Magnetic Circular Dichroism Studies of Succinate Dehydrogenase

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Department of Biochemistry and Biophysics, University of California, Sun Francisco, California ... This work was supported by National Institutes of Health Grant.
CHEMISTRY THEJOURNALOF BIOLOGICAL

Vol. 260,No. 12, Issue of June 25, pp. 7368-7378,1985 Printed in U.S.A.

Magnetic Circular Dichroism Studies of Succinate Dehydrogenase EVIDENCE FOR [2Fe-2S], [SFe-xS], AND [4Fe-4S] CENTERS IN RECONSTITUTIVELY ACTIVE ENZYME* (Received for publication, December 6, 1984)

Michael K. Johnson, JoyceE. Morningstar, andDeborah E. Bennett From the Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803

Brian A. C. Ackrell and Edna B. Kearney From the Molecular Biology Division, Veterans Administratwn Medical Center, Sun Francisco, California 94121 andthe Department of Biochemistry and Biophysics, University of California, Sun Francisco, California94143

Reconstitutively active and inactive succinate dehy- the reduction of ubiquinone (Q’) (4-6). drogenase have been investigated by low temperature Purified preparations of soluble succinate dehydrogenase magnetic circular dichroism (MCD) and EPR spectros- contain on average 1covalently bound FAD, 8 nonheme irons, copy and room temperature CD and absorption spec- and 8 acid-labile sulfides/molecule. The number, type, and troscopy. Reconstitutively active succinate dehydrosubunit location of the Fe-S clusters in succinate dehydrogengenase is found to contain threespectroscopically dis- ase has been, and stillis, the subject of considerable disagreetinct Fe-S clusters: S1,S2, and53.In agreement with previous studies, MCD and CD spectroscopy confirm ment in the literature. The arguments have recently been that center S1 is a succinate-reducible [2Fe-2S]2+v’+ reviewed (7-9). There is general agreement on the presence S1 (10, l l ) , which has a redox potential center. The MCD characteristics of center 52 identify of a [2Fe-2SI2+*’+, around 0 mV in soluble succinate dehydrogenase (12) and is it as a dithionite-reducible [4Fe-4S]2+*’+similarto those in bacterial ferredoxins. EPRpower saturation reducible by succinate giving a rhombic EPR signal with studies and the weakness of the EPR signal from re- principalg values around 2.025, 1.930, and 1.910 (13, 14). The duced 52 indicate that there isa weak magnetic inter- signal corresponds to approximately 1 spin/FAD inboth action between centers S1 and 5 2 in their paramag- succinate-reduced Complex I1 and soluble succinate dehydronetic, S = %,reduced states. Center 53 is identified genase preparations. Its spin relaxation is relatively slow, both by the form of the MCD spectrum and the char- such that the EPR signal is readily power saturated at temacteristic magnetization behavior as a reduced [3Fe- peratures below 30 K and significant line broadening only xS] center in both succinate- and dithionite-reduced occurs at temperatures above 100 K (12). reconstitutively active succinate dehydrogenase. ArOne major source of controversy concerns the existence of guments are presented in favor of centers S2 and 53 a second binuclear Fe-S cluster, S2. The presence of S2 was being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive first proposed by Ohnishi et al. (12, 15, 16) on the basis of a succinate dehydrogenase is found to be deficient in 1.5- to 2-fold increase in the intensity of the S1 EPR signal below20 K on dithionite reduction of partially reconstitucenter 53. These results resolve many of the controversies con- tively active succinate-reduced succinate dehydrogenase prepcerning theFe-S cluster contentof succinate dehydro- arations. Subsequent experiments on submitochondrial pargenase and reconcile published EPR data with analyt- ticles (17) and reconstitutively active succinate dehydroical and core extrusion studies. Moreover, they indi- genase’ failed to find evidence for any significant additional cate that center 53 is a necessary requirement for EPR intensity, over and above that of succinate-reduced S1, reconstitutive activity and suggest that it is able to on dithionite reduction, a finding which led to doubts consustain ubiquinone reductase activity as a [3Fe-xS] cerning the existence of cluster S2 (17). However, the marked center. difference in the EPRrelaxation properties of S1 between the succinate- and dithionite-reduced enzyme, a phenomenon that is observed in submitochondrial particles, Complex 11, The succinate:ubiquinone oxidoreductase segment, Com- and soluble succinate dehydrogenase, would still require an plex 11,of the mammalian mitochondrial respiratory chain explanation, if cluster S2 were nonexistent. The enhancement contains four subunits (1).Two nonidentical subunits, a fla- of the S1 spin relaxation has been shown to coincide with an voprotein of 70,000 molecular weight and asmaller protein of n = 1 redox process with a midpoint potential of approxi27,000 molecular weight constitute the enzyme succinate de- mately -400 mV in soluble succinate dehydrogenase prepahydrogenase (2). The remaining two smaller subunits, which rations (12). This redox process presumably involves producare associated with a b-type cytochrome (3), are required for tion of a rapidly relaxing paramagnet in close proximity to binding the enzyme to the mitochondrial membrane and for SI. Whether this reduced species is an Fe-S cluster, S2, or * This work was supported by National Institutes of Health Grant some other group in the protein hasyet to be ascertained. FeGM-33806 (M. K. J.) and by National Institutes of Health Grant HL- S core extrusion and interprotein core transfer studies (18) 16251 and the Veterans Administration (B. A. C. A. and E. B. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.



The abbreviations used are: Q, ubiquinone; MCD, magnetic circular dichroism; DTT, dithiothreitol; W, watt. S. P. J. Albracht, H. Beinert, B. A. C. Ackrell, and T. P. Singer, unpublished data quoted in Ref. 9.

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Magnetic Circular Dichroism Studies of Succinate Dehydrogenase have been interpreted as supportingthe existence of a second binuclear Fe-S center, S2. A second distinct type of Fe-S EPR signal is observed in the fully oxidized form of intact mitochondria, in Complex 11, and in succinate dehydrogenase that is reconstitutively active, i.e. capable of recombining with submitochondrial particles, depleted of succinate dehydrogenase activity by alkaline treatment, to restore electron transport from succinate to oxygen, or with the two small subunits from Complex I1 to restore Q reductase activity. It is not observed in reconstitutively inactive preparations (19-22). This cluster, termed S3, exhibits a relatively isotropic (g = 2.015, 2.014, and 1.990) and rapidly relaxing EPR signal that is observable only below 20 K. The redox potential of S3 is +65 mV in Complex I1 and >+120 mV in mitochondria (8). Cluster S3 is stable in membranebound enzyme and in Complex 11, where the spin concentration is found to be approximately equal to that of FAD (2123). In soluble succinate dehydrogenase, however, it is extremely labile toward oxidants (20, 22, 23), and this presumably accounts for absence of its EPRsignal in reconstitutively inactive preparations. The ability of soluble succinate dehydrogenase preparations to show the characteristic EPR signal of center S3 correlates with their reconstitutive ability and their "low K,"-ferricyanide reductase activity (23, 24); the latter serves as aconvenient measure of reconstitutive capacity. Before the discovery of [3Fe-xS] centers ( x = 3 or 4) (2527), the EPR signal of oxidized S3 was attributed to an oxidized highpotential Fe-S cluster, [4Fe-4SI3+.It is now well established that of all known Fe-S clusters, [BFe-xS] centers have EPR characteristics most similar to those of center S3. Recent measurements of the linear electric field effect on this EPR signal in oxidized Complex I1 confirm the assignment of S3 as an oxidized [BFe-xS] center (28). Thus the possibility must be considered that, as in the case of aconitase (27, 29), the oxidized [3Fe-xS] center could be a product of oxidative degradation of a [4Fe-4S] cluster originally present in the enzyme. Similarly, by analogy with aconitase(29), cluster conversion to a [4Fe-4SI2+s1+ center may occur on reduction of the center. At present there is no evidence available to assess the validity of these possibilities. In this work we present a fresh approach to the complex problem of cluster determination in succinate dehydrogenase by using low temperature magnetic circular dichroism (MCD) spectroscopy. The controversies and uncertainty in the Fe-S cluster composition of succinate dehydrogenase originate for the most part from centers that may be paramagnetic but exhibit no EPR signals for reasons of zero-field splittings (e.g. reduced [3Fe-xS] centers) or weak coupling between paramagnetic centers (e.g. S1 and S2). MCD spectroscopy performed at cryogenic temperatures provides an optical probe for paramagnetic chromophores and is an effective method for determining Fe-S cluster type in multicomponent enzymes (30). Smallzero-field splittings orweak magnetic interactions between centers will generally not interfere with the increase of MCD intensity for a paramagnetic chromophore that accompanies a decrease in temperature (MCD C-term (31)). Furthermore, estimatesof electronic ground state parameters such as spin state and g values can be made by accurately monitoring MCD intensity as a function of magnetic fields up to 5 tesla and temperatures down to 1.5 K and plotting MCD magnetization curves (30, 32, 33). This information is especially useful for paramagnetic EPR-silent Fe-S clusters, e.g. reduced [3Fe-xS] (34) andoxidized P clusters in nitrogenase (35). WhereEPR spectra areobserved from chromophoric species, analysis of magnetization curves facilitates correla-

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tion of EPR signals with optical transitions. In thispaper we report low temperature MCD spectra and magnetization curves at discrete wavelengths, together with parallel EPR studies, for both reconstitutively active and reconstitutively inactive succinate dehydrogenase. Our results point to theexistence of three Fe-S clusters, SI, S2, and S3, in reconstitutively active succinate dehydrogenase. Moreover, the form of the MCD spectra and magnetization curves for these centers confirm S1 to be a [2Fe-2SI2+.'+center, identify S2 as a [4Fe-4SI2+*"center, and show S3 to be a [SFe-xS] center present inthe reduced form in both the succinate- and dithionite-reduced enzymes. These results resolve many of the controversies concerning the EPR and core-extrusion properties and provide the first definitive assessment of the nature of all the Fe-S clusters in this complex multicomponent enzyme. In addition, they address the question of whether center S3 is able to sustain Q reductase activity as a [3Fe-xS] center. EXPERIMENTALPROCEDURES

Purification and Assay of Succinate Dehydrogenase-Complex I1 was prepared from beef heart mitochondria according to published procedures (36). Reconstitutively active succinate dehydrogenase was extracted from butanol-treated Complex I1 at pH 9.2 under strictly anaerobic conditions (under argon),with 20 mM succinate and 1 mM DTT present, and purified as in previous work (BS-succinate dehydrogenase) (37). The enzyme was also extracted fromComplex I1 essentially according to Davis and Hatefi (2) by sequential treatment with 400 and 800 mM sodium perchlorate, under argon, with 1 mM DTT present, but, intentionally, without succinate, and further purified in the absence of succinate (P-succinate dehydrogenase). Such enzyme preparations are expected to be largely reconstitutively inactive since succinate was omitted and strictanaerobiosis is difficult to maintain during homogenization steps in the procedure. Both BSsuccinate dehydrogenase and P-succinate dehydrogenase were stored as (NH&SOI pellets in liquid nitrogen prior to use. Protein determinations were made on trichloroacetic acid-precipitated material by the Lowry method. The content of bound flavin and nonheme iron and enzyme activities were determined as in previous work (21,37).Enzyme concentrations were equated with the flavin content of the preparations. Reconstitutive activity was assessed hy the "low &"-ferricyanide reductase assay (37). MCD Spectroscopy-MCD spectra were recorded in the range 290800 nm at temperatures between 1.54 and 110 K and magnetic fields between 0 and 4.5 tesla, using an Oxford Instruments SM3 split-coil superconducting magnet mated to a Jasco J500C spectropolarimeter. Spectra were recorded digitally with an OK1 IF800 model 30 microcomputer interfaced via a JascoIF500 interface. Sample temperatures were measured with calibrated carbon-glass resistors (Lake Shore Cryogenics) placed both directly above and below the sample and controlled by a Rh/Fe resistor and heater connected to an Oxford Instruments DTC2 temperature controller. Temperatures below 4.22 K were obtained by pumping on a bath of liquid helium with a two-stage rotary pump connected to a manostatto achieve a constant reduced pressure. Magnetic field calibration was carried out with a transverse Hall probe (Lake Shore Cryogenics). All samples were handled anaerobically under nitrogen on a gas handling line and placed in specially constructed anaerobic MCD cells (30) of measured pathlength (0.15 to 0.17 cm) using gas-tight Hamilton syringes. MCD spectra are corrected for natural CD and expressed as AE, (AE = EL - t ~ ) which , is the difference between the absorption coefficients for left and right circularly polarized light, respectively, in units of M" cm". The magnetic fields used are indicated in the figure legends.All samples for MCD spectroscopy contained 50% (v/v) ethylene glycol to enable optical quality glasses to be formed on freezing MCD samples. Any depolarization of the light beam by the sample was assessed and corrected for by measuring the natural CD of a standard sample of D-tris(ethy1endiamine) cobalt 111 chloride placed after the sample. In all cases depolarization corrections were less than 10%. The presence of 50% (v/v) ethylene glycol had no effect on the UV-visible absorption, CD, or EPR characteristics of the enzyme and did not cause inactivation, as determined in subsequent assays of phenazine methosulfate and "low &"-ferricyanide reductase activities.

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MCD magnetization plots were measured by monitoring MCD intensity a t several fixed temperatures as a function of magnetic field strength. The data are presented as plots of magnetization % uersus @B/2kTwhere magnetization % refers to the MCD intensity as a percentage of the intensity at magnetic saturation, p is the Bohr magneton, B is the magnetic flux density, k is Boltzmann’s constant, and T is the absolute temperature. Theoretical magnetization curves for two special cases, i.e. an axial S = % ground state, and an axial doublet originating from a S > 1 and even ground state with gl = 4 S and g l = 0, were formulated as described previously (30, 32-34). The parameter mz/m+,quoted in the theoretical magnetization data, is a measure of the polarization of the transition and refers to the ratio of the transition dipole moments along z and in the x,y plane for an axial chromophore. AbsorptionSpectroscopy-Room temperature absorption spectra were recorded anaerobically in 1-mm quartz cuvettes with a Cary 219 UV-visible absorption spectrometer. EPR Spectroscopy-EPR spectra were recorded on a Varian E-line X-band spectrometer interfaced to an Apple IIc microcomputer to facilitate quantitation and fitted with an Air-Products Helitranslowtemperature cryostat. The spectra were quantified by double integration according to published procedures (38,39) using l mM CuEDTA as standard, unless otherwise indicated. Care was taken to ensure that both sample and standard were under nonsaturating conditions for all spin quantitations. RESULTS

TABLE I1 EPR datafor samples of BS-succinate dehydrogenase and P-succinate dehydrogenase used in MCD studies g values taken a t peak maxima, cross-over, and peak minima, accurate to fO.OO1. Spectra recorded at 70 K and 1 mW microwave power. Spin quantitations are performed under the same conditions using 1 mM CuEDTA as standard unless otherwise indicated. BS-succinate P-succinate dehydrogenase dehydrogenase As isolated (no succinate)“ g valuesb Isotropic, g = 2.02 Spins/FAD* lo mW). These EPR experiments will be reported in detail elsewhere? For samples of dithionitereduced enzyme, the observed variation in the spin quantitation and spin relaxation rate of the S1 EPR signal (apparent in the previous literature) and the variation in the form of the EPR signal from S2 presumably reflect subtle differences in the magnitude or angularorientation of the magnetic interaction in different types of preparation. J. J. Maguire, B. A. C. Ackrell, and E. B. Kearney, unpublished results. M. K. Johnson, J. E. Morningstar, B. A. C. Ackrell, and E. B. Kearney, unpublished results. 'J. J. Maguire, M. K. Johnson, J. E. Morningstar, B. A. C. Ackrell, and E. B. Kearney, manuscript in preparation.

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In addition to centers S1 and S2, the MCD results attest to the presence of a higher redox potential Fe-S center,S3. It is present as a reduced [SFe-xS] center in both types of preparation investigated but occurs in much greater abundance in the reconstitutively active form, BS-succinate dehydrogenase. Theseresults confirm the linear electric field effect EPR measurements on the isotropic g = 2.01 signal observed in oxidized Complex 11, which indicated that this signal originated from an oxidized [3Fe-xS] center rather than an oxidized tetranuclear high potential Fe-Scluster, [4Fe-4SI3+(28). Moreover, the MCD data suggest that center S3 is a requirement for reconstitutive activity and indicate that bulk conversion of this center to a [4Fe-4S] cluster does not occur on addition of substrate. Partial cluster conversion on reduction by substrate to yield a diamagnetic [4Fe-4Slz+center remains a possibility. It cannot be completely ruled out by our data since the magnitude of the low temperature MCD spectrum of reduced S3 is less than thatgenerally observed for reduced [SFe-xS] centers in bacterial ferredoxins (49). An alternative rationalization of less than stoichiometric amounts of reduced S3 lies in the extreme oxygen lability of this center in solubilized preparations, although the BS-succinate dehydrogenase preparation used here gave no indication of oxygen damage in the"low K,"-ferricyanide reductase assay. Preliminary MCD experiments do confirm that intentional oxygen damage of BS-succinate dehydrogenase results in loss of reduced S3 MCD signals and anincrease in the g = 4.3 rhombic Fe signal in the EPR, yielding a form of the enzyme similar to Psuccinate dehydrogenase? Although we cannot unambiguously conclude that no [3Fe-xS]to [4Fe-4S] cluster conversion is occurring on addition of substrate, it nevertheless seems likely that the [SFe-xS] center in succinate dehydrogenase is able to sustain Q reductase activity. Succinate dehydrogenase would then provide the second example of a metalloenzyme that exhibits enzymatic activity with a [3Fe-xS] center present, thefirst being nitrate reductase from Escherchia coli (49). To address thisquestion more directly, future experiments on succinate dehydrogenase will focus on cluster conversion experiments while monitoring MCD and activity. Finally we wish to discuss whether S2 and S3 are distinct clusters or represent the interconversion products of the same cluster, a possibility suggested in Ref. 28. The results presented here are best interpreted in terms of separate clusters. Our reasoning is as follows. First, if S2 and S3 refer to [4Fe4S] and [3Fe-xS] forms of the same center then substantial spin relaxation enhancement of reduced S1 would beexpected in succinate-reduced BS-succinate dehydrogenase, since S1 presumably would be in close proximity to a paramagnetic, S = 2, reduced [3Fe-xS] center. Moreover, no significant difference in spin relaxation of S1 was observed for succinatereduced BS-succinate dehydrogenase and P-succinate dehydrogenase despite their differing amounts of reduced [3Fe-xS] clusters. Second, preliminary low temperature MCD experiments on intentionally oxygen-damaged BS-succinate dehydrogenase indicate no significant loss in centers S1 and S2 accompanying the breakdown of center S3. [SFe-xS] centers are known to be generated via aerial oxidative damage of [4Fe-4S] clusters in bacterial ferredoxins (41) and aconitase (29, 42). Third, the MCD spectra and magnetization data for succinate- and dithionite-reduced BS-succinate dehydrogenase give no indication that the spectral changes induced by dithionite result from a decrease in the MCD contribution from reduced [3Fe-xS] centers. (In aconitase, conversion of [3Fe-xS] to [4Fe-4S] on reduction with dithionite in the absence of additional Fehas been invoked to explain spectroscopic data (29).) Fourth, the extensive analytical and extru-

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of Succinate Dehydrogenase

sion data reported in the literature are only consistent with 23. Beinert, H., Ackrell, B. A. C., Vinogradov, A. D., Kearney, E. B., and Singer, T. P. (1977) Arch. Bioehem. Bwphys. 182,95-106 distinct clusters for S2 and S3. Pure succinate dehydrogenase A.D., Gavrikova, E. V., and Goloveshkina, V. G . preparations contain approximately 8 nonheme irons and 8 24. Vinogradov, (1975) Biochem. Biophys. Res. Commun. 6 5 , 1264-1269 acid-labile sulfides/molecule. Assuming one of each type of 25. Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Ormecenter (Sl, S2, and S3)/FAD, the cluster composition proJohnson, W.H., and Miinck, E. (1980) J. Bwl. Chem. 2 5 5 , 1793-1796 posed in this work accounts for up to 9 nonheme irons/FAD. The slightly lower experimental data couldbe accounted for 26. Stout, C. D., Ghosh, D., Pattabhi, B., and Robbins, A. H. (1980) J.Bwl. Chem. 256,1797-1800 by less than stoichiometric quantities of center S3. Careful 27. Beinert, H., and Thomson, A. J. (1983) Arch. Biochem. Biophys. core extrusion and interprotein core transfer experimentson 222,333-361 BS-succinate dehydrogenase samples yielded approximately 28. Ackrell, B. A. C., Kearney, E. B., Mims, W. B., Peisach, J., and two [2Fe-2S] and one [4Fe-4S] center (18). However, the Beinert, H. (1984) J. Biol. Chem. 259,4015-4018 [3Fe-rS] center in aconitase as isolated is known to extrude 29. Kent, T. A., Dreyer, J.-L., Kennedy, M.C., Huynh, B. H., Emptage, M. H., Beinert, H., and Munck, E. (1982) Proc. Natl. as a [2Fe-2S] cluster (51), and this type of center is yet to be Acad. Sei. U. S. A . 79, 1096-1100 extruded intact from any metalloprotein. In light of this, the M. K., Robinson, A.E., and Thomson, A. J. (1982) in extrusion results are consistent with the presence of distinct 30. Johnson, iron-Sulfur Proteins(Spiro, T. G., ed) pp. 367-406, John Wiley [ 2Fe-2S1, [SFe-xS], and [4Fe-4S] centers in succinate dehyand Sons, New York drogenase. 31. Stephens, P. J. (1976) Adu. Chem. Phys. 3 5 , 197-265 REFERENCES 1. Capaldi, R. A., Sweetland, J., and Merli, A. (1977) Biochemistry 16,5707-5710 2. Davis, K. A., and Hatefi, Y. (1971) Biochemistry 1 0 , 2509-2516 3. Davis, K.A., Hatefi, Y., Poff, K.L., and Butler, W.L. (1972) Biochem. Biophys. Res. Commun. 4 6 , 1984-1990 4. Yu, C. A., Yu, L., and King, T. E. (1977) Biochem. Biophys. Res. Commun. 78, 259-265 5. Ackrell, B. A. C., Ball, M. B., and Kearney, E. B. (1980) J. Biol. Chem. 255,2761-2769 6. Hatefi, Y., and Galante, Y. M.(1980) J. Biol. Chem. 255,55305537 7. Ohnishi, T. (1981) in Mitochondria and Microsomes (Lee, C. P., Schatz, G., and Dallner, G., eds) pp. 191-216, Addison-Wesley, Reading, MA 8. Ohnishi, T., and Salerno, J. C. (1982) in Iron-Sulfur Proteins (Spiro, T. G., ed) pp. 285-327, John Wiley and Sons, New York 9. Beinert, H., and Albracht, S. P. J. (1982) Biochim. Biophys. Acta 683,245-277 10. Salerno, J. C., Ohnishi, T., Blum, H., and Leigh, J. S. (1977) Biochim. Biophys. Acta 494, 191-197 11. Albracht, S. P. J., and Subramanian, J. (1977) Biochim. Biophys. Acta 462.36-48 12. Ohnishi, T., Salerno, J. C., Winter, D. B., Lim, J., Yu, C. A., Yu, L., and King. T. E. (1976) J. Biol. Chem. 251,2094-2104 13. Beinert, H., and Sands, R. H. (1960) Biochem. Biophys. Res. Commun. 3,41-46 14. Salerno, J. C., Lim, J., King, T. E., Blum, H., and Ohnishi, T. (1979) J. Biol. Chem. 254,4828-4835 15. Ohnishi, T.,Winter, D. B., Lim, J., and King, T. E. (1973) Biochem. Biophys. Res. Commun. 53,231-237 16. Ohnishi, T., Leigh, J. S., Winter, D. B., Lim, J., and King, T. E. (1974) Biochem. Biophys. Res. Commun. 61,1026-1035 17. Albracht, S. P. J. (1980) Biochim. Biophys. Acta 6 1 2 , 11-28 18. Coles, C. J., Holm, R. H., Kurtz, D. M., Orme-Johnson, W. H., Rawlings, J., Singer, T. P., and Wong, G . B. (1979) Proc. Natl. Acad. Sci. U. S. A . 76,3805-3808 19. Beinert, H., Ackrell, B. A. C., Kearney, E. B., and Singer, T. P. (1974) Biochem. Bwphys. Res. Commun. 58, 504-572 20. Ohnishi, T., Winter, D. B., Lim, J., and King, T. E. (1974) Biochem. Biophys. Res. Commun. 6 1 , 1017-1025 21. Beinert, H., Ackrell, B. A. C., Kearney, E. B., and Singer, T. P. (1975) Eur. J. Biochem. 54, 185-194 22. Ohnishi, T., Lim, J., Winter, D.B., and King, T. E. (1976) J. Biol. Chem. 251,2105-2109

32. Schatz, P. N.,Mowery,R. L., and Krausz, E. R. (1978) Mol. Physiol. 35,1535-1557 33. Thomson, A. J., and Johnson, M.K. (1980) Biochem. J. 1 9 1 , 411-420 34. Thomson, A. J., Robinson, A. E., Johnson, M. K., Moura, J. J. G., Moura, I., Xavier, A.V., and LeGall, J. (1981) Biochim. Biophys. Acta 670,93-100 35. Johnson, M. K., Robinson, A. E., Thomson, A. J., and Smith, B. E. (1981) Biochim. Biophys. Acta 671,61-70 36. Baginsky, M. L., and Hatefi, Y. (1969) J.Biol. Chem. 244,53135319 37. Ackrell, B. A. C., and Kearney, E. B., and Coles, C. J. (1977) J. Biol. Chem. 252,6963-6965 38. Aasa, R., and Viinngird, T. (1976) J. Magn. Reson. 19,308-315 39. Wyard, S.J. (1965) J. Sci. Instrum. 42,769-770 40. Johnson, M. K., Thomson, A. J., Robinson, A. E., Rao, K. K., and Hall, D. 0.(1981) Biochim. Biophys. Acta 6 6 7 , 433-451 41. Thomson, A. J., Robinson, A. E., Johnson, M. K., Cammack, R., Rao, K. K., and Hall, D. 0.(1981) Biochim. Biophys. Acta6 3 7 , 423-432 42. Johnson, M. K., Thomson, A. J., Richards, A. J. M., Peterson, J., Robinson, A. E., Ramsay, R. R., and Singer, T. P. (1984) J. Biol. Chem. 259,2274-2282 43. Vickery, L. E., Salmon, A. G., and Sauer, K. (1975) Biochim. Biophys. Acta 386,87-98 44. Springall, J. P., Stillman, M. J.,and Thomson, A. J. (1976) Bioehim. Biophys. Acta453,494-501 45. Thomson, A. J., Cammack, R., Hall, D. O., Rao, K. K., Briat, B., Rivoal, J. C., and Badoz, J. (1977) Biochim. Biophys. Acta4 9 3 , 132-141 46. Yachandra, V. K., Hare, J., Gewirth, A., Czernuszewicz,R.S., Kimura, T., Holm, R. H., and Spiro, T. G. (1983) J.Am. Chem. SOC. 105,6462-6468 47. Stephens, P. J., Thomson, A. J., Dunn, J. B. R., Keiderling, T. A., Rawlings, J., Rao, K. K., and Hall, D. 0.(1978) Biochemistry 17,4770-4778 48. Hatchikian E. C., Cammack, R., Patil, D. S., Robinson, A.E., Richard, A. J. M.,George, S., and Thomson, A. J. (1984) Biochem. Biophys. Acta 784,40-47 49. Johnson, M. K., Bennett, D. E., Morningstar, J. E., Adams, M. W. W., and Mortenson, L. E. (1985) J. Biol. Chem. 260,54565463 50. Palmer, G . (1980) in Methods for Determining Metal Ion Enuironment in Proteins (Darnall, D. W., and Wilkins, R. G., eds) pp. 153-182, Elsevier/North-Holland, New York 51. Kurtz, D. M., Holm, R. H., Ruzicka, F. J., Beinert, H., Coles, C. J., and Singer, T. P. (1979) J. Bid. Chem. 254.4967-4969