Isolation and Characterization of Bound Iron-Sulfur Proteins from Bacterial Photosynthetic Membranes

In an investigation concerned with the solubilization and characterization of membrane-bound iron-sulfur proteins of photosynthetic bacteria, succinate dehydrogenase was solubilized from Rhodospirillum rubrum chromatophores by the detergent lauryl dimethylamine oxide (LDAO) or by an alkaline washing treatment. The LDAO-solubilized enzyme was purified by a combination of DEAE-cellulose and Sephadex G-150 chromatography. The solubilized R. rubrum succinate dehydrogenase, although similar in most respects to other solubilized preparations of the enzyme, had some novel electron paramagnetic resonance and thermodynamic features. Similarities to other solubilized succinate dehydrogenase preparations included a molecular weight of about 90,000 (monomeric form), optical absorption spectra, a flavin:non-heme iron:acid-labile sulfur ratio of approximately 1:8:8, two iron-sulfur centers of the ferredoxin type that are paramagnetic in the reduced state (centers 1 and 2), and one ironsulfur center of the high potential type that is paramagnetic in the oxidized state (center 3). A new feature of our R. rubrum succinate dehydrogenase was the presence of an additional iron-sulfur center of the ferredoxin type that was detected by optical potentiometric titrations and identified by EPR spectroscopy. Potentiometric titrations of both the LDAO-solubilized and the alkaline-solubilized succinate dehydrogenase showed three components, one (reducible by succinate) with a midpoint potential of about +50 mV (and n = 2), the second with a midpoint potential of about -160 mV (it = l), and the third with a midpoint potential of about -380 mV tn = 1). The second and third components were not reducible by succinate but were reduced by dithionite. EPR spectra were recorded for samples of the enzyme poised at each of the optical titration end points. All three oxidation-reduction components gave signals of the ferredoxin type, with g values of 2.03, 1.93, and 1.91. Two of these ferredoxin type EPR spectra are identified with the well documented centers 1 and 2 of succinate dehydrogenase and the third ferredoxin type EPR spectrum, corresponding to the oxida-