Thermodynamic and EPR Characteristics of a HiPIP-type Iron-Sulfur Center in the Succinate Dehydrogenase of the Respiratory Chain*

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-l and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidized state analogous to the bacterial high potential iron-sulfur protein, has been detected in the reconstitutively active soluble succinate dehydrogenase preparation. Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 =t 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1: 1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations so far examined give rise to no Center S-3 spectra or, if any, highly modified. These observations indicate that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating

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Thermodynamic and EPR Characteristics of a HiPIP-type Iron-Sulfur Center in the Succinate Dehydrogenase of the Respiratory Chain* (Received for publication, September 24, 1975)  In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-l and S-2), a third iron-sulfur center (Center S-3), which is paramagnetic in the oxidized state analogous to the bacterial high potential iron-sulfur protein, has been detected in the reconstitutively active soluble succinate dehydrogenase preparation.
Midpoint potential (at pH 7.4) of Center S-3 determined in a particulate succinate-cytochrome c reductase is +60 =t 15 mV. In soluble form, Center S-3 becomes extremely labile towards oxygen or ferricyanide plus phenazine methosulfate similar to reconstitutive activity of the dehydrogenase. Thus, even freshly prepared reconstitutively active enzyme preparations show EPR spectra of Center S-3 which correspond approximately to 0.5 eq per flavin; in particulate preparations this component was found in a 1: 1 ratio to flavin. All reconstitutively inactive dehydrogenase preparations so far examined give rise to no Center S-3 spectra or, if any, highly modified. These observations indicate that Center S-3 is an innate constituent of succinate dehydrogenase and plays an important role in mediating electrons from the flavoprotein subunit to most probably ubiquinone and then to the cytochrome chain.
In the preceding paper (I), characterization of two distinct ferredoxin-type iron-sulfur centers (Centers S-l and S-2) in the succinate dehydrogenase segment of the respiratory chain has been described. Both centers exhibit EPR signals in the reduced state at g, = 2.03, g, = 1.93, and g, = 1.91 with similar line shape; both centers are present in approximately equivalent concentrations to that of flavin, but only Center S-l is directly involved in the electron transfer reaction. EPR absorbance of the third iron-sulfur center has been detected (2) in a particulate succinate-ubiquinone reductase (Complex II (3)), which contains 8 non-heme iron atoms and 8 mol of acid-labile sulfide per flavin. This center is paramagnetic in the oxidized state analogous to the bacterial high potential iron-sulfur protein (4) and is present in an equivalent concentration to that of flavin (2). This HiPIP-type iron-sulfur center is reported to be nearly or completely absent in the soluble succinate dehydrogenase preparations which contain either 8 or 4 non-heme iron atoms and equivalent amount of acid-labile sulfide per flavin (2, 5). * This work was SuDDOrted bv United States Public Health Service Grants GM-12202, GM-16767, -and HL-12576, and National Science Foundation Grants GB-42817 and BO-35207.
The present paper describes EPR and thermodynamic characteristics of a HiPIP-type iron-sulfur center (designated as Center S-3) in various soluble succinate dehydrogenase preparations (cf. Table I in Ref. l), which differ in their content of non-heme iron and acid-labile sulfide, and in their ability to reconstitute with purified cytochrome b.c, complex or the alkali-treated submitochondrial particles (both of which are free from the dehydrogenase) for the reformation of antimycin A-sensitive succinate-cytochrome c reductase (6) and the succinate respiratory chain, respectively (cf. Ref. 1 and references cited therein).
Upon removal of the dehydrogenase from the mitochondrial membrane, Center S-3 becomes extremely labile toward oxygen or toward ferricyanide; rapid diminution of Center S-3 signals accompanies the parallel decline of the reconstitutive activity of the enzyme, but not of the artificial dye reductase activity as conventionally employed. These observations indicate that this center plays an important role in electron transfer in the following sequence: succinate -+ iron-flavoprotein subunit (F,) + iron-sulfur protein subunit (I,) (proposed to contain Center S-3) --+ most probably ubiquinone and eventually to the cytochrome system (the arrows symbolize the flow A HiPIP-type Iron-Sulfur Center in Succinate Dehydrogenase of electron or hydrogen). A preliminary report of this investigation has appeared (7).

EXPERIMENTAL PROCEDURES
All enzyme preparations and experimental methods used in this investigation were described in detail in the preceding paper (1).

Typical
EPR spectra of the iron-sulfur center S-3 in particulate succinate-ubiquinone reductase (Complex II) and in a soluble reconstitutively active succinate dehydrogenase (BS-SDH) are presented in Fig. 1 was completely oxidized (Fig. 1). Analogous to the S-3 signals, the initial (<45 s) enzymic activity of the reconstitutively active BS-SDH, but not the particulate reductase, has been found also very labile to both ferricyanide and phenazine methosulfate (9, 10). In Fig. 4  were simultaneously taken out and oxidized with 100 PM ferricyanide and 10 PM phenazine methosulfate and rapidly frozen. Peak heights at g = 2.02 from the base-line of the lower magnetic field were also plotted as a function of the aging time of the debydrogenase. EPR operating conditions were the same as in Fig. 3.
with air at 0". The peak height at g = 2.02 and reconstitutive activity decayed in parallel, with half-time of approximately 35 min in either case. On the other hand, more than about 70% of artificial dye reductase activity (measured as electron transfer from succinate to 2,6-dichlorophenolindophenol mediated by phenazine methosulfate) remained even after both Center S-3 signals (7, 11) and reconstitutive activity (see p. 182, Ref. 12) were practically lost. EPR-detectable Centers S-1 and S-2 showed less than 30% decrease in the time period of the experiment (125 min) for the measurement of decay of Center S-3 signals. Fig. 5 compares EPR spectra of succinate dehydrogenase preparations which differ in their content of non-heme iron and acid-labile sulfide, as well as in their reconstitutive activity toward the cytochrome b.c, complex (6). None of the inherently reconstitutively inactive dehydrogenases exhibit EPR absorbance of Center S-3. Spectrum B demonstrates that B-SDH, which was prepared by the same procedure as BS-SDH except without succinate in the preincubation mixture, exhibits no resonance absorbance of Center S-3. This mol of acid-labile sulfide per flavin, the same as the reconstitutively active BS-SDH. These observations further indicate that even a subtle change of the molecular configuration around Center S-3 results in a loss of the reconstitutive activity simultaneously with the EPR spectrum of this HiPIP-type iron-sulfur center. Center S-3 is stable in the particulate reductase but very labile in the soluble enzyme. This characteristic resembles the reconstitutive property of succinate dehydrogenase, which is very stable in particulate preparations but extremely labile once solubilized (12, 13 and references cited therein). Double integration of Center S-3 spectra obtained with particulate preparations (such as succinate-cytochrome c reductase and Complex II) shows that this center is present in a 1:l ratio to the flavin, in agreement with previously reported results (2). In the soluble enzyme (BS-SDH) about one-half of the Center S-3 remains EPR-active (Table I). This is evidently due to an inactivation of this center during the purification and subsequent manipulation, since Center S-3 is extremely labile, as is the reconstitutive activity of the dehydrogenase (cf. Figs. 3 and 4). Fig. 6 represents a potentiometric oxidation-reduction titration of Center S-3 conducted with particulate antimycin A-sensitive succinate-cytochrome c reductase (6). Midpoint potential measured at pH 7.4 was obtained as +60 + 15 mV. The midpoint potential of Center S-3 has been reported to be + 120 mV in mitochondria (14). This difference in values serves to emphasize again the sensitivity of Center S-3 to its environment. Center S-3 is classified as a HiPIP-type iron-sulfur center (15), because this center is found to be paramagnetic in the oxidized state, similar to bacterial high potential iron-sulfur protein (4) in contrast with ferredoxin-type iron-sulfur centers which are paramagnetic in the reduced state. Thus, this nomenclature does not refer to the oxidation-reduction