[19] Desulfofuscidin: Dissimilatory, high-spin sulfite reductase of thermophilic, sulfate-reducing bacteria

276

DISSIMILATORY SULFATE REDUCTION

[19]

heme and [Fe-S] cluster is maintained in all oxidation-reduction states examined. A solitary iron center not associated with either the sirohemes or [4Fe-4S] clusters was detected. In the as-isolated state this solitary iron was high-spin ferric whereas in the reduced state it was high-spin ferrous. Acknowledgment This work was supportedby NIH Grant GM34903.

[19] D e s u l f o f u s c i d i n : D i s s i m i l a t o r y , H i g h - S p i n Sulfite Reductase of Thermophilic, Sulfate-Reducing Bacteria By E.

CLAUDE

HATCHIKIAN

Four different types of dissimilatory sulfite reductase, including desulfoviridin, 1desulforubidin,2 P-582,3 and desulfofuscidin,4,5have been found in various genera of sulfate-reducing bacteria: Their substrate is actually the bisulfite ion as deduced from the optimum pH of activity of these enzymes 4,5,7 Desulfofuscidin, which is the subject of this chapter is present only in the extreme thermophilic sulfate reducers of the genus Thermodesulfobacterium. 8 As with the other dissimilatory sulfite reductases, 1,2,9 desulfofuscidin catalyzes the reduction of sulfite mainly to trithionate with concomitant formation of thiosulfate and sulfide under assay conditions. 4

I j..p. Lee and H. D. Peck, Jr., Biochem. Biophys. Res. Commun. 45, 83 (1971). 2 j..p. Lee, C.-S. Yi, J. LeGall, and H. D. Peck, Jr., J. Bacteriol. 115, 453 (1973). 3 p. A. Trudinger, J. Bacteriol. 104, 158 (1970). 4 E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983). 5 G. Fauque, A. R. Lino, M. Czechowski, L. Kang, D. V. DerVartanian, J. J. G. Moura, J. LeGall, and I. Moura, Biochim. Biophys. Acta 1040, 112 (1990). 6 j. LeGall and G. Fanque, in "Biology of Anaerobic Microorganisms" (A. J. B. Zehnder, ed.), p. 587. Wiley, New York, 1988. 7 B. Suh and J. M. Akagi, J. Bacteriol. 99, 210 (1969). 8 F. Widdel, in "The Prokaryotes" (A. Balows, H. G. Tr0per, M. Dworkin, W. Harder, and K.-H. Schleicher, eds.), 2nd Ed., Vol. 4, p. 3390. Springer-Verlag, New York, 1992. 9 j. M. Akagi and V. Adams, J. Bacteriol. 116, 392 (1973).

METHODS IN ENZYMOLOGY, VOL. 243

Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

[ 19]

DESULFOFUSCIDIN

277

Characteristics of Genus Thermodesulfobacteriurn The genus Thermodesulfobacterium includes only two species, namely Thermodesulfobacterium commune (DSM 2178, ATCC 33708) 1°and Thermodesulfobacterium mobile (DSM 1276), formerly called Desulfovibrio thermophilus, ll Both strains have been isolated from extreme environments and are only distantly related to other sulfate-reducing eubacteria. 8 They are identified by their high-temperature optimum for growth (65 to 70°), rod-shaped cells that do not form spores, the presence of unusual nonisoprenoid ether-linked lipids, and the utilization of H2, formate, lactate, and pyruvate as electron donors for sulfate reduction. They contain desulfofuscidin as specific dissimilatory sulfite reductase 8 and a tetrahemic cytochrome c3, which appears to be homologous to the Desulfovibrio species tetrahemic cytochrome c3.1z'13

Assay Method Principle. Bisulfite reductase activity can be determined manometrically as hydrogen uptake in a system containing hydrogenase, bisulfite reductase, methyl viologen (MV), and sulfite.l This assay requires saturating concentration of reduced methyl viologen, which is achieved under the assay conditions by using a large excess of hydrogenase as compared to bisulfite reductase. The reduced dye then serves as the electron donor for the bisulfite reductase as well as other reductascs present in the extracts. The rate of hydrogen consumption is proportional to the amount of bisulfite reductase added to the reaction mixture when enzyme activity is calculated from the initial rate of hydrogen utilization. Except for the concentrations of substrates, similar reaction conditions are used to determine other reductase activities present in the extracts, such as thiosulfate reductase and trithionate reductase. 4 These will interfere with the activity measurements in cell extracts (Table I). Reagents Potassium phosphate buffer (0.5 M), pH 6.0 Desulfovibrio gigas pure hydrogenase, 1 mg/ml (specific activity, 180 units/mg) 10j. G. Zeikus, M. A. Dawson, T. E. Thompson, K. Ingvorsen, and E. C. Hatchikian, J. Gen. Microbiol. 129, 1159(1983). i1 E. P. Rozanovaand T. A. Pivovarova, Mikrobiologiya 57, 102 (1988). iz E. C. Hatchikian,P. Papavassiliou,P. Bianco, and J. Haladjian,J. Bacteriol. 159, 1040 (1984). ~3G. Fauque, J. LeGall, and L. L. Barton, in "Variations in Autotrophic Life" (J. M. Shively and L. L. Barton, eds.), p. 271. AcademicPress, London, 1991.

278

DISSIMILATORY SULFATE REDUCTION

[19]

Methyl viologen, 80 mM Bovine serum albumin, 10 mg/ml Sodium sulfite, 110 mM, prepared just before use NaOH, 10 N Procedure. The main compartment of the manometric vessel contains 150/zmol of phosphate buffer (pH 6.0), 2.1 tzmol of MV, 3 mg of bovine serum albumin, 50 tzg of pure D. gigas hydrogenase, and a variable amount of enzyme. The side arm contains 22 /zmol of sodium sulfite (freshly prepared), and the center well receives 0.1 ml of 10 N NaOH. The enzymatic activity was routinely measured at 45° and the final volume is 3 ml. After preincubation under hydrogen for 30 min, the reaction is started by tipping in 0.2 ml of 0.11 M sulfite from the side arm. Hydrogen sulfide produced during the reaction is scavenged by I0 N NaOH in the center well. It can be determined using the method of Fogo and Popowsky. 14 Trithionate and thiosulfate, which accumulate in the reaction mixture, can be estimated according to the procedure of Kelly et al. 15 Definition o f Unit and Specific Activity. One unit of enzyme is the enzyme activity catalyzing the consumption of 1 /zmol of hydrogen per minute under the assay conditions. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Lowry et al.16 with bovine serum albumin as standard. Procedure for Purification of Thermodesulfobacterium commune Desulfofuscidin Growth Conditions and Preparation o f Cell Extract. The stock cultures are kept in the low phosphate-buffered basal medium with 0.1% (w/v) yeast extract, 0.4% (w/v) Na2SO4, and 0.4% (w/v) sodium lactate in Hungate tubes under an atmosphere of nitrogen.l° For large-scale cultures in a 300-liter fermentor (model 674 - 300 - 72; Chemap) Thermodesulfobacterium commune is grown in a basic lactate-sulfate medium containing sodium lactate [60% solution (v/v), 7.5 ml], NH4CI (1 g), MgSO 4 • 7H20 (2 g), Na2SO4" 10 H20 (4 g), KEHPO4 (0.5 g), trace elements solution (1 ml), 17 distilled water (1000 ml). The pH is adjusted to 7.2. After sterilization, the medium is supplemented with 2 ml/liter of a 10% (w/v) sodium sulfide solution kept in tubes under vacuum and the bacteria arc grown 14j. K. Fogo and M. Popowsky, Anal. Chem. 21, 732 (1949). 15 D. P. Kelly, L. A. Chambers, and P. A. Trudinger, Anal. Chem. 41, 898 (1969). 16 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J, Randall, J. Biol. Chem. 193, 265 (1951). i7 T. Bauchop and S. R. Elsden, J. Gen. Microbiol. 23, 457 (1960).

[19]

DESULFOFUSCIDIN

279

at 65 ° under an atmosphere of nitrogen. The cells are harvested at the stationary phase after 48 hr of growth, by centrifugation in a Sharpies centrifuge (model AS 16). The growth medium is cooled at 10° in a refrigerated container prior to centrifugation. Freshly thawed cells of T. commune (430 g wet weight) are suspended in 250 ml of 20 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl) buffer, pH 7.6, containing 1 /.tM deoxyribonuclease and are passed twice through a French pressure cell at 15,000 lb/in 2. The extract is centrifuged at 30,000 g for 30 min, and the supernatant fluid is then centrifuged for 2 hr at 198,000 g. The pellet is washed once with 100 ml of 20 mM Tris-HC1 buffer, pH 7.6, and centrifuged under the same conditions. The soluble extract constituted by the mixture of the two 198,000 g supernatants is used as the source of the enzyme. All subsequent steps are carried out at 4° and all buffers are at pH 7.6. Bisulfite reductase is monitored in the extracts both by its enzymatic activity and its typical absorption peak at 576 nm. Silica Gel Fractionation. A settled volume of silica gel [Merck (Rahway, N J) type 60] equal to 70 ml, equilibrated with 20 mM Tris-HCl (pH 7.6), is added to the soluble extract and the mixture is stirred for 3 hr. This suspension is subsequently poured into a column, and the unadsorbed proteins are collected. After washing the gel with 100 ml of the same buffer, the adsorbed protein fraction, which contains the tetrahemic cytochrome c3 (Mr 13,000), is eluted with 1 M K2HPO 4 containing 1 M NaC1 and stored at - 8 0 ° . 12 DEAE-Cellulose Fractionation. The unadsorbed proteins are subsequently treated with DEAE-cellulose (DE-52; Whatman, Clifton, N J) by a batchwise technique. About 100 ml of DEAE-cellulose equilibrated with 20 mM Tris-HC1 buffer (pH 7.6) and previously dried in a Bi]chner funnel in oacuo is added to the extract, and the mixture is stirred for 5 hr. 18The suspension is then poured into a column and, after washing the resin with the equilibration buffer, the adsorbed acidic proteins are eluted with 1 M Tris-HC1 buffer. The acidic extract is then dialyzed overnight against 10 liters of distilled water. First DEAE-Cellulose Column Chromatography. The dialyzed fraction from the previous step is applied to a column (4.2 × 16 cm) of DEAEcellulose equilibrated with 20 mM Tris-HC1 buffer. After washing, the proteins are eluted with a discontinuous gradient (1000 ml) with 100-ml aliquots of Tris-HC1 buffer (in 50 mM steps from 50 to 500 raM). The bisulfite reductase exhibiting a brown color is eluted with 300 mM TrisHCI buffer, and the acidic electron carriers (rubredoxin and ferredoxin) 18j. Le Gall and N. Forget, this series, Vol. 53, p. 613.

280

DISSIMILATORY SULFATE REDUCTION

[19]

are eluted with 350 and 450 mM Tris-HCl buffers, respectively. 19 At this stage, the extract containing the enzyme (21 mg/ml) is divided into three parts of equal volume, and each fraction is purified separately. First Ultrogel AcA 34 Gel Filtration. The previous bisulfite reductasecontaining fraction is concentrated to 45 ml by ultrafiltration using a PM30 membrane (Amicon, Danvers, MA) and filtered through an Ultrogel AcA 34 (IBF) column (5 x 100 cm) equilibrated with 20 mM Tris-HCl. The protein is collected in 8-ml fractions and the tubes that have a purity index (A279 nm/A389 nm) lower than 2.7 are combined. Second DEAE-Cellulose Column Chromatography. The bisulfite reductase is subsequently adsorbed on a second DEAE-cellulose column (3 × 12 cm) equilibrated with 20 mM Tris-HCl. The column is eluted with a discontinuous gradient (500 ml), with 70 ml aliquots of Tris-HCl buffer (in 50 mM steps from 50 to 350 mM). The enzyme elutes at about 300 mM with a purity index of 2.46. Second Ultrogel AcA 34 Gel Filtration. The active fraction is concentrated to 30 ml by ultrafiltration as indicated previously and filtered once again through an Ultrogel AcA 34 column (5 x 100 cm) equilibrated with 20 mM Tris-HC1. Hydroxylapatite Column Chromatography. Finally, the protein is adsorbed onto a hydroxylapatite (BioGel HTP; Bio-Rad, Richmond, CA) column (2.8 x 8 cm) equilibrated with 20 mM Tris-HCl buffer and eluted with a discontinuous gradient (660 ml) of potassium phosphate buffer (pH 7.6) with 60 ml of 2, 10, 2 0 . . . up to 100 mM. This procedure yields a dark brown-colored bisulfite reductase preparation exhibiting a purity index of 2.14. The protein fractions are pooled and stored at - 8 0 °. The purified enzyme has a specific activity of 0.20 /zmol of H2 utilized per minute per milligram protein at 45° and the yield is 117 mg of pure bisulfite reductase. Typical data obtained at each purification step are summarized in Table I. The fivefold purification of desulfofuscidin as compared to the specific activity of acidic protein fraction (Table I) is indicative of the abundance of this enzyme in the crude extract of T. commune. The apparently higher specific activity observed with fractions from steps 1 and 2 as compared to the acidic protein extract from step 3 (Table I) is due to the presence in these fractions of trithionate and thiosulfate reductase activities that subsequently reduce the products of bisulfite reduction (trithionate and thiosulfate) to sulfide. After the batch treatment with DEAE-cellulose, trithionate and thiosulfate reductase activities are 19p. Papavassiliou and E. C. Hatchikian, Biochim. Biophys. Acta 810, 1 (1985).

[19]

DESULFOFUSCIDIN

281

TABLE I PURIFICATION OF BISULFITE REDUCTASEFROM Thermodesulfobacterium c o m m u n e

Step 1. Soluble extract a 2. Silica gel fractionation 3. DEAE-cellulose fractionation 4. First DEAE-cellulose column 5. First Ultrogel AcA34 6. Second DEAE-ceUulose chromatography 7. Second Ultrogel AcA34 8. Hydroxylapatite chromatography

Volume (ml)

Total protein (mg)

Total activity (units)

Specific activity (units/mg)

Recovery (%)

380 385 543

17,100 15,732 8,688

1,075 1,070 363.8

0.063 0.068 0.042

100 b

180

3,780

215

0.057

59.1

218 51

810.9 525.3

81 64

0.100 0.122

22.1 17.6

240 195

216 117

37.6 23.4

0.174 0.2

10.3 6.4

From 430 g of wet cell paste of T. c o m m u n e . In practice, steps 5-8 of the purification procedure were performed from one-third of the original volume of the eluate of step 4. b The activity of the eluate of step 3 is considered as 100% because it is devoid of trithionate and thiosulfate reductase activities, in contrast to the fractions from steps 1 and 2.

separated from the bisulfite reductase as shown by the presence of these activities in the unadsorbed protein fraction. 4 Purification of Thermodesulfobacterium mobile Desulfofuscidin

This procedure is taken from that described by Fauque et al. 5 Thermodesulfobacterium mobile is grown at 65 ° on a standard lactate-sulfate medium. 2° The cells (450 g of wet cell paste) are suspended in 10 mM Tris-HC1 buffer (pH 7.6) and disrupted by passing twice through a French pressure cell at 7000 lb/in 2. The extract is centrifuged at 35,000 g for 2 hr. The crude extract is obtained after centrifugation of the supernatant twice at 40,000 g for 2 hr. All the purification procedures are carried out at 0-4 ° and Tris-HC1 or phosphate buffers (pH 7.6) are used. The enzyme has been purified in four chromatographic steps. The crude extract is applied on a DEAE-BioGel A (Bio-Rad) column (7 × 40 cm) equilibrated with 10 mM Tris-HC1. The proteins are eluted using a continuous Tris-HC1 gradient (2 liters of 10 mM Tris-HC1 and 2 liters of 20 R. L. Starkey, Arch. Mikrobiol. 8, 268 (1938).

282

DISSIMILATORY SULFATE REDUCTION

[19]

500 mM Tris-HC1). The fraction containing mainly desulfofuscidin (400 ml) is concentrated to 16 ml on an ultrafilter (Diaflo; Amicon) using a YM30 membrane and passed twice through a Sephacryl S-200 column (5 × 77 era) equilibrated with 50 mM Tris-HCl. At this stage, the active fraction exhibits a purity index (.4280nm/A392nm) of 4.78. It is dialyzed overnight and adsorbed onto a second DEAE-BioGel A column (5 x 21 cm) equilibrated with I0 mM Tris-HC1. The bisulfite reductase is eluted with a linear gradient of Tris-HCl (600 ml of 10 mM and 600 ml of 350 mM) and concentrated to 12 ml by ultrafiltration using a YM-30 membrane. The previous fraction is then adsorbed onto a hydroxylapatite (BioGel HTP; Bio-Rad) column (4.5 × 15 cm) equilibrated with 300 mM Tris-HCl. The Tris concentration is decreased to 5 mM after washing the column with a descending gradient. A continuous potassium phosphate gradient (400 ml of 5 mM and 400 ml of 200 mM) is subsequently applied for elution. The desulfofuscidin elutes at about 75 mM with a purity coefficient of 2.31. The protein is finally submitted to preparative electrophoresis using a 6% (w/v) polyacrylamide gel. 21 The brown-colored protein band that is collected exhibits a purity coefficient equal to 2.18. This procedure yields 51 mg of pure bisulfite reductase from 450 g of cells. Properties Purity. After polyacrylamide disk gel electrophoresis of the purified T. commune bisulfite reductase, one brownish band is observed in the

gels, and staining with Coomassie blue on a duplicate run gives a single band with the same Rf as the brownish pigment. The protein band in the gels directly catalyzes the sulfite-dependent oxidation of reduced methyl viologen. 4 The purified bisulfite reductase at a protein concentration of I mg/ml in 20 mM Tris-HCl (pH 7.6) containing 0.1 M NaC1 sedimented in the ultracentrifuge as a single symmetrical boundary and the brown color appeared to sediment with the single peak. Molecular Weight and Subunit Structure. The molecular weight of desulfofuscidin from T. commune, determined by analytical ultracentrifugation at equilibrium sedimentation,22is found to be 167,000, using a partial specific volume (0.736) derived from amino acid analysis. The molecular weight of the subunits of bisulfite reductase is estimated to be 47,000 by sedimentation equilibrium centrifugation of the enzyme extensively dialyzed against 4 M guanidine-hydrochloride containing 0.1 M 2-mercaptoethanol, whereas from polyacrylamide gel electrophoresis in sodium zl j. M. Brewer and R. B. Ashworth, J. Chem. Educ. 46, 41 (1969). zz D. A. Yphantis, Biochemistry 3~ 297 (1964).

[19]

DESULFOFUSCIDIN

283

dodecyl sulfate a molecular weight of 48,000 is obtained. The enzyme appears to be composed of four similar but not identical subunits as shown by the N-terminal sequence of the protein, which is indicative of the presence of two different polypeptidic chains in the molecule (see Amino Acid Composition and N-Terminal Amino Acid Sequence, below). The native protein therefore is a tetramer exhibiting a quaternary structure of an ad32 type. Desulfofuscidin from T. mobile exhibits similar molecular weight and subunits structure. 5 Its molecular mass was found to be 175 kDa by gel filtration and 190 kDa by sedimentation equilibrium whereas on sodium dodecyl sulfate (SDS)-gel electrophoresis the enzyme presents a band of 44 to 48 kDa indicative of the presence of four subunits with very close molecular masses per molecule of native protein. Absorption Spectra and Coefficients. The absorption spectra of oxidized and dithionite-reduced desulfofuscidin from T. commune are shown in Fig. 1. The spectrum of the oxidized form is typical of a sirohemecontaining protein and exhibits maxima at 576, 389, and 279 rim, with a weak absorption band at about 693 nm and a shoulder at 532 nm. After addition of dithionite, the peaks at 576 and 693 nm decrease, and a new absorption band appears around 605 nm. Furthermore, the Soret peak

1.2 3~9

..

, m

1.6

i

~B

i

l.2

~

~ 0.4

m 0.8

o; 300

400

500

600

700

WAVELENGTH (nm ) FIG. 1. Absorption spectra of T. c o m m u n e desulfofuscidin [from E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 12211 (1983)].(--) Oxidized desulfofuscidin, 50 mM in TrisHC1 (pH 7.6), 2.6 /zM; ( - - - ) reduced with dithionite. Inset: oxidized (--) and reduced ( - - - ) desulfofuscidin, 13.4 ftM.

284

DISSIMILATORY SULFATE REDUCTION

[19]

shifts from 389 to 391 nm, and a shoulder appears in the 420-nm region. At room temperature, the spectrum reaches stability 30 rain after the addition of dithionite. The oxidized spectrum is regenerated by gently shaking the enzyme preparation with air. The molar extinction coefficients of the purified enzyme at 576,389, and 279 nm are determined to be 89,000, 310,000, and 663,000 M-1 cm-~, respectively. The reduced desulfofuscidin reacts with various ligands to give complexes that induce modifications of the optical spectrum of the protein. In the presence of dithionite, CO reacts with the enzyme to give a green complex exhibiting a typical spectrum with maxima at 593 and 548 nm, with a Soret peak shifted to 395 nm (Fig. 2). Thermodesulfobacterium mobile desulfofuscidin exhibits absorption maxima at 578.5, 392.5, and 281 nm, with a small band around 700 nm. The molar extinction coefficients at 578.5 and 392.5 nm are 94,200 and 323,600 M -~ cm -1, respectively) After photoreduction of the oxidized enzyme, a decrease in absorption at 578.5 nm and a concomitant increase in absorption at 607 nm are observed) In addition to CO complex, which shows absorption maxima at 592.5, 550, and 395 nm (Fig. 3D), CN-, S 2-, and SO32- react with the photoreduced bisulfite reductase. CN- and S 2-

0.8 i

395

i 0.6

i0.4

~ 593

0

J

I 400

,

I

500

,

I

,

600

I

I

700

WAVELENGTH ( n m ) FIG. 2. Absorption spectrum of the CO complex of T. c o m m u n e desulfofuscidin [from E. C. I-Iatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983)]. (--) Oxidized desulfofus¢idin, 2.5/.tM in 50 mM potassium phosphate (pH 7.7); ( - - - ) complex between reduced desulfofuscidin and carbon monoxide.

[19]

DESULFOFUSCIDIN 0.5

0.5

A

0.4

0.4 ~ X 1 0.3-

0.3

0.2 -

~\

i

I

I

0.5

"'t [

0.0

400 500 600 700

0.5

0.4

C

x~tl

D

0.4

0.3

0.3

0.2

,-- ~

0.2

0.1 0.0

B

0.1 400 500 600 700

0

I/~X1 ]

0.2

0ol-0.0

285

X1

r.r~ X3 xx• ",~ff\\

0.1 i

I

I

400 500 600 700

0.0

WAVELENGTH

400 500 60O 71111 ( nm )

FIc. 3. Absorption spectra of T. mobile desulfofuscidin. Complex between photoreduced desulfofuscidin (7 ftM) in the presence of deazaflavin and sulfite (A), cyanide (B), sulfide (C), and carbon monoxide (D). [From G. Fauque, A. R. Lino, M. Czechowski, L. Kang, D. V. DerVartanian, J. J. G. Moura, J. LeGall, and L Moura, Biochim. Biophys. Acta 1040, 112 (1990).]

give complexes with absorption maxima at 605 and 603 nm, respectively (Fig. 3B and C), whereas the complex between the reduced enzyme and SO32- presents absorption maxima at 567 and 375 nm (Fig. 3A). Identification of Heine Prosthetic Group as Siroheme. The spectrum of the supernatant obtained after centrifugation of the extracted chromophore from T. commune desulfofuscidin with acetone hydrochloride (0.015 M)23 exhibits typical absorption peaks at 371 and 594 nm (Fig. 4A). The absorption spectrum of the extracted heme on transfer to pyridine exhibits the characteristic siroheme absorption spectrum, with wavelength maxima at 401 and 557 nm and a shoulder at about 520 nm (Fig. 4B). Desulfofuscidin from both species of Thermodesulfobacterium 4,5 contains 4 moles of siro23M. J° Murphy, L. M. Siegel, and H. Kamin, J. Biol. Chem. 248, 2801 (1973).

286

DISSIMILATORY SULFATE REDUCTION

oO1 1 ~

[19]

A 0.6

0.4

0.8

20.4

400

500

400 800 600 700 WAVELENGTH (nm )

600

700

FIG. 4. Absorption spectrum of the extracted heine chromophore of T. commune desulfofuscidin in acetone-hydrochloride (A) and pyridine (B). [From E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983).]

heme per mole of enzyme, based on the a peak absorption of acetonehydrochloride-extracted heme. 24 Composition. Desulfofuscidin, like the other sulfite reductases, is an iron-containing enzyme. 4,5 Murphy et al. 24 have identified the heme prosthetic group in both assimilatory and dissimilatory sulfite reductases as siroheme, an iron tetrahydroporphyrin of the isobacteriochlorin type with eight carboxylic acid-containing side chains. This heme is common to ferredoxin-nitrite reductases. 25 The average values of iron and acidlabile sulfur content of T. c o m m u n e desulfofuscidin are found to be 20 and 16 atoms per molecule of enzyme, respectively, based on the concentration of the protein estimated from amino acid analysis. On the other hand, desulfofuscidin from T. mobile contains 32 - 2 iron atoms per molecule. 5 Taking into account the content of siroheme, the composition of active sites of T. c o m m u n e enzyme is indicated to be four sirohemes and four [4Fe-4S] centers per molecule. 4 In the case of T. mobile desulfofuscidin, which also contains four sirohemes per molecule, the number of [4Fe-4S] clusters per molecule has been reported to be twice that of T. c o m m u n e desulfofuscidin. 5 The nature of the iron core is substantiated by the EPR characteristics of the ironsulfur signals detected. 24 M. J. Murphy, L. M, Siegel, H. Kamin, D. V. DerVartanian, J.-P. Lee, J. LeGall, and H. D. Peck, Jr., Biochem. Biophys. Res. Commun. 54, 82 (1973). 25 M. J. Murphy, L. M. Siegel, S. R. Tove, and H. Kamin, Proc. Natl. Acad. Sci. U.S.A. 71, 612 (1974).

[ 19]

DESULFOFUSCIDIN

287

Electron Paramagnetic Resonance Spectra of Desulfofuscidin. The electron paramagnetic resonance (EPR) spectrum of T. commune desulfofuscidin as isolated (Fig. 5) exhibits resonances typical of a rhombically distorted high-spin ferric heme with gx = 7.02, gy = 4.81, and gz = 1.93. In addition, it shows minor EPR signals at g values of 6, 4.3, and 2.02. The signal at g = 6 is probably due to contaminant free heme, and the resonances at g = 4.3 and g = 2.02 are assigned to "adventitious" ferric iron and oxidized [3Fe-4S] ~÷ clusters, respectively. Only minor changes in the EPR spectra are observed after an 8-min reaction with dithionite. On addition of dithionite plus methyl viologen to desulfofuscidin, highspin ferric heme resonances disappear within 1 min, owing to reduction to the diamagnetic ferrous state (not shown). In the g = 2 region, a small, complex "g = 1.94" signal appears that can be assigned to [4Fe4S] 1÷ clusters. Oxidized T. mobile desulfofuscidin shows an EPR spectrum with resonance absorption at g values 7.26, 4.78, and 1.92 (Fig. 6A). 5 The heme EPR signals, as well as the g = 4.3 line, disappear when the sample is anaerobically photoreduced in the presence of deazaflavin with the concomitant appearance of a "g = 1.94"-type signal characteristic of a [4Fe-4S] cluster (Fig. 6B). The EPR spectrum of the desulfofuscidin CO complex shows minor modifications in the g = 2 region (Fig. 6C). g -value 876 I

I

I

I

5

4

3

2

[

I

I

I

7.02

6.0

481 2.02

ILSa

I

I

0.1

I

I

0.2 Magnetic

I

I

0.3 field ( T )

I

I

0.4

FIG. 5. EPR spectrum of T. c o m m u n e desulfofuscidin as isolated [by permission of R. Cammack, unpublished data (1982)]. Experimental conditions: temperature, 10 K; modulation amplitude, 10 G; microwave frequency, 9.175 GHz; microwave power, 20 mW; protein concentration, 59.6/zM in 250 mM Tris-HC1 (pH 8).

288

DISSIMILATORY SULFATE REDUCTION

[19]

7.26

t

6.33 4.78

c

f

MAGNETIC

FIELD

FIG. 6. EPR spectra of T. mobile desulfofuscidin [from G. Fauque, A. R. Lino, M. Czechowski, L. Kang, D. V. DerVartanian, J. J. G. Moura, J. LeGall, and I. Moura, Biochim. Biophys. Acta 1040, 112 (1990)]. Experimental conditions: temperature, 10 K; modulation amplitude, 1 mT; microwave frequency, 9.239 GHz; microwave power, 1 mW. Spectrum A, as isolated enzyme; spectrum B, photoreduced enzyme; spectrum C, enzyme photoreduced as in spectrum B and anaerobically reacted with CO. A m i n o A c i d Composition a n d N-Terminal A m i n o A c i d Sequence. The amino acid composition of desulfofuscidin from T. c o m m u n e and T. mobile are presented in Table II. The two proteins contain a preponderance o f acidic amino acids. The ratio (Asx + Glx)/(Lys + Arg) is equal to 1.7 for T. c o m m u n e enzyme. The number o f cysteine residues of each protein, 36 and 52 residues, is higher than the number required to link four [4Fe-4S] or eight [4Fe-4S] clusters reported to be present in T. com-

[ 19]

DESULFOFUSCIDIN

289

TABLE II AMINO ACID COMPOSITION OF DESULFOFUSCIDIN FROM Thermodesulfobacterium c o m m u n e AND Thermodesulfobacterium mobile

Desulfofuscidin Amino acid a

T. c o m m u n e b

T. mobile c

Aspartic acid Threonine Serine Glutamic acid Proline Cysteine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan

160 92 60 200 120 36 144 100 108 36 136 104 68 72 60 128 84 ND d

136 83 68 163 98 52 127 93 112 23 l01 109 60 65 40 109 81 34

a Moles of amino acid per mole of enzyme. b From E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983). c From G. Fauque, A. R. Lino, M. Czechowski, L. Kang, D. V. DerVartanian, J. J. G. Moura, J. Le Gall, and I. Moura, Biochim. Biophys. A c t a 1040, l l 2 (1990). d ND, Not determined.

mune and T. mobile bisulfite reductase, respectively. 4,5 The N-terminal sequence of T. c o m m u n e bisulfite reductase indicates the presence of two

distinct polypeptide chains in the molecule, with threonine and serine as N-terminal residues, respectively (Table III). This N-terminal sequence shows strong homologies with that of desulfofuscidin from T. mobile (Table III). Catalytic Properties. Thermodesulfobacterium commune desulfofuscidin reduces sulfite but not thiosulfate, trithionate, or tetrathionate. It also catalyzes the reduction of nitrite or hydroxylamine to NH3 as reported

290

DISSIMILATORY SULFATE REDUCTION

[19]

TABLE II1 N-TERMINAL SEQUENCINGDATA OF DESULFOFUSCIDIN FROM Thermodesulfobacterium commune AND Thermodesulfobacterium mobile

Residues identified Step

T. commune ~

1 2 3 4 5 6 7 8 9 10

Thr, Set Glu, Ile Val, Glu Lys, Lys Phe, Lys Lys, Lys Glu, Asp Leu, Thr Asp, Asp Pro, Lys

T. mobile b

Gly, Pro Glu, Ile Val, Glu Lys, Lys Phe, Tyr Lys, Lys

a From E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983). b From G. Fauque, A. R. Lino, M. Czechowski, L. Kang, D. V. DerVartanian, J. J. G. Moura, J. Le Gall, and I. Moura, Biochim. Biophys. Acta 111411, 112 (1990). Residues 7 to 10 have not been determined.

with other sulfite reductases. 26 This is related to the activity of siroheme, which is the active site common to sulfite and nitrite reductases. 25 Enzymatic activity is obtained with methyl or benzyl viologen. Desulfofuscidin shows a sharp optimum of activity around pH 5.8-6.0, 4,5 indicating that bisulfite is probably the active species in sulfite reduction. The specific activity of T. commune bisulfite reductase is 2 ~mol of H 2 consumed per minute per milligram of protein at 65 ° as compared to 1.48 units/mg at 60 ° for T. mobile enzyme. 4'5 Effect of Temperature on Bisulfite Reductase Activity. The effect of temperature on T. commune bisulfite reductase activity is shown in Fig. 7. The enzymatic activity increased from 35 ° to nearly 70 ° , and no deflection point is observed in the slope of the curve. The maximum activity occurs between 65 and 70 °. The effect of temperature on bisulfite reductase activity is associated with unusually high Q10 values of 3.6 and 3.2 in the 26 j..p. Lee, J. LeGall, and H. D. Peck, Jr., J. Bacteriol. 115, 529 (1973).

[ 19]

DESULFOFUSCIDIN

291

"~ 1.5

~0.5 t~ O

o

2.8

2.9

3

3.1 xl0

3.2

3.3

3

T FIG. 7. Arrhenius plot of T. commune desulfofuscidin. [From E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983).]

35-50 and 50-65 ° temperature ranges, respectively. The value of the energy of activation, calculated from the slope of the data for the Vmaxplots, between 35 and 65°, was 99.58 J/mol. The enzyme exhibits very low activity below 35 °, whereas the activity becomes less dependent on temperature above 65 °. Thermostability. Thermodesulfobacterium commune bisulfite reductase shows higher stability than desulforubidin, the homologous protein from the mesophilic sulfate-reducing bacterium Desulfomicrobium baculatum Norway 4 strain. 2 With the T. commune enzyme, no absorbance change of the chromophore occurs until 70° whereas such a change occurs above 50° with desulforubidin. The greater thermostability of desulfofuscidin may be related to its higher content of siroheme (four sirohemes per molecule) as compared to desulforubidin (two sirohemes per molecule). 27 The discrepancy existing between the unstability temperature of the pure bisulfite reductase (70°) and the maximum growth temperature of T. com27 C. L. Liu, D. V. DerVartanian, and H. D. Peck, Jr., Biochem. Biophys. Res. Commun. 91, 962 (1979).

292

[19]

DISSIMILATORY SULFATE REDUCTION

TABLE IV PRODUCTS OF BISULFITE REDUCTIONCATALYZEDBY BISULFITE REDUCTASEOF Thermodesulfobacterium commune °,b

Amount of product (~mol) SO32- added (/.~mol)

H2 consumed (tzmol)

S3062-

S2032-

H2S

S (%)

22 l0

8.1 3.4 3.8 1.8

7.0 2.8 2.9 1.1

0.30 0.20 0.21 0.15

0.10 0.11 0.14 0.09

98 92 93 92

4

a From E. C. Hatchikian and J. G. Zeikus, J. Bacteriol. 153, 1211 (1983). b Bisultite reductase activity is measured by the manometric assay after hydrogen uptake. The reaction employs 2.2 mg of pure enzyme at 45 ° and the incubation time is 45 min. When no more hydrogen is consumed by the reaction, the products are determined colorimetrically.

(800) 10 may be attributed to the higher stabilization of this protein within the cell. Intracellular Location. Desulfofuscidin from T. commune is recovered mainly in the soluble protein extract after cell disruption and centrifugation.4 Immunocytochemical localization experiments indicate that desulfofuscidin from T. mobile, like the homologous bisulfite reductases from Desulfovibrio mesophilic species, is located in the cytoplasm. 28 Product Ambiguity. At its optimum pH, T. commune desulfofuscidin catalyzes the reduction of sulfite mainly to trithionate. 4 An average value of 88% of sulfite sulfur appears as trithionate whereas trace amounts of thiosulfate and sulfide are produced (Table IV). Sulfide and thiosulfate slightly increase when low sulfite concentrations are used. No change in the products of the reaction is observed between 35 and 70 ° . The homologous dissimilatory bisulfite reductases including desulfoviridin, 1desulforubidin, 2 and P-582, 9 either produce trithionate as the sole product 1or trithionate as the major product with thiosulfate and sulfide as minor products.2,9 It was shown that the proportions of the products of sulfite reduction are dependent on the pH and the electron donor and electron acceptor concentration. 29-31The mechanism of dissimilatory sulfite reduction is still mune

2s D. R. Kremer, M. Veenhuis, G. Fauque, H. D. Peck, Jr., J. LeGall, J. Lampreia, J. J. G. Moura, and T. A. Hansen, Arch. Microbiol. 150, 296 (1988). 29 K. Kobayashi, Y. Seki, and M. Ishimoto, J. Biochem. (Tokyo) 75, 519 (1974). 30 H. E. Jones and G. W. Skyring, Biochim. Biophys. Acta 377, 52 (1975). 3l H. L. Drake and J. M. Akagi, J. Bacteriol. 126, 733 (1976).

[ 19]

DESULFOFUSCIDIN

293

a matter of debate.6,32 Two mechanisms of dissimilatory bisulfite reduction to sulfide have been proposed: 80322e 35032-

1'[

6e-

) S3062-

(1)

) S 2-

2e -

2e -

) 52032-

;

) S 2-

(2)

$

SO32- <_. . . . . . . . . SO32-

The first mechanism is the six-electron reduction of sulfite to H2S in one step [Eq. (1)], 33-35 catalyzed by bisulfite reductase without any free intermediates, as was demonstrated with the assimilatory-type sulfite red u c t a s e s . 36'37 In the second postulated mechanism [Eq. (2)], sulfite is reduced to sulfide in three steps via the free intermediates trithionate and thiosulfate. 3s The trithionate pathway requires three separate enzymes, namely sulfite reductase, trithionate reductase, and thiosulfate reduct a s e . 7'29'30'39 A specific thiosulfate reductase has been purified from various sulfate-reducing bacteria, 4°-42 and a trithionate reductase system has been isolated in Desulfovibrio vulgaris Hildenborough. 43 Evidence for the trithionate pathway as opposed to a direct six-electron reduction of sulfite to sulfide has been obtained by Fitz and Cypionka, 44 using deenergized cells of Desulfovibrio desulfuricans Essex 6. Whole cells of this sulfatereducing bacterium produce thiosulfate and trithionate during sulfite reduction coupled to oxidation of physiological electron donors. Most of their observations can be explained on the basis of the redox potentials of the intermediates. Further evidence for intermediate formation during sulfite reduction was obtained from proton translocation coupled to the reduction of trithionate, inhibition of thiosulfate reductase by trithionate, and reverse trithionate reductase activity. 44 The question of whether additional free intermediates occur between 32 H. D. Peck, Jr., and J. LeGall, Philos. Trans. R. Soc. London B 298, 443 (1982). 33 p. A. Trudinger and L. A. Chambers, Biochim. Biophys. Acta 293, 26 (1973). 34 L. M. Siegel, in "Metabolic Pathways. Metabolism of Sulfur Compounds" (D. M. Greenberg, ed.), Vol. 7, p. 217. Academic Press, New York, 1975. 35 L. A. Chambers and P. A. Trudinger, J. Bacteriol. 123, 36 (1975). 36 K. Asada, G. Tamura, and R. S. Bandurski, J. Biol. Chem. 244, 4904 (1969). 37 L. M. Siegel, P. S. Davis, and H. Kamin, J. Biol. Chem. 249, 1572 (1974). 3s K. Kobayashi, S. Tachibana, and M. Ishimoto, J. Biochem. (Tokyo) 65, 155 (1969). 39 j. M. Akagi, M. Chan, and V. Adams, J. Bacteriol. 120, 240 (1974). 40 W. Nakatsukasa and J. M. Akagi, J. Bacteriol. 98, 429 (1969). 41 R. H. Haschke and L. L. Campbell, J. Bacteriol. 106, 603 (1971). 42 E. C. Hatchikian, Arch. Microbiol. 105, 249 (1975). 43 J.-H. Kim and J. M. Akagi, J. Bacteriol. 163, 472 (1985). 44 R. M. Fitz and H. Cypionka, Arch. Microbiol. 154, 400 (1990).

294

DISSIMILATORY SULFATE REDUCTION

[19]

g

~,~

oo,.~

,~'~

.--~,

~-~+

M

~.~ ~~; -~ -;'7,

~ ,-a >:

~.

~-

e~

~.

.1

.~

. ] ~

•

6 o

10i

,.1

I°',~ i~;> [-" o

= ~,

~.,

~+-@~

i-, < i< r..)

•~. ~: ~.i

Z <

u

m m

~0.~--~

-~.~. o

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0

,.e

~=

~=~=~-.~ E ~ . ~

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~

Z

~

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[19]

DESULFOFUSCIDIN

295

sulfite and sulfide remains open. Several modes of dissimilatory reduction of bisulfite may be possible in the sulfate-reducing bacteria. One pathway would be the trithionate pathway. 3s An alternate pathway functioning in different environmental conditions may involve another mechanism such as the six-electron reduction of bisulfite catalyzed either by the wellknown bisulfite reductase or by a distinct enzyme. Isolation of mutants that will either be altered with respect to both reductase activities, or to only one of them, would afford definitive information on the bisulfite reduction pathway. Thiosulfate and trithionate reductase activities appear to be present in the crude extract of T. commune.4 These enzymatic activities are separated from bisulfite reductase during the DEAE-cellulose fractionation step of the purification procedure (Table I). These observations, together with the formation of trithionate as the main product of sulfite reduction by desulfofuscidin, are indicative of the presence of the trithionate pathway of bisulfite reduction in this microorganism. Conclusion Desulfofuscidin is the specific dissimilatory sulfite reductase of extreme thermophilic sulfate-reducing eubacteria (maximum temperature for growth, 85°) as compared to desulfoviridin and desulforubidin isolated from mesophilic species of the genus Desulfovibrio and P-582 isolated from the moderate thermophile spore-forming species Desulfotomaculum nigrificans. The main physicochemical properties of the four types of dissimilatory sulfite reductase are reported in Table V. They exhibit similar molecular weights and quaternary structure but they differ by their absorption spectrum. Electron paramagnetic resonance spectroscopy indicates that all these enzymes contain high-spin siroheme and [4Fe-4S] clusters as prosthetic groups. In contrast to desulfoviridin, which yields a siroporphyrin, the other bisulfite reductases yield a siroheme on acetone-acid extraction. 4-6 The content of siroheme in desulfofuscidin is twice that of the other bisulfite reductases. Desulfofuscidin shows higher thermostability than desulforubidin and exhibits its maximum activity between 65 and 70° . Acknowledgments The author wishes to thank Professor J. G. Zeikus, who initiated the study on desulfofuscidin from Thermodesulfobacterium commune, and Professor R. Cammack for his critical review of the manuscript and valuable comments. Thanks are also due to N. Forget for skillful technical assistance.