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A COMPARISON of CLOSTRIDIAL FERREDOXINS* Mortenson VOL. 49, 1963 BIOCHEMISTRY: BUCHANAN ET AL. 345 19 Gest, H., in Phosphorus Metabolism, ed. W. D. McElroy and B. Glass (Baltimore: Johns Hopkins Press, 1952), vol. 2, p. 522. 2 Bove, J., C. Bove, F. R. Whatley, and D. I. Arnon, presented by D. I. Arnon at the Ninth International Botanical Congress, Montreal, Canada (August, 1959). 21 The term "poise," as applied to oxidation-reduction phenomena, has been frequently used incorrectly as denoting the position or value of the potential of a redox system. The verb, poise, in the sense of "to balance, to keep in equilibrium" was originally used by Clark22 as follows: "A solution may be said to be poised when it tends to resist change in Eh on addition of an oxidiz- ing or reducing agent." 22 Clark, W. M., Public Health Reports (U.S.), 38, 443 (1923); Reprint No. 823; Oxidation- Reduction Potentials of Organic Systems (Baltimore: Williams and Wilkins Co., 1960), p. 145. 23 Several types of evidence24, 25 indicate that the photochemical apparatus of R. rubrum is normally integrated with the cytoplasmic membrane, or membranous extensions penetrating the cytoplasm. Disengagement of the pigment system, with its associated electron transfer carriers, from the native cellular matrix might be expected6. 26 to cause a number of changes, including disturbance of redox balance. 24 Tuttle, A. L., and H. Gest, these PROCEEDINGS, 45, 1261 (1959). 25 Giesbrecht, P., and G. Drews, Arch. Mikrobiol., 43, 152 (1962). 26 Gest, H., and M. D. Kamen, in Encyclopedia of Plant Physiology, ed. W. Ruhland (Berlin- Gottingen-Heidelberg: Springer-Verlag, 1960), vol. V/2, p. 568. 27 Gest, H., J. G. Ormerod, and K. S. Ormerod, Arch. Biochem. Biophys., 97, 21 (1962). A COMPARISON OF CLOSTRIDIAL FERREDOXINS* BY BOB B. BUCHANAN, WALTER LOVENBERG, AND JESSE C. RABINOWITZ DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF CALIFORNIA, BERKELEY Communicated by H. A. Barker, January 18, 1963 Mortenson, Valentine, and Carnahan' have recently isolated a non-heme iron-containing protein from Clostridium pasteurianum that is required for the for- mation of acetyl phosphate and hydrogen from pyruvate by extracts of that or- ganism. They have named this protein "ferredoxin". Valentine, Jackson, and Wolfe2 have reported that ferredoxin is also necessary for hydrogen formation in the fermentation of xanthine by Micrococcus lactilyticus. Tagawa and Arnon' subsequently crystallized C. pasteurianum ferredoxin and found it to have a mo- lecular weight of about 12,000, 10 atoms of iron per molecule, and a redox potential of -418 mV at pH 7.11. Ferredoxin was also shown to be reversibly oxidized and reduced and to be similar to the "photosynthetic pyridine nucleotide reductase" originally isolated from spinach by San Pietro and Lang.4 Because of our interest in the metabolism of pyruvate by Clostridium acidi-urici and Clostridium cylindrosporum,5 we examined these purine-fermenting organisms for ferredoxin and found them to contain relatively high concentrations of this pro- tein. In the present communication, the isolation of crystalline ferredoxin from C. acidi-urici is described and its properties are compared with those of crystalline ferredoxin isolated from C. pasteurianum. Materials and Methods.-Organisms: C. acidi-uridi6 was grown on uric acid as previously described.7 Clostridium butyricum was grown on glucose as described by Wolfe and O'Kane,8 and C. pasteurianum was grown on sucrose with nitrogen gas as the sole nitrogen source as described by Carnahan and Castle.9 Downloaded by guest on September 25, 2021 346 BIOCHEMISTRY: BUCHANAN ET AL. PROC. N. A. S. Determination of ferredoxin activity: Ferredoxin was assayed by measuring its stimulation of acetyl phosphate formation from pyruvate with a ferredoxin-free C. pasteurianum "elastic system" based on that described by Mortenson, Valentine, and Carnahan.' The assay mixture (1 ml) contained 10 pmoles of sodium pyruvate, 25 ,umoles of potassium phosphate buffer, pH 6.5, 0.05 ,Amoles of CoA, 8 mg of the lyophilized DEAE-cellulose treated bacterial extract, and ferredoxin as indicated. The assay mixture was incubated at 370 for 10 min when the reaction was stopped by the addition of 0.5 ml of neutralized hydroxylamine,10 and after an additional 10 min period, 1.5 ml of ferric chloride reagent was added.10 The mixture was centrifuged and the absorbancy of the supernatant solution was determined at 540 miA. The assay was linear using up to 5 JAg of the crystalline C. pasteurianum ferredoxin, with a proportionality constant of approximately 0.055 absorbancy units per 1 jug protein" per ml incubation mixture per 10 min. A unit of ferredoxin activity is defined as the amount needed to effect a change of 1.0 in absorbancy at 540 m~uwhen corrected for the absorption of the control tube incubated in the absence of ferredoxin, and is equivalent to the formation of approximately 5 uomoles of acid hydroxamate per 10 min. Other methods: Total iron was determined by the o-phenanthroline method12 in a final volume of 2.5 ml after hydrolysis of the sample for 10 min in 1% HCl at 800. "Labile sulfide" was determined by the method of Fogo and Popowsky,13 using a final assay volume of 2.5 ml instead of 250 ml and measuring the absorbancy of the methylene blue formed at 670 miA. Control experiments with cysteine, oxidized and reduced glutathione, insulin, and bovine serum albumin were negative and indicated that the method was specific for free sulfide. For the analysis of amino acids other than tryptophan, ferredoxin was hydrolyzed for 12 hr in 6.0 N HC1 at 1200. The amino acid composi- tion of the hydrolysate was determined with the Spinco automatic analyzer. Tryptophan was determined by fluorescence analysis following alkaline hydrolysis.14 Isolation and crystallization of ferredoxin: The following procedure was used for purifying ferredoxin from C. acidi-urici and is typical of that used for the isolation of ferredoxin from other organisms. All steps of the purification procedure were done at 2°-3°. Preparation of cell-free extract: 230 gm of a frozen cell paste of C. acidi-urici were thawed over- night and suspended in water with the aid of a magnetic stirrer to a final volume of about 750 ml. Small amounts of deoxyribonuclease and ribonuclease were added to obtain a free-flowing suspen- sion. 80 ml aliquots of this suspension were subjected to a 10 kc Raytheon Sonic Oscillator for 10 min; cell debris was removed by a 15 min centrifugation at 13,000 X g. The precipitate was washed with 100 ml of water by centrifugation and discarded. The wash water was added to the initial extract. First DEAE-cellulose column: This step was based on the procedure of Mortenson, Valentine, and Carnahan.' The DEAE-cellulose was equilibrated with 1.0 M potassium phosphate buffer, pH 6.5 and was washed with water before use. A column containing approximately 75 ml of DEAE-cellulose (2.8 X 12 cm) was packed under pressure (4.5 lbs/in2). Subsequent operations were carried out under this pressure. The sonic extract was applied to the column, which was then washed with water until the eluate was colorless (approximately 10 column volumes). The major portion of the ferredoxin was adsorbed as a black band near the top of the column. The column was then washed with 10 volumes of 0.05 M potassium phosphate buffer at pH 6.5. The ferredoxin was then eluted with 0.15 M Tris HCl buffer, pH 7.3, containing 0.67 M sodium chlo- ride3 in a volume of 120 ml. First G-25 Sephadex column: Sixty ml aliquots of the eluate obtained in the previous step were desalted on a column of G-25 Sephadex (2.8 X 38 cm) with water. Ferredoxin was collected as a single black fraction following the elution of a contaminating reddish-brown protein. Second DEAE-cellulose column: This step was based on a procedure reported by Tagawa and Arnon. The desalted ferredoxin in a volume of 125 ml obtained in the previous step was applied to a DEAE-cellulose column (2.8 X 24 cm) equilibrated with 0.15 M Tris HCl buffer, pH 7.3, containing 0.15 M sodium chloride. The black ferredoxin fraction was eluted with this same buffer directly following an inactive reddish-brown protein fraction. Second G-25 Sephadex column: The previous fraction was desalted by passage through another column of G-25 Sephadex as described above. The aqueous solution of ferredoxin was then lyo- philized and dissolved in water to a volume of 22.5 ml. In the following steps, crystallization was effected by adding solid ammonium sulfate to the Downloaded by guest on September 25, 2021 VOL. 49, 1963 BIOCHEMISTRY: BUCHANAN ET AL. 347 extract while continuously mixing with a magnetic stirrer. Precipitates were removed by centri- fugation for 10 min at 39,100 X g. First crystallization: Ammonium sulfate was slowly added to the extract to 60% saturation. Brown ferredoxin crystals were observed microscopically at this concentration of ammonium sul- fate, but they were associated with a large amount of colorless, noncrystalline material. The precipitate was removed by centrifugation and discarded. Ammonium sulfate was added to the supernatant solution to 69% saturation. The crystals were collected by centrifugation, washed three times with 75% saturated ammonium sulfate solution, and dissolved in water to a volume of 11.5 ml. This solution was dialyzed 21/2 hr against 15 liters of water; 18 ml of ferredoxin solu- tion were recovered. Second crystallization: Ammonium sulfate was added to the dialyzed solution to 65% satu- ration and the crystals were collected by centrifugation, washed twice with 70% saturated am- monium sulfate solution, and dissolved in water to a volume of 8.5 ml.
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