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[29] Reverse siroheme sulfite reductase from Thiobacillus denitrificans
422
OXIDATION OF REDUCED SULFUR COMPOUNDS
[29]
[29] R e v e r s e Siroheme Sulfite Reductase from Thiobacillus denitrificans By HANS G. TROPER
Within the chemotrophic dissimilatory sulfur-oxidizing bacteria the Gram-negative, single-cell, short motile rods have been traditionally grouped into the genus Thiobacillus. 1 On the basis of 16S rRNA-based phylogenetic taxonomy this concept is no longer valid, as different species of "Thiobacillus" have been shown to belong to rather diverse branches within the two subclasses alpha and beta of the class Proteobacteria. 2,3 The type species, Thiobacillus thioparus, belongs to the beta subclass and is closely related only to Thiobacillus denitrificans. All other" Thiobacillus" species are not closely related to these two and will have to be assigned to other genera in the future. The phylogenetic biodiversity of the thiobacilli is paralleled by an almost exotic diversity in their oxidation pathways for reduced sulfur compounds. 4 Adenylylsulfate reductase (EC 1.8.99.2) has been found to occur only in the anaerobe T. denitrificans 5 and in the aerobes T. thioparus 6 and "Thiobacillus" thiooxidansT: however, it occurs in intracellular concentrations of around 3% of the total cell protein. These high concentrations, which are also typical of dissimilatory sulfate-reducing bacteria, indicate the active participation of the enzyme in dissimilatory sulfur oxidation in these thiobacilli. 1 D. P. Kelly and A. P. Harrison in "Bergey's Manual of Systematic Bacteriology, (J. T. Staley, M. P. Bryant, N. Pfennig, and J. G. Holt, eds.), Vol. 3, p. 1842, Williams & Wilkins, Baltimore, Maryland, 1989. 2 D. J. Lane, A. P. Harrison, Jr., D. Stahl, B. Pace, S. J. Giovannoni, G. J. Olsen, and N. R. Pace, J. Bacteriol. 174, 269 (1992). 3 S. Takakuwa, in "Organic Sulfur Chemistry: Biochemical Aspects" (S. Oae and T. Okuyarea, eds.), p. 1. CRC Press, Boca Raton, Florida, 1992. 4 For review cf. S. Takakuwa as cited above, and D. P. Kelly, in "The Nitrogen and Sulphur Cycles" (J. A. Cole and S. J. Ferguson, eds.), p. 65. Cambridge Univ. Press, Cambridge, 1988. 5 T. J. Bowen, F. C. Happold, and B. F. Taylor, Biochim. Biophys. Acta 118, 566 (1966); M. Aminuddin and D. J. D. Nicholas, Biochim. Biophys. Acta 325, 81 (1973). 6 R. M. Lyric and I. Suzuki, Can. J. Biochem. 48, 334 (1970); K. Adachi and I. Suzuki, Can. J. Biochem. 55, 91 (1977). 7 H. D. Peck, Jr., J. Bacteriol. 82, 933 (1961); H. D. Peck, Jr., T. E. Deacon, and J. T. Davidson, Biochim. Biophys. Acta 96, 429 (1965).
METHODS IN ENZYMOLOGY, VOL. 243
Copyright 6) 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[29]
SULFITE REDUCTASEFROM Thiobacillus
423
A siroheme-containing sulfite reductase (EC 1.8.99.1) in similar high intracellular concentration was found in T. denitrificans. 8'9 In contrast to the sulfite reductases in sulfate-reducing bacteria, in T. denitrificans the enzyme must function in the oxidative--or "reverse' '--direction, oxidizing sulfane sulfur to sulfite. So far, however, owing to a lack of electron acceptors that would not react with sulfide directly, no appropriate test system has been developed that would allow testing of the enzyme in the oxidative direction, that is, at the present state of the art, sulfite reductase can be tested only in the reductive direction. The overall sulfur metabolism of T. denitrificans has been studied in thiosulfate-grown strain RT (DSM 807) and shown to proceed as follows 10: 1. Thiosulfate is split to sulfite and sulfane sulfur by a thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1). 2. The sulfane moiety is transiently stored inside the cells in the form ofpolysulfides, most probably in the periplasm. No microscopically visible "sulfur" globules appear inside or outside the cells. 3. The polysulfide is oxidized to sulfite by reverse sulfite reductase. 4. Sulfone as well as sulfane-derived sulfite is oxidized to adenylylsulfate by adenylylsulfate reductase. 5. Sulfate is released from adenylylsulfate by ATP-sulfurylase or ADPsulfurylase, both of which are present in this organism. In addition to adenylylsulfate reductase, an AMP-independent sulfite oxidase has been found in a different strain ("Oslo") and studied in detail. 1~ Enzyme Assay
Principle. Sulfite reductase is measured in a manometric assay in the direction of sulfite reduction with enzymatically reduced methyl viologen as electron donor. The reduction of methyl viologen by hydrogen gas is catalyzed by purified hydrogenase (flee from sulfite reductase and thiosulfate reductase activities) from Desulfovibrio gigas. The consumption of hydrogen is recorded manometrically. Procedure. The reaction is carried out in Warburg flasks under hydrogen at 30°. The main compartment contains (in a total volume of 2.5 ml) 1 ml of hydrogenase solution (1-2 U), 0.1 ml of 75 mM methyl viologen, 0.1 ml of 1 M potassium phosphate (pH 7.0), and 0.1-1.3 ml of enzyme 8 M. Schedel, J. LeGall, and J. Baldensperger, Arch. Microbiol. 105, 339 (1975). 9 M. Schedel and H. G. Triiper, Biochim. Biophys. Acta 568, 454 (1979). 10 M. Schedel and H. G. Triiper, Arch. Microbiol. 124, 205 (1980); A. Schug, H. Ulbricht, and H. G. Trtiper, unpublished results, 1982. lJ M. Aminuddin and D. J. D. Nicholas, J. Gen. Microbiol, 82, 103 (1974).
424
OXIDATION OF REDUCED SULFUR COMPOUNDS
[29]
solution. The center well contains 0.2 ml of 5 N NaOH. The reaction is started by adding 0.3 ml of 50 mM sodium sulfite from the side arm. One enzyme unit (U) is defined as that amount of protein that consumes 1 /zmol of hydrogen per minute. 12
Preparation and AssaY of Hydrogenase from Desulfooibrio gigas. Desulfooibrio gigas (DSM 496) is grown in a medium (pH 7.0) that contains (in 1 liter) 12 ml of 45% sodium lactate solution, 2 g of NHaCI, 2 g of MgSO4 • 7 H20, 4.4 g of Na2SO4, 1 g of yeast extract, 2 ml of 10-fold concentrated trace element solution SL4, ~3and 20 ml of 1 M potassium phosphate. The latter is sterilized separately and added to the autoclaved medium after cooling. Cells are incubated at 30° under anoxic conditions for 2 days, harvested by centrifugation, and stored at - 18° under hydrogen. For preparation of hydrogenase, aliquots of the cell paste are thawed, mixed with 1 vol of hydrogen-saturated 30 mM potassium phosphate (pH 7.0) (HSPP) at 0°, and stirred in an ice bath under hydrogen for 5 min. The suspension is then centrifuged and the dark-red supernatant is collected. This procedure for washing the cell pellet is repeated four times. The combined supernatants are supplied with HSPP-suspended alumina Cy-gel (Serva, Heidelberg, Germany). After centrifugation the hydrogenase-containing yellow supernatant is concentrated under hydrogen in a Diaflo cell (membrane PM10; Amicon, Danvers, MA) and filtered through an HSPP-equilibrated Sephadex G-75 column. Hydrogenase-containing fractions of the eluate are combined, concentrated under hydrogen in a Diaflo cell, and stored at - 18°. Hydrogenase activity is measured manometrically in Warburg flasks under hydrogen at 30°: the main compartment contains 1.55 ml of hydrogenase solution and 0.15 ml of 1 M potassium phosphate (pH 7.0). The reaction is started by the addition of 0.3 ml of 4% methyl viologen from the side arm. Hydrogenase activity is calculated from the initial rate of hydrogen consumption. One enzyme unit (U) is defined as the amount of protein that consumes 1 ~mol of hydrogen per minute.
Purification Frozen cell paste is thawed and suspended in 50 mM potassium phosphate (pH 7.0). The cells are broken by passing the suspension two or three times through a French pressure cell at about 138 MPa. The crude 12 M. Schedel and H. G. Triiper, cf. Ref. 9; modified after A. Yoshimoto and R. Sato, Biochim. Biophys. Acta 153, 555 (1968). 13 N. Pfennig and K. D. Lippert, Arch. Microbiol. 55, 245 (1966).
SULFITEREDUCTASEFROMThiobacillus
[29]
425
extract is adjusted to pH 4.5 by adding 1 vol of 0.3 M ammonium acetate (pH 4.1). After centrifugation the precipitated material is discarded. This step is necessary to remove the dissimilatory nitrite reductase (cytochrome cd) of T. denitrificans, which like the siroheme sulfite reductase will reduce sulfite with viologen dyes as electron donors. In crude extracts it may contribute up to 50% of the total sulfite reductase measured. The supernatant is adjusted to pH 5.5 with 1 M K2HPO4 and then fractionated with (NH4)2SO4. The protein precipitating between 45 and 70% saturation is collected by centrifugation, dissolved in 50 mM potassium phosphate (pH 7.0), and applied to Ecteola cellulose (column size, 3 × 100 cm, Serva, Heidelberg, Germany) previously equilibrated with the same buffer. The protein that adsorbs to the gel is eluted with a linear gradient between 50 and 350 mM potassium phosphate (pH 7.0). The fractions containing sulfite reductase are eluted at about 220 mM and show an intense green color; they are combined, dialyzed against 50 vol of 50 mM potassium phosphate, and then stirred into an appropriate volume of alumina C3,-gel slurry suspended in water. The gel to which sulfite reductase adsorbs is centrifuged, washed once with 50 mM potassium phosphate, and then eluted with stepwise increasing concentrations of potassium phosphate (pH 7.0; 80, 110, 140, 170, and 210 mM). The eluates at 110, 140, and 170 mM are combined and brought to 80% saturation with solid (NH4)2SO4. The precipitated protein is collected by centrifugation, dissolved in 50 mM potassium phosphate (pH 7.0) and applied to a Sephadex G-200 column (diameter, 3 cm; length, 100 cm), that has been previously equilibrated with the same buffer. The green fractions of the eluate are combined and frozen at - 2 0 °. Under these conditions the enzyme is stable for 3 months. A typical purification is summarized in Table I.
TABLE I PURIFICATION OF SULFITE REDUCTASE FROM Thiobacillus denitrificans RT
Step 1. 2. 3. 4. 5. 6.
Crude extract Ammonium acetate fraction 45-70% (NH4)2SO 4 Ecteola eluate Alumina C3, eluate Sephadex G-200 filtrate
Protein (rag)
Activity (units)
Specific activity (mU/mg)
18,400 5,280 3,540 386 312 254
258 121 71 113 106 84
14 31 20 293 339 331
426
OXIDATION OF REDUCED SULFUR COMPOUNDS
[30]
Properties
Molecular Properties. The enzyme obtained by the procedure given above is electrophoretically homogeneous. 9 Determined by gel filtration with protein standards as markers, the molecular mass of the purified enzyme is 160 kDa. The isoelectric point is at pH 4.8. The enzyme has a n 0/2~ 2 subunit structure; the a subunit has a molecular mass of 38 kDa, and the molecular mass of the/3 subunit is 43 kDa. Absorption spectra. The oxidized and reduced spectra are almost identical. The oxidized spectrum shows absorption peaks at 274,393, and 594 nm. The molar extinction coefficients at these wavelengths are 280 × 103, 181 × 10 3, and 60 × 10 3 cm z mmol -j, respectively. The siroheme content indicated by these spectral properties may be extracted with HCI in acetone 15; in this extract it shows typical absorption peaks at 370 and 594 nm. Iron-Sulfur Clusters. The enzyme contains 24 mol of iron and 20 mol of (acid-labile) sulfur per mole of enzyme. These data indicate that the enzyme contains six [4Fe-4S] clusters per a2/32 molecule. Unlike assimilatory sulfite reductases the enzyme does not contain flavin groups. Catalytic Properties. The enzyme reduces sulfite, but not thiosulfate, dithionate, trithionate, or tetrathionate. As electron donors in the test system methyl or benzyl viologen is suitable, whereas NADH 2 and NADPHz are inactive. The pH optimum of the enzyme is about 6.0.14 14 M. Schedel, Doctoral Thesis, University of Bonn, G e r m a n y (1977). 15 M. J. M u r p h y and L. M. Siegel, J. Biol. Chem. 248, 6911 (1973).
[30] P u r i f i c a t i o n a n d P r o p e r t i e s o f C y t o c h r o m e c-555 f r o m Phototrophic Green Sulfur Bacteria
By T.
E. MEYER
Background The green sulfur bacteria are strictly anaerobic, obligately photosynthetic, and autotrophic bacteria not closely related to any other group. 1 They utilize reduced sulfur compounds such as sulfide, sulfur, and less commonly thiosulfate as electron donors and carbon dioxide as the primary carbon source. Acetate, but no other organic compound, is assimi1 j. F. Imhoff, in " P h o t o s y n t h e t i c P r o k a r y o t e s " (N. H. M a n n and N. G. Carr, eds.), p. 53. P l e n u m , N e w York, 1992.
METHODS IN ENZYMOLOGY, VOL. 243
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
OXIDATION OF REDUCED SULFUR COMPOUNDS
[29]
[29] R e v e r s e Siroheme Sulfite Reductase from Thiobacillus denitrificans By HANS G. TROPER
Within the chemotrophic dissimilatory sulfur-oxidizing bacteria the Gram-negative, single-cell, short motile rods have been traditionally grouped into the genus Thiobacillus. 1 On the basis of 16S rRNA-based phylogenetic taxonomy this concept is no longer valid, as different species of "Thiobacillus" have been shown to belong to rather diverse branches within the two subclasses alpha and beta of the class Proteobacteria. 2,3 The type species, Thiobacillus thioparus, belongs to the beta subclass and is closely related only to Thiobacillus denitrificans. All other" Thiobacillus" species are not closely related to these two and will have to be assigned to other genera in the future. The phylogenetic biodiversity of the thiobacilli is paralleled by an almost exotic diversity in their oxidation pathways for reduced sulfur compounds. 4 Adenylylsulfate reductase (EC 1.8.99.2) has been found to occur only in the anaerobe T. denitrificans 5 and in the aerobes T. thioparus 6 and "Thiobacillus" thiooxidansT: however, it occurs in intracellular concentrations of around 3% of the total cell protein. These high concentrations, which are also typical of dissimilatory sulfate-reducing bacteria, indicate the active participation of the enzyme in dissimilatory sulfur oxidation in these thiobacilli. 1 D. P. Kelly and A. P. Harrison in "Bergey's Manual of Systematic Bacteriology, (J. T. Staley, M. P. Bryant, N. Pfennig, and J. G. Holt, eds.), Vol. 3, p. 1842, Williams & Wilkins, Baltimore, Maryland, 1989. 2 D. J. Lane, A. P. Harrison, Jr., D. Stahl, B. Pace, S. J. Giovannoni, G. J. Olsen, and N. R. Pace, J. Bacteriol. 174, 269 (1992). 3 S. Takakuwa, in "Organic Sulfur Chemistry: Biochemical Aspects" (S. Oae and T. Okuyarea, eds.), p. 1. CRC Press, Boca Raton, Florida, 1992. 4 For review cf. S. Takakuwa as cited above, and D. P. Kelly, in "The Nitrogen and Sulphur Cycles" (J. A. Cole and S. J. Ferguson, eds.), p. 65. Cambridge Univ. Press, Cambridge, 1988. 5 T. J. Bowen, F. C. Happold, and B. F. Taylor, Biochim. Biophys. Acta 118, 566 (1966); M. Aminuddin and D. J. D. Nicholas, Biochim. Biophys. Acta 325, 81 (1973). 6 R. M. Lyric and I. Suzuki, Can. J. Biochem. 48, 334 (1970); K. Adachi and I. Suzuki, Can. J. Biochem. 55, 91 (1977). 7 H. D. Peck, Jr., J. Bacteriol. 82, 933 (1961); H. D. Peck, Jr., T. E. Deacon, and J. T. Davidson, Biochim. Biophys. Acta 96, 429 (1965).
METHODS IN ENZYMOLOGY, VOL. 243
Copyright 6) 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[29]
SULFITE REDUCTASEFROM Thiobacillus
423
A siroheme-containing sulfite reductase (EC 1.8.99.1) in similar high intracellular concentration was found in T. denitrificans. 8'9 In contrast to the sulfite reductases in sulfate-reducing bacteria, in T. denitrificans the enzyme must function in the oxidative--or "reverse' '--direction, oxidizing sulfane sulfur to sulfite. So far, however, owing to a lack of electron acceptors that would not react with sulfide directly, no appropriate test system has been developed that would allow testing of the enzyme in the oxidative direction, that is, at the present state of the art, sulfite reductase can be tested only in the reductive direction. The overall sulfur metabolism of T. denitrificans has been studied in thiosulfate-grown strain RT (DSM 807) and shown to proceed as follows 10: 1. Thiosulfate is split to sulfite and sulfane sulfur by a thiosulfate sulfurtransferase (rhodanese, EC 2.8.1.1). 2. The sulfane moiety is transiently stored inside the cells in the form ofpolysulfides, most probably in the periplasm. No microscopically visible "sulfur" globules appear inside or outside the cells. 3. The polysulfide is oxidized to sulfite by reverse sulfite reductase. 4. Sulfone as well as sulfane-derived sulfite is oxidized to adenylylsulfate by adenylylsulfate reductase. 5. Sulfate is released from adenylylsulfate by ATP-sulfurylase or ADPsulfurylase, both of which are present in this organism. In addition to adenylylsulfate reductase, an AMP-independent sulfite oxidase has been found in a different strain ("Oslo") and studied in detail. 1~ Enzyme Assay
Principle. Sulfite reductase is measured in a manometric assay in the direction of sulfite reduction with enzymatically reduced methyl viologen as electron donor. The reduction of methyl viologen by hydrogen gas is catalyzed by purified hydrogenase (flee from sulfite reductase and thiosulfate reductase activities) from Desulfovibrio gigas. The consumption of hydrogen is recorded manometrically. Procedure. The reaction is carried out in Warburg flasks under hydrogen at 30°. The main compartment contains (in a total volume of 2.5 ml) 1 ml of hydrogenase solution (1-2 U), 0.1 ml of 75 mM methyl viologen, 0.1 ml of 1 M potassium phosphate (pH 7.0), and 0.1-1.3 ml of enzyme 8 M. Schedel, J. LeGall, and J. Baldensperger, Arch. Microbiol. 105, 339 (1975). 9 M. Schedel and H. G. Triiper, Biochim. Biophys. Acta 568, 454 (1979). 10 M. Schedel and H. G. Triiper, Arch. Microbiol. 124, 205 (1980); A. Schug, H. Ulbricht, and H. G. Trtiper, unpublished results, 1982. lJ M. Aminuddin and D. J. D. Nicholas, J. Gen. Microbiol, 82, 103 (1974).
424
OXIDATION OF REDUCED SULFUR COMPOUNDS
[29]
solution. The center well contains 0.2 ml of 5 N NaOH. The reaction is started by adding 0.3 ml of 50 mM sodium sulfite from the side arm. One enzyme unit (U) is defined as that amount of protein that consumes 1 /zmol of hydrogen per minute. 12
Preparation and AssaY of Hydrogenase from Desulfooibrio gigas. Desulfooibrio gigas (DSM 496) is grown in a medium (pH 7.0) that contains (in 1 liter) 12 ml of 45% sodium lactate solution, 2 g of NHaCI, 2 g of MgSO4 • 7 H20, 4.4 g of Na2SO4, 1 g of yeast extract, 2 ml of 10-fold concentrated trace element solution SL4, ~3and 20 ml of 1 M potassium phosphate. The latter is sterilized separately and added to the autoclaved medium after cooling. Cells are incubated at 30° under anoxic conditions for 2 days, harvested by centrifugation, and stored at - 18° under hydrogen. For preparation of hydrogenase, aliquots of the cell paste are thawed, mixed with 1 vol of hydrogen-saturated 30 mM potassium phosphate (pH 7.0) (HSPP) at 0°, and stirred in an ice bath under hydrogen for 5 min. The suspension is then centrifuged and the dark-red supernatant is collected. This procedure for washing the cell pellet is repeated four times. The combined supernatants are supplied with HSPP-suspended alumina Cy-gel (Serva, Heidelberg, Germany). After centrifugation the hydrogenase-containing yellow supernatant is concentrated under hydrogen in a Diaflo cell (membrane PM10; Amicon, Danvers, MA) and filtered through an HSPP-equilibrated Sephadex G-75 column. Hydrogenase-containing fractions of the eluate are combined, concentrated under hydrogen in a Diaflo cell, and stored at - 18°. Hydrogenase activity is measured manometrically in Warburg flasks under hydrogen at 30°: the main compartment contains 1.55 ml of hydrogenase solution and 0.15 ml of 1 M potassium phosphate (pH 7.0). The reaction is started by the addition of 0.3 ml of 4% methyl viologen from the side arm. Hydrogenase activity is calculated from the initial rate of hydrogen consumption. One enzyme unit (U) is defined as the amount of protein that consumes 1 ~mol of hydrogen per minute.
Purification Frozen cell paste is thawed and suspended in 50 mM potassium phosphate (pH 7.0). The cells are broken by passing the suspension two or three times through a French pressure cell at about 138 MPa. The crude 12 M. Schedel and H. G. Triiper, cf. Ref. 9; modified after A. Yoshimoto and R. Sato, Biochim. Biophys. Acta 153, 555 (1968). 13 N. Pfennig and K. D. Lippert, Arch. Microbiol. 55, 245 (1966).
SULFITEREDUCTASEFROMThiobacillus
[29]
425
extract is adjusted to pH 4.5 by adding 1 vol of 0.3 M ammonium acetate (pH 4.1). After centrifugation the precipitated material is discarded. This step is necessary to remove the dissimilatory nitrite reductase (cytochrome cd) of T. denitrificans, which like the siroheme sulfite reductase will reduce sulfite with viologen dyes as electron donors. In crude extracts it may contribute up to 50% of the total sulfite reductase measured. The supernatant is adjusted to pH 5.5 with 1 M K2HPO4 and then fractionated with (NH4)2SO4. The protein precipitating between 45 and 70% saturation is collected by centrifugation, dissolved in 50 mM potassium phosphate (pH 7.0), and applied to Ecteola cellulose (column size, 3 × 100 cm, Serva, Heidelberg, Germany) previously equilibrated with the same buffer. The protein that adsorbs to the gel is eluted with a linear gradient between 50 and 350 mM potassium phosphate (pH 7.0). The fractions containing sulfite reductase are eluted at about 220 mM and show an intense green color; they are combined, dialyzed against 50 vol of 50 mM potassium phosphate, and then stirred into an appropriate volume of alumina C3,-gel slurry suspended in water. The gel to which sulfite reductase adsorbs is centrifuged, washed once with 50 mM potassium phosphate, and then eluted with stepwise increasing concentrations of potassium phosphate (pH 7.0; 80, 110, 140, 170, and 210 mM). The eluates at 110, 140, and 170 mM are combined and brought to 80% saturation with solid (NH4)2SO4. The precipitated protein is collected by centrifugation, dissolved in 50 mM potassium phosphate (pH 7.0) and applied to a Sephadex G-200 column (diameter, 3 cm; length, 100 cm), that has been previously equilibrated with the same buffer. The green fractions of the eluate are combined and frozen at - 2 0 °. Under these conditions the enzyme is stable for 3 months. A typical purification is summarized in Table I.
TABLE I PURIFICATION OF SULFITE REDUCTASE FROM Thiobacillus denitrificans RT
Step 1. 2. 3. 4. 5. 6.
Crude extract Ammonium acetate fraction 45-70% (NH4)2SO 4 Ecteola eluate Alumina C3, eluate Sephadex G-200 filtrate
Protein (rag)
Activity (units)
Specific activity (mU/mg)
18,400 5,280 3,540 386 312 254
258 121 71 113 106 84
14 31 20 293 339 331
426
OXIDATION OF REDUCED SULFUR COMPOUNDS
[30]
Properties
Molecular Properties. The enzyme obtained by the procedure given above is electrophoretically homogeneous. 9 Determined by gel filtration with protein standards as markers, the molecular mass of the purified enzyme is 160 kDa. The isoelectric point is at pH 4.8. The enzyme has a n 0/2~ 2 subunit structure; the a subunit has a molecular mass of 38 kDa, and the molecular mass of the/3 subunit is 43 kDa. Absorption spectra. The oxidized and reduced spectra are almost identical. The oxidized spectrum shows absorption peaks at 274,393, and 594 nm. The molar extinction coefficients at these wavelengths are 280 × 103, 181 × 10 3, and 60 × 10 3 cm z mmol -j, respectively. The siroheme content indicated by these spectral properties may be extracted with HCI in acetone 15; in this extract it shows typical absorption peaks at 370 and 594 nm. Iron-Sulfur Clusters. The enzyme contains 24 mol of iron and 20 mol of (acid-labile) sulfur per mole of enzyme. These data indicate that the enzyme contains six [4Fe-4S] clusters per a2/32 molecule. Unlike assimilatory sulfite reductases the enzyme does not contain flavin groups. Catalytic Properties. The enzyme reduces sulfite, but not thiosulfate, dithionate, trithionate, or tetrathionate. As electron donors in the test system methyl or benzyl viologen is suitable, whereas NADH 2 and NADPHz are inactive. The pH optimum of the enzyme is about 6.0.14 14 M. Schedel, Doctoral Thesis, University of Bonn, G e r m a n y (1977). 15 M. J. M u r p h y and L. M. Siegel, J. Biol. Chem. 248, 6911 (1973).
[30] P u r i f i c a t i o n a n d P r o p e r t i e s o f C y t o c h r o m e c-555 f r o m Phototrophic Green Sulfur Bacteria
By T.
E. MEYER
Background The green sulfur bacteria are strictly anaerobic, obligately photosynthetic, and autotrophic bacteria not closely related to any other group. 1 They utilize reduced sulfur compounds such as sulfide, sulfur, and less commonly thiosulfate as electron donors and carbon dioxide as the primary carbon source. Acetate, but no other organic compound, is assimi1 j. F. Imhoff, in " P h o t o s y n t h e t i c P r o k a r y o t e s " (N. H. M a n n and N. G. Carr, eds.), p. 53. P l e n u m , N e w York, 1992.
METHODS IN ENZYMOLOGY, VOL. 243
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.