FERREDOXIN OF CLOSTRIDIUM THERMOSACCHAROLYTICUM

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1 FERREDOXIN OF CLOSTRIDIUM THERMOSACCHAROLYTICUM MARTIN WILDER, R. C. VALENTINE,' AND J. M. AKAGI Department of Bacteriology, University of Kansas, Lawrence, Kansas, and The Rockefeller Institute, New York, New York Received for publication 2 June 1963 WILDER, MARTIN (University of Kansas, Lawrence), R. C. VALENTINE, AND J. M. AKAGI. Ferredoxin of Clostridium thermosaccharolyticum. J. Bacteriol. 86: An electron-transferring agent has been isolated from Clostridium thermosaccharolyticum. This factor was found to participate as an electron carrier in the phosphoroclastic reaction of pyruvate, with the subsequent formation of acetyl phosphate and molecular hydrogen. It can be employed interchangeably with the ferredoxin of C. pasteurianum in various reactions. Thermal-stability studies indicated that this factor from C. thermosaccharolyticum was comparatively more heat-resistant than the carrier obtained from C. pasteurianum. It was concluded that this carrier was ferredoxin or a ferredoxin-like substance. A natural electron carrier capable of replacing the redox dye methyl viologen in the phosphoroclastic reaction of Clostridium pasteurianum was obtained by Mortenson, Valentine, and Carnahan (1962). It was named ferredoxin and was found to be a nonheme, iron-containing protein. This factor was found to function between pyruvic dehydrogenase and hydrogenase with the subsequent production of molecular hydrogen and acetyl phosphate. In addition, it was able to substitute for methyl viologen in the hydrogenevolution assay of Peck and Gest (1956). Further studies demonstrated that ferredoxin can transfer electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP+), which results in the production of reduced NADP+ (Valentine, Brill, and Wolfe, 1962a). Tagawa and Arnon (1962) demonstrated the presence of a similar type of carrier in spinach chloroplasts. This factor was not identical to the ferredoxin of C. pasteurianum but was designated as ferredoxin, since both carriers were found to 1 Present address: The Rockefeller Institute, New York, N.Y. be interchangeable with each other. Valentine, Jackson, and Wolfe, (1962b) reported the presence of ferredoxin in Mficrococcus lactilyticus (Veillonella alcalescens), which was found to participate in the oxidation of hypoxanthine to xanthine. Whiteley and Woolfolk (1963) extended these studies and showed that ferredoxin from M. lactilyticus was involved in the reduction of various inorganic and organic substrates. This communication concerns the isolation of ferredoxin from the thermophilic anaerobe C. thermosaccharolyticum. By utilizing the phosphoroclastic and the hydrogenase systems of this organism, we found the ferredoxin isolated to be similar to those already mentioned in the literature. MATERIALS AND METHODS Organisms. C. thermosaccharolyticum strain 3814 was kindly supplied by R. F. Shriver, National Canners Association, Washington, D.C. C. pasteurianum was obtained through the courtesy of R. S. Wolfe, Department of Microbiology, University of Illinois, Urbana. Media. C. thermosaccharolyticum was cultivated in a medium containing Tryptone (Difco), 1. g; yeast extract (Difco), 1. g; K2HPO4, 1.25 g; KH2PO4,.9 g; sodium thioglycolate, 1. g; CaCO3,.25 g; and distilled water, 1 liter. Stock cultures were maintained by freezing test tubes containing actively growing cells. For large-scale cultivation, the CaCO3 was omitted from the medium to avoid collecting extraneous particulate matter during harvesting of the cells. C. pasteurianum was grown in the synthetic medium described by Carnahan and Castle (1958). Preparation of cell-free extracts. For cultivation of C. thermosaccharolyticum on a large scale, 12- liter Florence flasks, containing 9 liters of medium (without glucose), were autoclaved at 121 C for 1 hr. After cooling to 55 C, 1 ml of a filtersterilized 1% Fe(NH4)2(SO4)2 solution and a 861

2 862 WILDER, VALENTINE, AND AKAGI J. BACTERI OL. sterile glucose solution were added to each flask. Then, 1 liter of actively growing cells was added to each Florence flask, and the inoculated flasks were incubated at 55 C. By use of this procedure, the cells were ready to harvest within 1 hr. C. pasteurianum was cultivated in a similar manner at 3 C in the synthetic medium described above. Both organisms were harvested with a Sharples centrifuge. Cell-free extracts of C. thermosaccharolyticum were obtained by crushing freshly harvested cells in a Hughes press. The crushed cells were suspended in.5 M potassium phosphate buffer (ph 7.6) and centrifuged at 27, X g for 2 min. Maximal levels of hydrogenase activity were obtained when the extracts were handled under a constant atmosphere of hydrogen, which was supplemented by the addition of cysteine to a final concentration of.1%. Storage of crude extracts under hydrogen at -2 C for several days resulted in little loss of activity. Cell-free extracts of C. pasteurianum were obtained by the autolysis of vacuum-dried cells according to the method of Carnahan et al. (196). Enzymatic assays. Hydrogenase activity was routinely measured by the evolution assay described by Peck and Gest (1956). Hydrogenutilization studies were performed at 5 C according to the procedure described by Akagi and Campbell (1961). The phosphoroclastic reaction was performed according to the method of Mortenson et al. (1962). The standard assay mixture contained: sodium pyruvate, 1,umoles; phosphate buffer (ph 6.5), 1 Amoles; coenzyme A (CoA),.9,umole; methyl viologen, 1.,mole (or.1 ml of ferredoxin); and.1 ml of extract in a total volume of 1. ml. The reaction mixture was incubated at the desired temperature for 15 min, and the formation of acetyl phosphate was determined by the method of Lipmann and Tuttle (1945). Preparation and assay of ferredoxin. Ferredoxin was isolated from C. pasteurianum and C. thermosaccharolyticum by the diethylaminoethyl (DEAE)-cellulose chromatography technique described by Mortenson et al. (1962). After dialysis of the ferredoxin-containing fraction,.1 ml was incorporated into the standard assay. Rechromatography of the dialyzed ferredoxin preparation on DEAE-cellulose was carried out to determine the concentration of phosphate which eluted the carrier. This was accomplished by employing a stepwise gradient with phosphate buffer (ph 6.5). Special reagents. Methyl viologen was purchased from the Mann Research Laboratories. CoA was obtained from Pabst Laboratories. Protein determinations were performed by the method of Bucher (1947). RESULTS Hydrogenase activity. Evolution of molecular hydrogen from reduced methyl viologen by crude extracts was found to be proportional to protein concentration. The ability of these extracts to utilize molecular hydrogen, in the presence of a suitable acceptor, was found to be considerably weaker than that of the evolving system. This discrepancy in the hydrogenase activities might possibly be attributed to the differences in the reducing conditions expected to be present in the two assays. Phosphoroclastic reaction. Koepsell and Johnson (1942) first established that the butyric acid clostridia decarboxylate pyruvic acid to carbon dioxide and hydrogen without the formation of formic acid. When crude extracts of C. thermosaccharolyticum were incubated with pyruvate and a suitable electron carrier, such as methyl viologen or C. pasteurianum ferredoxin, a rapid evolution of molecular hydrogen was observed. In contrast, no activity was detected when formate was substituted for pyruvate. Isolation of ferredoxin from C. thermosaccharolyticum. Passage of a crude extract of C. thermosaccharolyticum through a DEAE-cellulose column (1.5 by 2 cm) resulted in the accumulation of a dark-brown substance at the top of the column. The fraction representing the phosphoroclastic system passed unadsorbed through the DEAE-cellulose. As with C. pasteurianum ferredoxin, the brown band was eluted from the column with 1 M phosphate buffer (ph 6.5). The amount of acetyl phosphate formed by the phosphoroclastic system, i.e., the portion which passed unadsorbed through the column, was found to be proportional to the amount of 1 M eluate added (Fig. 1). Since this fraction substituted for methyl viologen and C. pasteurianum ferredoxin in the reconstituted phosphoroclastic system (Table 1), it was concluded that it was ferredoxin or a ferredoxin-like substance. Reconstituted phosphoroclastic system of C.

3 VOL. 86, 1963 FERREDOXIN OF C. THERMOSACCHAROLYTICUUM a.7 a..5 for spinach chloroplasts (Tagawa and Arnon, 1962). This factor was eluted from DEAE-cellulose at a concentration of.25 M phosphate buffer (ph 6.5). It appeared reddish-brown in the oxidized state and became light yellow upon reduction with dithionite. The absorption spec- 4.3 a MILLIGRAMS FERREDOXIN FIG. 1. Effect of Clostridium thermosaccharolyticum ferredoxin on acetyl phosphate formation from pyruvate. Reaction mixtures contained 1,umoles of sodium pyruvate,.9,mole of CoA, 1,umoles of phosphate buffer (ph 6.7), 1. mg of DEAE-treated C. thermosaccharolyticum extract, indicated amounts of ferredoxin, and water in a final volume of 1. ml. Incubated for 2 min at 55 C; incubation was terminated by the addition of 1 ml of 2 M neutral NH2H. Acetyl phosphate was measured as acetohydroxamic acid (Lipmann and Tuttle, 1945). thermosaccharolyticum. Since crude extracts of C. thermosaccharolyticum contain sufficient amounts of natural carrier, the ability of crude extracts to catalyze the oxidative decarboxylation of pyruvate was determined. It was observed that molecular hydrogen and acetyl phosphate were produced without any added carrier. When crude extracts were passed through a DEAE-cellulose column, this activity dropped significantly, indicating the removal of some endogenous electron carrier. This activity was found to be reconstituted when ferredoxin obtained from the extracts (or methyl viologen) was added to the DEAEtreated extracts (Table 2). The slightly lower values for acetyl phosphate formation, compared with the amount of hydrogen evolved, were consistently obtained. This may have been due to the hydrolysis of some of the acetyl phosphate at 5 C or to the presence of acetokinase in these preparations. Properties of C. thermosaccharolyticum ferredoxin. The electron carrier obtained from C. thermosaccharolyticum exhibited certain properties similar to those reported for C. pasteurianum (Mortenson et al., 1962), for M. lactilyticus (Valentine et al., 1962b; Whiteley and Woolfolk, 1963), and TABLE 1. Participation of Clostridium thermosaccharolyticum ferredoxin in phosphoroclastic system of C. pasteurianum Additions* Acetyl phosphate formed JAmoles None....8 C. thermosaccharolyticum ferredoxin 4.6 C. pasteurianum ferredoxin Methyl viologen * The basic mixture contained 5 mg of ferredoxin-free extract of C. pasteurianum (see Materials and Methods), 1,umoles of sodium pyruvate,.9 umole of CoA, 1 /moles of phosphate buffer (ph 6.8), carrier, and water, in a total volume of 1. ml. Concentrations of ferredoxin of C. thermosaccharolyticum and C. pasteurianum were.5 and.25 mg, respectively. Concentration of methyl viologen was.8,umole. Incubation time, 15 min; temperature, 3 C. TABLE 2. Formation of hydrogen and acetyl phosphate from pyruvate by the reconstituted phosphoroclastic system of Clostridium thermosaccharolyticum Acetyl System* phosphate H2 evolved formed jumoles ;&moles DEAE-treated extract DEAE-treated extract + 1.,umole of methyl viologen DEAE-treated extract +.5 mg of ferredoxin Crude extract * Reaction mixtures contained 2,Amoles of phosphate buffer (ph 6.8), 1,Amoles of sodium pyruvate,.9 MAmole of CoA, 2 mg of DEAEtreated or crude extract, methyl viologen or ferredoxin (as indicated), and water, in a total volume of 2. ml. Center well contained.2 ml of 2% KOH solution. Gas phase, helium; temperature, 5 C; time of incubation, 15 min. Immediately after the last readings were recorded, the content of each vessel was analyzed for acetyl phosphate.

4 864 WILDER, VALENTINE, AND AKAGI J. BACTERIOL. trum of the partially purified ferredo) xin is shown in Fig. 2. Two peaks are observed in lthe oxidized state at wavelengths of approximateely 38 and 49 mu. Upon reduction of this facto Ir, the peaks diminished rapidly..4 c,,4 VO F 2 _ I Thermal-stability studies indicated that the ferredoxin extracted from C. thermosaccharolyticum was a relatively stable protein. Comparing its stability with the ferredoxin of C. pasteurianum (Fig. 3), it was observed that the ferredoxin obtained from the thermophile could withstand exposures to elevated temperatures more effectively than its mesophilic counterpart. DISCUSSION,,2 _. The ability of ferredoxins to participate in various electron-transfer reactions has been.3 : -- - demonstrated by several investigators (Mortenson et al., 1962; Valentine et al., 1962a, b; Tagawa and Arnon, 1962; Whiteley and Woolfolk, 1963) The present experiments with the hydrogenase WAVE LENGTH of C. thernwsaccharolyticum support the view that FIG. 2. Absorption spectrum of thermostable the enzyme functions in close proximity with ferredoxin. The i25 M phosphate (p ) eluate ferredoxin in the formation of molecular hydrogen. was employed for this determination. 2P'his fraction represented I- a 68-fold purification over As with C. pasteurianum ferredoxin, the thermorthat of the crude 14 extract. The reduced spectrum stable ferredoxin participates in electron-transdithionite to port reactions, as evidenced by its ability to obtained by the addition of.1 mg of 1.8 mg of the partially purified ferredoxin in.1 8. ml. replace methyl viologen in the reconstituted The reduced spectrum was obtained mmnder an at- phosphoroclastic reaction. This was reinforced mosphere of N2 in a Zeiss spectrophotometer. by the finding that Eo values for C. pasteurianum ferredoxin and methyl viologen were almost TO Co identical, and in both cases the number of elec- Ato~~8 C trons participating in the oxidation-reduction waemlydfo hsdtemnto. 7 reactions, as determined from the slope of a potentiometric curve, was found to be one (Tagb awa and Arnon, 1962). In cell-free preparations 6\ of C. thermosaccharolyticum, the carrier appears wfooechroiytism gtwlm to transfer to hydrogenase the low-potential electrons with the subsequent evolution of molecular hydrogen. ACKNOWLEDGMENTS This study was supported in part by grant E-4672 from the National Institute of Allergy TIME IN MINUTES and Infectious Diseases, U.S. Public Health FIG. 3. Thermal-stability studies of ferredoxins Service, by the University of Kansas General of Clostridium thermosaccharolyticu,m and C. Research Fund, and by the Institutional NASA pasteurianum. Dialyzed concentrated ferredoxins grant CRES-3B. from C. pasteurianum and C. thermosaccharolyticum were placed in water baths at temperatures of 6, 7, LITERATURE CITED and 8 C. The protein concentrationc)f both ferre- a total vol- AKAGI, J. M., AND L. L. CAMPBELL Studies doxins was adjusted to $2.5 mg per ml in ume of 3. ml. After the indicated exzdosure times, on thermophilic sulfate-reducing bacteria..5 ml of ferredoxins was removed and incorporated II. Hydrogenase activity of Clostridium into the standard assay. The ferredoxrin-free phos- nigrificans. J. Bacteriol. 82: phoroclastic system employed was oibtained from BUCHER, T UCber ein phosphatubertragendes C. pasteurianum. A 6-mg amount of this extract Garungsferment. Biochim. Biophys. Acta was employed in the assay. 1:

5 VOL. 86, 1963 FERREDOXIN OF C. THE] RMOSACCHAROLYTICUM 865 CARNAHAN, J. E., AND J. E. CASTLE Some requirements of biological nitrogen fixation. J. Bacteriol. 75: CARNAHAN, J. E., L. E. MORTENSON, H. F. Mo- WER, AND J. E. CASTLE Nitrogen fixation in cell-free extracts of Clostridium pasteurianum. Biochim. Biophys. Acta 44: KOEPSELL, H. J., AND M. J. JOHNSON Dissimilation of pyruvic acid by cell-free preparations of Clostridium butylicum. J. Biol. Chem. 145: LIPMANN, F., AND L. C. TUTTLE A specific micromethod for the determination of acyl phosphates. J. Biol. Chem. 159: MORTENSON, L. E., R. C. VALENTINE, AND J. E. CARNAHAN An electron transport factor from Clostridium pasteurianum. Biochem. Biophys. Res. Commun. 7: PECK, H. D., JR., AND H. GEST A new procedure for assay of bacterial hydrogenases. J. Bacteriol. 71:7-8. TAGAWA, K., AND D. I. ARNON Ferredoxins as electron carriers in photosynthesis and in the biological production and consumption of hydrogen gas. Nature 195: VALENTINE, R. C., W. J. BRILL, AND R. S. WOLFE. 1962a. Role of ferredoxin in pyridine nucleotide reduction. Proc. Natl. Acad. Sci. U.S. 48: VALENTINE, R. C., R. L. JACKSON, AND R. S. WOLFE. 1962b. Role of ferredoxin in hydrogen metabolism of Micrococcus lactilyticus. Biochem. Biophys. Res. Commun. 7: WHITELEY, H. R., AND C. A. WOOLFOLK Ferredoxin dependent reactions in Micrococcus lactilyticus. Biochem. Biophys. Res. Commun. 9: Downloaded from on November 14, 218 by guest