (We estimate recoveries of better than 90% for Fe4S4 cores and better than 80% for Fe2S2 cores from control experiments; J. R. Bale, B. A. Averill, J. Rawlings, and W. H. Orme-Johnson, unpublished data.) In contrast to the NMR method, this method gives somewhat less complete recovery for Fe2S2 cores than for Fe4S4 cores, and the ratios of the two core types are less than 2 in the latter case. In passing, it is interesting that the EPR method requires only about 1/10th to 1/30th the amount of donor protein needed for the NMR procedure for a determination of comparable precision (Tables 1 and 2). However, the technology required for the NMR method is more widely distributed so that the advantages of the EPR method may not, in practice, be as great as this ratio would suggest, particularly if the amounts of protein available are not limiting. The data in Tables 1 and 2 collectively prove that mammalian succinate dehydrogenase contains two 2-Fe and one 4-Fe center per histidyl flavin, despite the fact that the concentration An extensive list of control experiments for the 19F NMR method is given in ref. 20.
Table 2. Core composition of succinate dehydrogenase by the interprotein core transfer methoda Products, ,uMb Initial conc., ,uM nd/flavin Total Fe nt/flavin 4-Fe Fdred 2-Fe Fdred Fe Flavin Exp. 1.1 1.8 ice 32 4.5 4.0 32 7 1.7 12 57 2df 7.1 0.92 6.5 7.1 57 3df 1.0 1.8 86 11.5 20 4d,e 89 11.1 a Experimental conditions: samples were prepared as described in the text. b Concentrations of the Fe-S centers were detected by EPR. Conditions for EPR measurements were: microwave power, 0.03 mW for 2-Fe measurements, 0.3 mW for 4-Fe measurements; 13 K; modulation amplitude, 10 G; field modulation, 100 kHz; time constant, 0.35; field sweep rate, 0.1 T min-'; Klystron frequency, 9.1 GHz. c Enzyme prepared by the method of Davis and Hatefi (6). d Enzyme prepared by the method of Ackrell et al. (13). e Both apoproteins present in sample. f Only one apoprotein in sample.
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Biochemistry:
Coles et al.
of centers S-1 plus S-2, as measured by EPR techniques after reduction by dithionite, is well below that predicted from this ratio (4, 9) and that, as noted above, in some preparations no evidence for center S-2 was found by EPR. It is particularly interesting that the sample of succinate dehydrogenase, prepared by the method of Ackrell et al. (13), in which quantitation of the EPR signal after dithionite reduction gave only one Fd-type (i.e., g t 1.94) center per mole of flavin (unpublished data), was the same as that used in the experiments of Tables 1 and 2. It appears, then, that in the most intact preparations the conformation of the enzyme renders center S-2 EPR-silent. We regard the characterization of Fe-S centers in succinate dehydrogenase reported here as direct and unequivocal and acknowledge agreement with the description of two 2-Fe centers and one 4-Fe center adduced from the inherently less explicit results of EPR investigations (4, 8-10, 29). This research was supported at the Molecular Biology Division, Veterans Administration Hospital, in part by National Institutes of Health Program Project HL-16251 and by National Science Foundation Grant PCM 76-03367, at Stanford University by National Institutes of Health Grant GM-22352, at the Stanford Magnetic' Resonance Laboratory by National Science Foundation Grant GR-23633 and National Institutes of Health Grant RR-00711, and at the University of Wisconsin by National Institutes of Health Grant GM 17170 and by
the Graduate Research Committee and the College of Agricultural and Life Sciences. D.M.K. is the recipient of a National Institutes of Health Postdoctoral Fellowship (1977-1979). 1. Singer, T. P., Kearney, E. B. & Zastrow, N. (1955) Biochim. Biophys. Acta 17, 154-155. 2. Kearney, E. B. & Singer, T. P. (1955) Biochim. Biophys. Acta 17, 596-597. 3. Massey, V. (1957) J. Biol. Chem. 229, 763-770. 4, Beinert, H., Ackrell, B. A. C., Vinogradov, A. D., Kearney, E. B. & Singer, T. P. (1977) Arch. Biochem. Biophys. 182,95-106. 5. Lusty, C. J., Machinist, J. M. & Singer, T. P. (1965) J. Biol. Chem. 240, 1804-1810. 6. Davis, K. A. & Hatefi, Y. (1971) Biochemistry 10, 2509-2516.
Proc. Natl. Acad. Sci. USA 76 (1979) 7. Coles, C. J., Tisdale, H. D., Kenney, W. C. & Singer, T. P. (1972) Physiol. Chem. Phys. 4,301-316. 8. Beinert, H., Ackrell, B. A. C., Kearney, E. B. & Singer, T. P. (1975) Eur. J. Biochem. 54,185-194. 9. Ohnishi, T., Salerno, J. C., Winter, D. B., Lim, J., Yu, C. A., Yu, L. & King, T. E. (1976) J. Biol. Chem. 251, 2094-2104. 10. Ohnishi, T., Lim, J., Winter, D. B. & King, T. E. (1976) J. Biol. Chem. 251, 2105-2109. 11. Dus, K., DeKlerk, H., Sletten, K. & Bartsch, R. G. (1967) Biochim. Biophys. Acta 140, 291-311. 12. Ohnishi, T., Leigh, J. S., Winter, D. B., Lim, J. & King, T. E. (1974) Biochem. Biophys. Res. Commun; 61, 1026-1035. 13. Ackrell, B. A. C., Kearney, E. B. & Coles, C. J. (1977) J. Biol. Chem. 252, 6963-6965. 14. Gillum, W. O., Mortenson, L, E., Chen, J.-S. & Holm, R. H. (1977) J. Am. Chemh. Soc. 99,584-595. 15. Holm, R. H. (1977) in Biological Aspects of Inorganic Chemistry, eds. Addison, A. W., Cullen, W. R., Dolphin, D. & James, B. R. (Wiley, New York), pp. 71-111. 16. Averill, B. A., Bale, J. R. & Orme-Johnson, W. H. (1978) J. Am. Chem. Soc. 100, 3034-3043. 17. Bale, J. R. (1974) Dissertation (Univ. Wisconsin, Madison,
WI). 18. Walker, W. H. & Singer, T. P. (1970) J. Biol. Chem. 245, 4224-4225. 19. Kurtz, D. M., Jr., Wong, G. B. & Holm, R. H. (1978) J. Am. Chem. Soc. 100, 6777-6779. 20. Wong, G. B., Kurtz, D. M., Jr., Holm, R. H., Mortenson, L. E. & Upchurch, R. G. (1979) J. Am. Chem. Soc., 101, 3078-3090. 21. Penefsky, H. S. (1977) J. Biol. Chem. 252,2891-2899. 22. Stombaugh, N. A., Burris, R. H. & Orme-Johnson, W. H. (1973) J. Biol. Chem. 248,7951-7956. 23. Orme-Johnson, W. H. & Beinert, H. (1969) J. Biol. Chem. 244, 6143-6148. 24. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) J. Biol. Chem. 177, 751-766. 25. Singer, T. P. (1974) in Methods of Biochemical Analysis, ed. Glick, D. (Wiley, New York), Vol. 22, pp. 123-175. 26. Brumby, P. E. & Massey, V. (1967) Methods Enzymol. 10, 463-474. 27. Aasa, R. & Vanngard, T. (1975) J. Magn. Reson. 19,308-315. 28. Wong, G. B. (1978) Dissertation (Stanford Univ., Stanford, CA). 29. Salerno, J. C., Ohnishi, T., Lim, J. & King, T. E. (1976) Biochem. Biophys. Res. Commun. 73,833-840.
Characterization of the iron-sulfur centers in succinate dehydrogenase (iron-sulfur proteins/core extrusion/core displacement/interprotein core transfer/iron-sulfur center structures)
CHRISTOPHER J. COLES*, RICHARD H. HOLMf, DONALD M. KURTZ, JR.f, WILLIAM H. ORME-JOHNSON*, JILL RAWLINGSf, THOMAS P. SINGER*, AND GEOFFREY B. WONGf *Department of Biochemistry and Biophysics, University of California, and Molecular Biology Division, Veterans Administration Hospital, San Francisco, California 94121; tDepartment of Chemistry, Stanford University, Stanford, California 94305; and tDepartment of Biochemistry, University of Wisconsin,
Madison, Wisconsin 53706 Contributed by Richard H. Holm, May 29, 1979
ABSTRACT Two techniques have been applied to the determination of the number and type (2-Fe, 4-Fe) of iron-sulfur centers in the iron-sulfur flavoprotein succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.11. One procedure uses p-CF3C6H4SH as an extrusion reagent and Fourier transform 19F nuclear magnetic resonance as the method of detection and quantitation of extruded cores of these centers in the form of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)42- (RF = PC6H4CF3). The second procedure, interprotein core transfer, involves thiol displacement of iron-sulfur cores followed by specific core transfer to the apoproteins of Bacillus polymyxa ferredoxin and adrenodoxin. Detection and quantitation are accomplished by electron paramagnetic resonance of reduced proteins at low temperatures. Both procedures clearly show that succinate dehydrogenase contains two dimeric (Fe2S2) and one tetrameric (Fe4S4) centers per mole of histidyl flavin, accounting for all eight nonheme iron and eight labile sulfur atoms found by chemical analysis. These results remove uncertainties created by the less than stoichiometric amounts of binuclear centers detected by electron paramagnetic resonance after dithionite reduction and provide secure characterization of the iron-sulfur centers -in this enzyme.
respectively) accounted for the chemically determined nonheme iron and labile sulfide content until it was observed§ that in succinate-cytochrome c reductase, a membrane preparation, and in the most intact form of the soluble enzyme available (13) reduction with dithionite generates the same amount of EPR signal at g = 1.94 as does succinate (S. P. J. Albracht, H. Beinert, B. A. C. Ackrell, and T. P. Singer, unpublished data). These findings have led to questions concerning the existence of center S-2. § Because of uncertainties in the EPR signal quantitations and the puzzling observations of Albracht§ on center S-2, we have undertaken characterization of the Fe-S centers in succinate dehydrogenase by entirely different means. Both techniques used in this study begin with reaction 1 (14-17). apoprotein
Fe-S protein + RSH Unfolding solvent
[Fe2S2(SR)4 12-
(I)
)
and/or [ Fe4
[1]
S4(SR)4 ]2- (II)J
Although the presence of nonheme iron and of labile sulfide in succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] has been recognized for nearly 25 years (1-3), the chemical nature of the iron-sulfur centers of the enzyme has remained unsettled (4, §) despite extensive studies in many laboratories. Most investigators agree that, in relatively intact preparations, the protein contains 8 mol each of nonheme iron and acid-labile sulfur and one covalently bound flavin per mol (5-7). Two electron paramagnetic resonance (EPR)-detectable iron-sulfur centers subject to reduction by the substrate have been recognized in particulate and soluble preparations of the enzyme in amounts stoichiometric with the flavin (4,8-10). One center, S-1, in the reduced state affords an EPR signal centered near g t 1.94, which is typical of binuclear (Fe2S2) centers. The other center, S-3, when oxidized exhibits a g k 2.01 EPR signal, similar to that of the oxidized high potential protein of Chromatium (11), and has, therefore, been assumed to be a tetranuclear (Fe4S4) center. Reduction with dithionite but not with succinate elicits the appearance of an additional EPR signal at g ; 1.94 with a different line shape which has been ascribed to a third center, S-2, also assumed to be binuclear (4, 9). Contrary to earlier claims (12), the concentration of center S-2 calculated from the EPR signal generated by dithionite is less than 0.5 mol per mol of flavin, possibly because of spin coupling between centers S-1 and S-2 (4, 9). The presence of two 2-Fe and of one 4-Fe center (with Fe2S2 and Fe4S4 core structures,
In a medium capable of unfolding protein tertiary structure, Fe2S2 and Fe4S4 cores of protein centers are extruded [alternatively, displaced (16)] as their thiolate complexes I and II in the presence of excess thiol. If interfering chromophores are absent, the extruded cores may be spectrophotometrically assayed (14, 16). This procedure is not readily applicable to succinate dehydrogenase owing to the presence of a histidyl-8aFAD prosthetic group (2, 18). Two further methods of detecting and quantitating iron-sulfur cores have recently been developed which obviate the problem of interfering chromophores. One of these uses p-CF3C6H4SH (RFSH) as the extrusion reagent. Products I and II (R = p-C6H4CF3), resulting from protein 2-Fe and 4-Fe centers, respectively, exhibit contact-shifted 19F resonances that are well separated from each other and from that of excess RFSH. These species can be identified and quantitated by Fourier transform 19F nuclear magnetic resonance (NMR) spectroscopy (19, 20). In the interprotein core
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: EPR, electron paramagnetic resonance; Fd, ferredoxin; Me6phosphoramide, hexamethylphosphoramide; RF, p-C6H4CF3. § Albracht, S. P. J. (1977) Abstracts, International Symposium on Membrane Bioenergetics, Spetsai, Greece, no. 37.
3805
I2-Fe, 4-Fe apo Fd
2-Fe, 4-Fe Fdox S Folding S2042-
[2]
2-Fe, 4-Fe Fdred
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Proc. Natl. Acad. Sci. USA 76 (1979)
transfer method, cores are displaced with an appropriate thiol, 2-Fe and 4-Fe oxidized ferredoxins (Fdox) are formed by specific core transfer to acceptor apoFd proteins, and the latter are folded to normal protein configurations by dilution with aqueous buffer. Reaction sequence 2 is completed by dithionite reduction to yield Fdred proteins, which are identified and quantitated by low-temperature EPR spectroscopy. MATERIALS AND METHODS Materials. Unless otherwise mentioned, succinate dehydrogenase was isolated from complex II by the method of Ackrell et al. (13) and stored as (NH4)2SO4 pellets in liquid nitrogen. In the 19F NMR experiments (Table 1), the pellets were taken up immediately before use in a minimum volume of 50 mM Tris-HCl buffer (pH 8.5), containing 20 mM succinate, under a stream of argon. Residual (NH4)2SO4 (insoluble in the reaction medium) was removed by rapid centrifugation through a small Sephadex G-50 column (21). The specific activity of the enzyme at 38°C in the phenazine methosulfate assay at Vmax was 110 ,umol of succinate per min per mg of protein, and the histidyl flavin and nonheme iron contents were 8.6 nmol/mg and 67 nmol/mg, respectively. In one experiment, in the core transfer method (Table 2) an -70% pure sample of the enzyme, isolated by the method of Davis and Hatefi (6), was used. The specific activity was 82 ,umol/min per mg, the histidyl flavin content was 6.5 nmol/mg, and the nonheme iron content was 53 nmol/mg of protein. (Et4N)2[Fe2S2(SC6H4p-CF3)4], (Et4N)2[Fe4S4(SC6H4-p-CF3)4], p-CF3C6H4SH, CrH5CFC12, and o-xylyldithiol were prepared as described (20). N,N-Dimethylformamide was distilled from barium oxide and stored under argon. Hexamethylphosphoramide (Me6phosphoramide) was distilled under reduced pressure at -600C from barium oxide, redistilled from P205 immediately before use, and handled anaerobically. N-Methylformamide and dimethyl sulfoxide were used as supplied (Aldrich). Ferredoxins from Bacillus polymyxa (22) and adrenal cortical mitochondria (23) were converted to apoproteins by saturating 0.3 mM solutions of proteins in 0.5 M Tris-HCI (pH 8) with 2,2'-bipyridyl and adding solid dithiothreitol and urea to 25 mM and 6 M, respectively. After 1 hr at room temperature, apoprotein was recovered by passage of the solution through a Sephadex G-25 column equilibrated with 50 mM TrisIHCl, pH 8/10 mM 2-mercaptoethanol. Apoproteins were concentrated by ultrafiltration to 0.3 mM and stored under liquid nitrogen.
Chemical Determinations and Enzyme Assays. Protein was determined by the biuret method (24); histidyl flavin (25), nonheme iron (26), and succinate dehydrogenase activity (25) were determined by published procedures. Core Identification. All manipulations and measurements were carried out under strictly anaerobic conditions. 19F NMR method. The procedures for this method have been described in detail elsewhere (19, 20). Extrusions were performed in Me6phosphoramide/buffer solutions, 4:1 (vol/vol) (80%), with
FIG. 1. 19F Fourier transform NMR spectra (338 MHz) of a Me6phosphoramide/H20, 4:1 (vol/vol), solution of succinate dehydrogenase at -15°C after completion of the active site core extrusion reactions and after addition of standards. (Lower) Spectrum of succinate dehydrogenase 0.103 mM in flavin, RpSH/Fe mol ratio of 430/1. (Upper) Spectrum of 0.5 ml of the preceding solution + 5 ,l of 18.2 mM [Fe4S4(SRF)412' (t2-) + 8 Al of 24.06 mM [Fe2S2(SRF)412- (d2-) in dimethylformamide. Acquisition time of both spectra was 14 min.
RFSH as the extrusion reagent. Dithiothreitol (5 mM) was added to the protein solution prior to introduction of Me6phosphoramide. Extrusions were allowed to proceed for 45 min at 25°C.
These conditions were selected on the basis of visible spectral changes of aliquots of similar extrusion solutions monitored with a Cary model 14 spectrophotometer. The completeness of extrusion of the Fe-S centers was assayed by passing an aliquot of each extrusion mixture through a column of Sephadex G-50 equilibrated with 80%. Me6phosphoramide so as to separate the extruded clusters from the protein, and the iron content of the excluded (i.e., protein) phase was then determined. All NMR spectra were obtained in the Fourier transform mode with instruments and under the conditions described (19, 20). Extruded Fe2S2 and Fe4S4 cores were quantitated by measurement of 19F intensity ratios of spectra obtained on solutions before and after the addition of small volumes of standard solutions of [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2-. Further experimental details are given in Table 1 and Fig. 1. Interprotein core transfer (EPR) method. Reagents were prepared and all reactions were carried out at room temperature in a double-septum seal apparatus (16) rendered 02 free
by repeated evacuation and flushing with purified argon. Typically, to 0.66 mg of the enzyme in 0.05 ml of 0.2 M TrisHCI (pH 8.5) was added 0.2 ml of dimethyl sulfoxide/methylformamide, 5:1 (vol/vol) containing 15 mM o-xylyldithiol. After 30 min, 0.25 ml of 150 ,uM apoferredoxin in 0.1 M TrisHC1 (pH 8.5) was added and, after an additional 60 min, 0.01 ml of an aqueous solution containing 1 M Na2S204, 1 M Tris base, and 0.25 mM methyl viologen was added to reduce the
Table 1. Core composition of succinate dehydrogenase by the 19F NMR methoda % Fe Initial conc., mM Products, mM Flavin Fe Total Fe extrudedef nd/flavin njflavin [Fe2S2(SRF)4]2- IFe4S4(SRF)4120.146 1.151b 0.112 86e 1.8 0.77 0.988 0.270 0.103 0.804c 0.080 0.786 98f 2.3 0.78 0.233 2.1 0.0516 0.402d 0.74 0.107 0.038 0.365 91f a Experimental conditions: Me6phosphoramide/H20, 4:1 (vol/vol); aqueous solution before Me6phosphoramide dilution contained enzyme, 50 mM Tris-HCl (pH 8.5), 20 mM succinate, and 5 mM dithiothreitol; extrusion reaction time was 45 min at 251C. b-d RFSH/Fe mol ratios in extrusion solutions: b 270/1, c 430/1, and d 950/1. e,f Percent initial Fe analytically determined in residual protein after extrusion: e 7% and f -0%.
Proc. Natl. Acad. Sci. USA 76 (1979)
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perimentsO with spinach Fdoxred and Clostridium pasteuri2-Fe Fdox,red under very similar experimental conditions have demonstrated, by spectrophotometric ass'ay, that both the oxidized and reduced proteins afford [Fe2S2(SRF)4]2- as the extrusion product in nearly quantitative recovery (19, 20). Similarly, extrusion of Chromatium high potential proteinx,red and C. pasteurianum 8-Fe Fd0x produces [Fe4S4(SRF)4]2- in anum
high yield (19, 20).
These observations show that Fe2S2
Fe4S4
conversions involving cores in the enzyme with those oxidation levels ([Fe2S2]2+,+, [Fe4S4]3+'2+) indicated by the presence (or absence) of EPR signals do not occur to an important extent under the present experimental conditions. Thus, the signals in Fig. 1 demonstrate unequivocally that succinate dehydrogenase contains both 2-Fe and 4-Fe centers. The results from three extrusion experiments using the 19F NMR method are compiled in Table 1. Extrusion of total iron approaches completeness, particularly in the experiment illustrated in Fig. 1. For nd = 2, the recovery of Fe2S2 cores is in each case within ±15%, the estimated error of the method, leaving no doubt that two 2-Fe centers are present per flavin. For nt = 1, the recovery of Fe4S4 cores is below the usual error limit but highly suggestive of one 4-Fe center per flavin. Our experience with this method has shown that quantitation of Fe4S4 cores is usually core
I
2.05
2.02
FIG. 2. EPR spectra of core transfer samples prepared as described in the text and Table 2. (Upper) Spectrum of reconstituted B. polymyxa ferredoxin formed in Exp. 2, representing Fe4S4 cores. (Lower) Spectrum of reconstituted adrenodoxin formed in Exp. 3, representing Fe2S2 cores. EPR conditions were as described in Table 2.
ferredoxin formed. Concentrations of ferredoxin were estimated from EPR spectra of the reduced reaction mixture, obtained at 13 K, by comparison of the heights of features near g = 2.05 (Fe4S4 cores) or 2.02 (Fe2S2 cores) to reduced ferredoxin standards. The concentrations of the latter were checked by double integration of the EPR signals (27). This method will be described in more detail elsewhere (J. R. Bale, B. A. Averill, J. Rawlings, and W. H. Orme-johnson, unpublished data). EPR measurements were performed with a Varian E-9 spectrometer operating at 9.1 GHz. By the quantitation procedures described, the number of FenSn cores of Fe-S centers extruded per histidyl flavin was calculated as follows: nd = [extruded Fe2S2]/[flavin] and nt = [extruded Fe4S4]/[flavin]. RESULTS AND DISCUSSION Shown in Fig. 1 are the 19F NMR spectra of a solution of succinate dehydrogenase 0.103 mM in flavin in 80% Me6phosphoramide after core extrusion of the Fe-S centers with RFSH and after the addition of standards. The signals at 3.7 and 6.4 ppm arise from [Fe2S2(SRF)4]2- and [Fe4S4(SRF)4]2-, respectively, as shown by separate measurements of these two species under the same conditions (19, 20) and by the coincidence of these signals with those of the added standards. Control ex-
less satisfactory than for Fe2S2 cores (28). The core transfer experiments were conducted by a similar method. In Fig. 2 are shown EPR spectra corresponding to the second and third experiments described in Table 2-i.e., for cases where only one type of apoferredoxin was present in each reaction mixture. As can be seen from the data presented, the precision of the method is probably better than +10%. (We estimate recoveries of better than 90% for Fe4S4 cores and better than 80% for Fe2S2 cores from control experiments; J. R. Bale, B. A. Averill, J. Rawlings, and W. H. Orme-Johnson, unpublished data.) In contrast to the NMR method, this method gives somewhat less complete recovery for Fe2S2 cores than for Fe4S4 cores, and the ratios of the two core types are less than 2 in the latter case. In passing, it is interesting that the EPR method requires only about 1/10th to 1/30th the amount of donor protein needed for the NMR procedure for a determination of comparable precision (Tables 1 and 2). However, the technology required for the NMR method is more widely distributed so that the advantages of the EPR method may not, in practice, be as great as this ratio would suggest, particularly if the amounts of protein available are not limiting. The data in Tables 1 and 2 collectively prove that mammalian succinate dehydrogenase contains two 2-Fe and one 4-Fe center per histidyl flavin, despite the fact that the concentration An extensive list of control experiments for the 19F NMR method is given in ref. 20.
Table 2. Core composition of succinate dehydrogenase by the interprotein core transfer methoda Products, ,uMb Initial conc., ,uM nd/flavin Total Fe nt/flavin 4-Fe Fdred 2-Fe Fdred Fe Flavin Exp. 1.1 1.8 ice 32 4.5 4.0 32 7 1.7 12 57 2df 7.1 0.92 6.5 7.1 57 3df 1.0 1.8 86 11.5 20 4d,e 89 11.1 a Experimental conditions: samples were prepared as described in the text. b Concentrations of the Fe-S centers were detected by EPR. Conditions for EPR measurements were: microwave power, 0.03 mW for 2-Fe measurements, 0.3 mW for 4-Fe measurements; 13 K; modulation amplitude, 10 G; field modulation, 100 kHz; time constant, 0.35; field sweep rate, 0.1 T min-'; Klystron frequency, 9.1 GHz. c Enzyme prepared by the method of Davis and Hatefi (6). d Enzyme prepared by the method of Ackrell et al. (13). e Both apoproteins present in sample. f Only one apoprotein in sample.
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Coles et al.
of centers S-1 plus S-2, as measured by EPR techniques after reduction by dithionite, is well below that predicted from this ratio (4, 9) and that, as noted above, in some preparations no evidence for center S-2 was found by EPR. It is particularly interesting that the sample of succinate dehydrogenase, prepared by the method of Ackrell et al. (13), in which quantitation of the EPR signal after dithionite reduction gave only one Fd-type (i.e., g t 1.94) center per mole of flavin (unpublished data), was the same as that used in the experiments of Tables 1 and 2. It appears, then, that in the most intact preparations the conformation of the enzyme renders center S-2 EPR-silent. We regard the characterization of Fe-S centers in succinate dehydrogenase reported here as direct and unequivocal and acknowledge agreement with the description of two 2-Fe centers and one 4-Fe center adduced from the inherently less explicit results of EPR investigations (4, 8-10, 29). This research was supported at the Molecular Biology Division, Veterans Administration Hospital, in part by National Institutes of Health Program Project HL-16251 and by National Science Foundation Grant PCM 76-03367, at Stanford University by National Institutes of Health Grant GM-22352, at the Stanford Magnetic' Resonance Laboratory by National Science Foundation Grant GR-23633 and National Institutes of Health Grant RR-00711, and at the University of Wisconsin by National Institutes of Health Grant GM 17170 and by
the Graduate Research Committee and the College of Agricultural and Life Sciences. D.M.K. is the recipient of a National Institutes of Health Postdoctoral Fellowship (1977-1979). 1. Singer, T. P., Kearney, E. B. & Zastrow, N. (1955) Biochim. Biophys. Acta 17, 154-155. 2. Kearney, E. B. & Singer, T. P. (1955) Biochim. Biophys. Acta 17, 596-597. 3. Massey, V. (1957) J. Biol. Chem. 229, 763-770. 4, Beinert, H., Ackrell, B. A. C., Vinogradov, A. D., Kearney, E. B. & Singer, T. P. (1977) Arch. Biochem. Biophys. 182,95-106. 5. Lusty, C. J., Machinist, J. M. & Singer, T. P. (1965) J. Biol. Chem. 240, 1804-1810. 6. Davis, K. A. & Hatefi, Y. (1971) Biochemistry 10, 2509-2516.
Proc. Natl. Acad. Sci. USA 76 (1979) 7. Coles, C. J., Tisdale, H. D., Kenney, W. C. & Singer, T. P. (1972) Physiol. Chem. Phys. 4,301-316. 8. Beinert, H., Ackrell, B. A. C., Kearney, E. B. & Singer, T. P. (1975) Eur. J. Biochem. 54,185-194. 9. Ohnishi, T., Salerno, J. C., Winter, D. B., Lim, J., Yu, C. A., Yu, L. & King, T. E. (1976) J. Biol. Chem. 251, 2094-2104. 10. Ohnishi, T., Lim, J., Winter, D. B. & King, T. E. (1976) J. Biol. Chem. 251, 2105-2109. 11. Dus, K., DeKlerk, H., Sletten, K. & Bartsch, R. G. (1967) Biochim. Biophys. Acta 140, 291-311. 12. Ohnishi, T., Leigh, J. S., Winter, D. B., Lim, J. & King, T. E. (1974) Biochem. Biophys. Res. Commun; 61, 1026-1035. 13. Ackrell, B. A. C., Kearney, E. B. & Coles, C. J. (1977) J. Biol. Chem. 252, 6963-6965. 14. Gillum, W. O., Mortenson, L, E., Chen, J.-S. & Holm, R. H. (1977) J. Am. Chemh. Soc. 99,584-595. 15. Holm, R. H. (1977) in Biological Aspects of Inorganic Chemistry, eds. Addison, A. W., Cullen, W. R., Dolphin, D. & James, B. R. (Wiley, New York), pp. 71-111. 16. Averill, B. A., Bale, J. R. & Orme-Johnson, W. H. (1978) J. Am. Chem. Soc. 100, 3034-3043. 17. Bale, J. R. (1974) Dissertation (Univ. Wisconsin, Madison,
WI). 18. Walker, W. H. & Singer, T. P. (1970) J. Biol. Chem. 245, 4224-4225. 19. Kurtz, D. M., Jr., Wong, G. B. & Holm, R. H. (1978) J. Am. Chem. Soc. 100, 6777-6779. 20. Wong, G. B., Kurtz, D. M., Jr., Holm, R. H., Mortenson, L. E. & Upchurch, R. G. (1979) J. Am. Chem. Soc., 101, 3078-3090. 21. Penefsky, H. S. (1977) J. Biol. Chem. 252,2891-2899. 22. Stombaugh, N. A., Burris, R. H. & Orme-Johnson, W. H. (1973) J. Biol. Chem. 248,7951-7956. 23. Orme-Johnson, W. H. & Beinert, H. (1969) J. Biol. Chem. 244, 6143-6148. 24. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) J. Biol. Chem. 177, 751-766. 25. Singer, T. P. (1974) in Methods of Biochemical Analysis, ed. Glick, D. (Wiley, New York), Vol. 22, pp. 123-175. 26. Brumby, P. E. & Massey, V. (1967) Methods Enzymol. 10, 463-474. 27. Aasa, R. & Vanngard, T. (1975) J. Magn. Reson. 19,308-315. 28. Wong, G. B. (1978) Dissertation (Stanford Univ., Stanford, CA). 29. Salerno, J. C., Ohnishi, T., Lim, J. & King, T. E. (1976) Biochem. Biophys. Res. Commun. 73,833-840.
