Dec 11, 1989 - cupric ion (Cu2+) was reduced enzymatically with elemental sulfur (SO) by .... amide) on the reduction of Cu2+ were studied to check the.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1990, p. 693-696
Vol. 56, No. 3
0099-2240/90/030693-04$02.00/0 Copyright C) 1990, American Society for Microbiology
Reduction of Cupric Ions with Elemental Sulfur by Thiobacillus ferrooxidans TSUYOSHI SUGIO,* YOSHIHIKO TSUJITA, KENJI INAGAKI, AND TATSUO TANO Division of Biological Function and Genetic Resources Science, Faculty of Agriculture,
Okayama University, 1-1-1 Tsushima Naka, Okayama 700, Japan Received 16 August 1989/Accepted 11 December 1989
In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu2+) was reduced enzymatically with elemental sulfur (SO) by washed intact cells of ThiobaciUus ferrooxidans AP19-3 to give cuprous ion (Cu+). The rate of Cu2' reduction was proportional to the concentrations of So and Cu2' added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70TC. The activity of Cu2' reduction with S° by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as a,a'-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(N, N-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S° with Fe3+ or Mo6+ as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu2' by So, and the Michaelis constant of SFORase for Cu2+ was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe3+ and Mo6+ but also Cu2+.
The iron-oxidizing bacterium Thiobacillus ferrooxidans has been used for commercial copper ore leaching by bacteria. As an indirect mechanism of bacterial leaching, it is known that uranous ions (UO2), cuprous ions (Cu+), stannous ions (Sn2+), and antimony ions (Sb3+) are oxidized by Fe3" chemically, and the Fe2+ thus produced is oxidized by iron oxidase of T. ferrooxidans to generate Fe3" (3). Enzymatic oxidation of cuprous ions by T. ferrooxidans has been known (2, 8, 10). However, there has been no report of enzymatic reduction of Cu2+ with reduced sulfur compounds by T. ferrooxidans. This may be due to a marked instability of Cu+ under aerobic conditions in the presence of various kinds of metal ions, in particular, Fe . We showed recently that T. ferrooxidans AP19-3 not only oxidizes ferrous ions and reduced sulfur compounds but also reduces metal ions with reduced inorganic sulfur compounds (11, 14-17, 21). It was found that a hydrogen sulfide:ferric ion oxidoreductase (SFORase) (11, 14, 16), which plays a crucial role in the oxidation of elemental sulfur in T. ferrooxidans AP19-3, directly reduces both Fe3+ and Mo6+ with elemental sulfur as an electron donor (19). Tetravalent manganese is also reduced with elemental sulfur by strain AP19-3, but Mn4+ seems to be chemically reduced by Fe2+ and sulfite, both of which are produced as intermediates during the oxidation of elemental sulfur by SFORase (18). In the course of kinetic studies of sulfur oxidation by SFORase, we have observed that Cu2+ competitively inhibited the reduction by SFORase of Fe3+ or Mo6+ with elemental sulfur (19, 20). These results strongly suggest that Cu2+ is also reduced by a SFORase with elemental sulfur. In this work, we show the enzymatic reduction of Cu2+ with elemental sulfur by both washed intact cells and a purified SFORase.
*
MATERIALS AND METHODS
Microorganism, media, and conditions of cultivation. T.
ferrooxidans AP19-3 was used throughout this study (12). The composition of iron-salts medium used for the largescale production of cells and the method of cultivation were described previously (15). Cupric ion-reducing activity. Cupric ion-reducing activity was measured with washed intact cells of iron-grown T. ferrooxidans AP19-3 or a purified SFORase (14, 16). The SFORase, which catalyzes the oxidation of both hydrogen sulfide and elemental sulfur with Fe3" as an electron acceptor in the presence of glutathione to give sulfite and Fe2 was purified from iron-grown T. ferrooxidans AP19-3 by a method described previously (16). The activity of Cu2+ reduction with washed intact cells was determined under aerobic conditions by measuring Cu+ in the reaction mixture. The reaction mixture contained 9 ml of 0.1 M i-alanine-SO42- buffer (pH 5.0), washed intact cells, 5 mg of protein, 50 ,umol of sodium cyanide, 100 ,umol of CUSO4 5H20, and 200 mg of elemental sulfur in ,
-
total volume of 10 ml. The reaction mixture used for the determination of Cu2+-reducing activity by a purified SFORase contained 2.8 ml of 0.1 M sodium phosphate buffer (pH 6.5), 1.2 ml of 0.1 M sodium citrate buffer (pH 6.5), 0.2 mg of bovine serum albumin, a purified SFORase, 100 mg of elemental sulfur, and 50 ,umol of CuSO4 5H20 (adjusted to pH 6.5 with dilute NaOH) in a total volume of 5.0 ml. The reaction was carried out at 30°C. A sample of the reaction mixture (0.5 ml) was withdrawn at intervals, and the concentration of Cu+ was determined by the method described below. The amount of Cu+ produced chemically was always checked by using a SFORase boiled for 10 min instead of native SFORase. Analysis. The amount of Cu+ was determined with cuproin or a specific chelating agent for Cu+ spectrophotometrically. A solution containing 0.1% cuproin in amyl alcohol (1 ml) a
-
Corresponding author. 693
694
APPL. ENVIRON. MICROBIOL.
SUGIO ET AL.
9-
5 z0
E
E
4
-
E
-
3
Go U
*0
Ec0
GD
0.
0
0.. I.)
1 0
0 Concentration ( M )
0
10"
I
l02
FIG. 1. Reduction of cupric ion with elemental sulfur by washed intact cells of T. ferrooxidans AP19-3 under aerobic conditions in the presence of 5 mM sodium cyanide. Symbols: 0, Cu2" reduction in a complete reaction mixture; A, Cu2" reduction by cells boiled previously for 10 min.
added to 0.5 ml of reaction mixture. The mixture was stirred vigorously for 20 s and was centrifuged at 15,000 x g for 2 min to discard solid elemental sulfur. The purplish red color that developed immediately was extracted into 1 ml of amyl alcohol, and the A546 was measured with a Shimadzu UV-140 spectrophotometer after 10 min of incubation at 30°C. A clear relationship was observed between absorbance (0.058 to 0.472 A546) and Cu+ concentration (0.01 to 0.10 ,umol of Cu+). Protein content was determined by the biuret method (7) or the method of Lowry et al. (9), with crystalline bovine serum albumin as the reference protein. was
60 40 Time( min)
80
FIG. 2. Effect of sodium cyanide and sodium azide on cupric ion reduction with elemental sulfur by washed intact cells of T. ferrooxidans AP19-3. Symbols: reduction of Cu2+ in the presence of sodium cyanide (0) or sodium azide (i) under aerobic conditions.
Many investigators have reported enzymatic Cu+ oxidation by T. ferrooxidans (2, 8, 10). However, there have been no reports on reduction of Cu2+ with elemental sulfur, probably because Cu+ is markedly labile and rapidly reoxidized by iron oxidase enzymatically or by Fe3" chemically to give
Cu2+.
The effects of ,sxa'-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts (Tiron), a chelator of Fe3+, and diazene-dicarboxylic acid bis-(N,N-dimethylamide) (Diamide) on the reduction of Cu2+ were studied to check the involvement of SFORase in Cu2+ reduction by elemental sulfur. Both Tiron and ot,o'-dipyridyl strongly inhibited SFORase activity at 2 and 20 mM, respectively (data not shown). Diamide, which is known to oxidize glutathione stoichiometrically in the cells to GSSG (1, 4-6), strongly
0.3
RESULTS AND DISCUSSION Reduction of cupric ion with elemental sulfur by washed intact cells of T. ferrooxidans AP19-3. Under aerobic conditions in the presence of 5 mM sodium cyanide, washed intact cells of T. ferrooxidans AP19-3 reduced Cu2+ with elemental sulfur (Fig. 1). The concentration of Cu+ produced in the reaction mixture gradually decreased after 40 min, suggesting an enzymatic reoxidation of Cu+ or a chemical reoxidation of Cu+ by Fe3" in iron-grown intact cells. No Cu24 reduction was observed in the absence of sodium cyanide and sodium azide, suggesting that Cu+ produced by the cells is rapidly oxidized by iron oxidase of the cells (Fig. 2). Cu2+ reduction was also observed under anaerobic conditions in the absence of sodium cyanide (5 mM), supporting the involvement of iron oxidase in Cu+ oxidation (Fig. 3). The pH optimum of Cu2+ reduction with elemental sulfur was at pH 5.0, and the activity was completely destroyed by 10 min of incubation at 70°C (data not shown). The rate of Cu2+ reduction was proportional to the concentrations of elemental sulfur and Cu2+ in the reaction mixture (Fig. 4).
20
E
0.2
O
0.1
0
0.
40 20 Time (min) FIG. 3. Reduction of cupric ion with elemental sulfur by washed intact cells of T. ferrooxidans AP19-3 under anaerobic conditions. Cu2' reduction was measured under anaerobic conditions in the absence of sodium cyanide. Anaerobic conditions were made by exchanging space in the reaction flask with nitrogen gas. Symbols: 0, Cu2' reduction in a complete reaction mixture; E, Cu2+ reduction by cells boiled previously for 10 min.
0
Cu2+ REDUCTION WITH So BY T. FERROOXIDANS
VOL. 56, 1990
I_C
(a)
8
( b)
-EE
0
101
B
6
E E
695
E
-
C
4
'a
V
c
00 U
0
Q
0.
0.
a-
0
10
20 30 40 50 ( mg/ ml )
so
0
jlO' 10 Conc. of Cu2# ( M )
FIG. 4. Effect of concentration of elemental sulfur and cupric ion on activity of cupric ion-reducing system of T. ferrooxidans AP19-3. The reaction was performed under aerobic conditions in the presence of 5 mM sodium cyanide. (a) Effect of elemental sulfur concentration on activity of Cu2" reduction; (b) effect of cupric ion concentration on activity of Cu2" reduction.
inhibited SFORase activity at 5 mM (14, 16). If Cu2+ reduction with elemental sulfur by this strain is catalyzed by SFORase, o,ot'-dipyridyl, Tiron, and Diamide should inhibit Cu2' reduction. The activity of Cu2+ reduction in the presence of 1 mM Tiron, a,cx'-dipyridyl, and diamide decreased to 13, 41, and 51%, respectively, strongly suggesting the involvement of SFORase in not only Fe3" and Mo6+ but also Cu2+ reduction (Fig. 5). Since the iron in the outer membrane of iron-grown cells could not be washed out completely, a very high concentration of a,a'-dipyridyl or Tiron seems to be required to inhibit cupric ion reduction. Reduction of cupric ion with elemental sulfur by a purified SFORase. A purified SFORase at the stage of Mono Q column chromatography (16) reduced Cu2+ with elemental sulfur under optimal conditions for a SFORase reaction (in the presence of glutathione at pH 6.5), which is similar to that obtained previously for Mo6+ (7.6 mM) (19). From these results, we concluded that SFORase catalyzes the reduction
C
5
E
4
E
of not only Fe3" and Mo6+ but also Cu2" by elemental sulfur (Fig. 6). This conclusion is also supported by results showing that (i) the reduction of Fe3" with elemental sulfur by washed intact cells of T. ferrooxidans AP19-3 was competitively inhibited by Cu2" and growth inhibition by Cu2" on sulfursalts medium was completely restored by adding Fe3" to the medium (20), and (ii) the reduction of Mo6+ with elemental sulfur by a purified SFORase was competitively inhibited by Cu2+ and Fe3" (19). These results indicate that SFORase can utilize a wide range of metal ions as electron acceptor for the oxidation of elemental sulfur. Though T. ferrooxidans AP19-3 utilizes not only Fe3" but also Cu2+ and Mo6+ as electron acceptors during the oxidation of elemental sulfur, Fe3+, but not both Cu2+ and Mo6+, seems to be a physiologically important electron acceptor of SFORase because the specific activities of Fe3+, Mo6+, Cu2+, and Mn4+ reduction with washed intact cells of T. ferrooxidans AP19-3 were 3.3, 0.13, 0.46, and 0.16 ,umol of
0 E-c
50
D.
40
2 E
c3 U2
a.
30
>
20
-~~~~
C0
0.
1
0-3 10 5 10-4 lo2 Concentration (M) FIG. 5. Effect of ot,a'-dipyridyl (A), Tiron (0), and Diamide (-) on activity of cupric ion-reducing system of T. ferrooxidans AP19-3. The reaction was performed under aerobic conditions in the presence of 5 mM sodium cyanide. 0
0.2 0.1 1/ [Cu2] (mM) FIG. 6. Effect of cupric ion on velocity of cuprous ion formation by SFORase purified from T. ferrooxidans AP19-3. The composition -0.1
0
of the reaction mixture is described in Materials and Methods. The reaction was performed under aerobic conditions.
696
APPL. ENVIRON. MICROBIOL.
SUGIO ET AL.
Fe2", molybdenum blue, Cu+, or per h, respectively.
Mn2"
per mg of protein
ACKNOWLEDGMENT This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. LITERATURE CITED 1. Apontoweil, P., and W. Berends. 1975. Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K12. Biochim. Biophys. Acta 399:10-22. 2. Imai, K., H. Sakaguchi, T. Sugio, and T. Tano. 1973. On the mechanism of chalcocite oxidation by Thiobacillus ferrooxidans. J. Ferment. Technol. 51:865-870. 3. Ingledew, W. J. 1982. Thiobacillus ferrooxidans. The bioenergetics of an acidophilic chemolithotroph. Biochim. Biophys. Acta 683:89-117. 4. Kosower, E. M., and N. S. Kosower. 1969. Lest I forget thee, glutathione.... Nature (London) 224:117-120. 5. Kosower, N. S., E. M. Kosower, B. Wertheim, and W. S. Correa. 1969. Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem. Biophys. Res. Commun. 37:593-596. 6. Kosower, N. S., G. A. Vanderhoff, E. M. Kosower, and P. C. Huang. 1965. Decreased glutathione content of human erythrocytes. Biochem. Biophys. Res. Commun. 20:469-474. 7. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3:447-454. 8. Lewis, A. J., and J. D. A. Miller. 1977. Stannous and cuprous ion oxidation by Thiobacillus ferrooxidans. Can. J. Microbiol. 23:319-324. 9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 10. Nielsen, A. M., and J. V. Beck. 1972. Chalcocite oxidation and coupled carbon dioxide fixation by Thiobacillus ferrooxidans. Science 175:1124-1126. 11. Sugio, T., C. Domatsu, 0. Munakata, T. Tano, and K. Imai. 1985. Role of a ferric ion-reducing system in sulfur oxidation of
12.
13.
14.
15.
16.
Thiobacillus ferrooxidans. Appl. Environ. Microbiol. 49:14011406. Sugio, T., C. Domatsu, T. Tano, and K. Imai. 1984. Role of ferrous ions in synthetic cobaltous sulfide leaching of Thiobacillus ferrooxidans. Appl. Environ. Microbiol. 48:461-467. Sugio, T., 0. Hamamoto, M. Mori, K. Inagaki, and T. Tano. 1988. Mechanism of inhibition by Co2+ of the growth of Thiobacillus ferrooxidans on sulphur-salts medium. J. Gen. Microbiol. 134:887-892. Sugio, T., T. Katagiri, K. Inagaki, and T. Tano. 1989. Actual substrate for elemental sulfur oxidation by sulfur:ferric ion oxidoreductase purified from Thiobacillus ferrooxidans. Biochim. Biophys. Acta 973:250-256. Sugio, T., T. Katagiri, M. Moriyama, Ye Li Zhen, K. Inagaki, and T. Tano. 1988. Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans. Appl. Environ. Microbiol. 54:153-157. Sugio, T., W. Mizunashi, K. Inagaki, and T. Tano. 1987. Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans. J. Bacteriol. 169:4916-
4922. 17. Sugio, T., W. Mizunashi, T. Tano, and K. Imai. 1986. Production of ferrous ions as intermediates during aerobic sulfur oxidation in Thiobacillus ferrooxidans. Agric. Biol. Chem. 50:2755-2761. 18. Sugio, T., Y. Tsujita, K. Hirayama, K. Inagaki, and T. Tano. 1988. Mechanism of tetravalent manganese reduction with elemental sulfur by Thiobacillus ferrooxidans. Agric. Biol. Chem. 52:185-190. 19. Sugio, T., Y. Tsujita, T. Katagiri, K. Inagaki, and T. Tano. 1988. Reduction of Mo6+ with elemental sulfur by Thiobacillus ferrooxidans. J. Bacteriol. 170:5956-5959. 20. Sugio, T., K. Wada, W. Mizunashi, K. Imai, and T. Tano. 1986. Inhibition site of cupric ions on the growth of Thiobacillus ferrooxidans on sulfur-salts medium. Agric. Biol. Chem. 50: 2917-2918. 21. Sugio, T., K. Wada, M. Mori, K. Inagaki, and T. Tano. 1988. Synthesis of an iron-oxidizing system during growth of Thiobacillus ferrooxidans on sulfur-salts medium. Appl. Environ. Microbiol. 54:150-152.
