from Desulfovibrio desulfuricans (Essex) and comparison with the enzyme from Desulfovibrio .... on the Immobilion support was carried out with the Applied.
Eur. J. Biochem. 233, 873-879 (1995) 0 FEBS 1995
Molecular properties of the dissimilatory sulfite reductase from Desulfovibrio desulfuricans (Essex) and comparison with the enzyme from Desulfovibrio vulgaris (Hildenborough) Julia STEUBER’, Alexander F. ARENDSEN’, Wilfred R. HAGEN’ and Peter M. H. KRONECK’
’
Universitat Konstanz, Fakultat fur Biologie, Konstanz, Germany Wageningen Agricultural University, Department of Biochemistry, Wageningen, The Netherlands
(Received 12 Mayll3 July 1995) - EJB 95 0763/4
The dissimilatory sulfite reductase desulfoviridin was purified from the membrane (mSiR) and the soluble fraction (sSiR) of the sulfate-reducing bacterium Desulfovibrio desulfuricans (Essex). Molecular and spectroscopic properties were determined and compared with the properties of the soluble desulfoviridin from Desuljovibrio vulgaris (Hildenboroughj. The enzymes were isolated as (n = 1-3) multimers with a relative molecular mass of 200 t 10 (gel filtration). Both mSiR and sSiR from D. desulfuricans contained 24 t 3 Fe and 18 2 3 labile sulfide/200 kDa, respectively, and showed identical EPR spectra. Quantification of the high-spin Fe(II1) heme resonances at g of approximately 6 indicated that close to 80% of the siroheme moiety in the enzyme from D. desulfuricans was demetallated. D. desulfuricans sulfite reductase showed S = 9/2 EPR signals with the highest apparent g value at g = 17 as reported for SiR from D. vulgaris. Antibodies raised against the a, p and y subunit of the D. vulgaris enzyme exhibited cross-reactivity with the subunits of mSiR and sSiR from D. desulfuricans. N-terminal sequences of a, p and y subunits of both mSiR and sSiR from D. desuyuricans were identical and showed a high degree of similarity with the sequences of the corresponding subunits obtained from the D. vulgaris enzyme. During gel filtration of sSiR from D. desulfuricans, under non-denaturing conditions, a small protein (molecular mass = I 1 kDa) was separated. This 11-kDa protein exhibited cross-reactivity with the antibody raised against the y subunit of D. vulgaris sulfite reductase. In the case of D. desulfuricans sulfite reductase, the 1 1-kDa y subunit seems not to be an integral part of the protein and can be obtained from the soluble fraction and during purification of the soluble enzyme.
Keywords ; dissimilatory sulfite reduction ; membranous sulfite reductase ; desulfoviridin ; subunit composition; EPR.
Desulfoviridins are enzymes containing siroheme and Fe-S prosthetic centers and are involved in the dissimilatory reduction of sulfite by sulfate-reducing bacteria (LeGall and Postgate, 1973; Siege1 et al., 1978; Peck and LeGall, 1982; Widdel, 1988; LeGall and Fauque, 1988). Sulfate-reducing bacteria generate a proton motive force by the reduction of oxidized sulfur compounds, such as sulfate, sulfite or thiosulfate (Badziong and Thauer, 1980; Fitz and Cypionka, 1989). Many redox proteins of these bacteria have been intensively studied and led to the discovery of novel prosthetic groups, such as the putative [6Fe6S] prismane cluster (Pierik et al., 1992b). Adenylylsulfate (adenosine-5’-phosphosulfate,APS) reductase and dissimilatory rulfite reductase represent the key enzymes in the respiratory chain of sulfate-reducing bacteria. Dissimilatory sulfite reductases (SIR) are classified according to their ultraviolethisible absorption spectra: desulforubidin (Lee et al., 1973a; Mourd et Correspondence to P. M. H. Kroneck, Universitat Konstanz, Fakultat fur Biologie, Postfach 5560 M665, D-78434 Konstanz, Germany Fax: +49 7531 882966. Abbreviations. SIR, dissimilatory sulfite reductase; mSiR, membranous sulfite reductase; sSiR, soluble sulfite reductase. Enzymes. Sulfite reductase (EC 1.8.99.3); adenylylsulfate reductase (EC 1.8.99.2). Dedication. Dedicated to Prof. Norbert Pfennig on the occasion of his 70th birthday.
al., 1988; Arendsen et al., 1993), desulfofuscidin (LeGall and Fauque, 1988), P-582 (Trudinger, 1970) and desulfoviridin (Lee and Peck, 1971; Lee et al., 1973b; Moura et al., 1988; Pierik and Hagen, 1991 ; Wolfe et al., 1994). With regard to desulfoviridin, controversial aspects of the stoichiometry and structure of the prosthetic siroheme and Fe-S centers have been discussed (Wolfe et al., 1994). In desulfoviridin from Desuljovibrio vulgaris (Hildenborough) the Fe-S site was described as a S = 9/2 system that probably does not have a cubane but a more complex structure. It was concluded that the model proposed for the active site in sulfite reductase, with a siroheme-iron center bridged to a nearby [4Fe-4S] cube, as derived for the assimilatory enzyme from Escherichia coli (Christner et al., 1981), does not hold for dissimilatory-type sulfite reductases (Pierik and Hagen, 1991). For their purest desulfoviridin sample from D. vulgaris, Wolfe and coworkers (1994) found two pairs of [4Fe-4S] and fully metallated siroheme units (stoichiometry Fe/enzyme of 10:lj. Desulfoviridin-type sulfite reductases proved to be more complex than originally thought, since Pierik et al. (1992a) discovered a hitherto unnoticed third subunit in desulfoviridin from D. vulgaris (Hildenborough) of approximately 11 kDa. Thus, a hexameric composition &y2 was assumed (Pierik et al., 1992a). Genetic analysis revealed that the y subunit of D. vulgaris was not encoded on the same operon as the a and p sub-
874
Steuber et al. ( E m J. Biochenz. 233)
units (Karkhoff-Schweizer et al., 1993). Another matter of debate is concerned with the localization of the dissimilatory sulfite reductase within the cell. Since sulfite reduction contributes to the generation of a proton motive force in whole cells (Badziong and Thauer, 1980; Fitz and Cypionka, 1989), one might expect the enzyme to be membrane bound. However, in three strains of sulfate-reducing bacteria [Desu(fovibriogigas, D. vulgaris (Hildenborough) and Desulfovibrio thermophilus] the enzyme was detected in the cytoplasma according to immunocytochemical experiments (Kremer et al., 1988). Previously, we reported on the purification of desulfoviridin from membranes of Desulfovibrio desulfuricans (Essex). The membranous dissimilatory sulfite reductase (mSiR) converted sulfite to sulfide with cytochrome c 3as electron carrier, in contrast to the cytoplasmic enzyme (sSiR) which did not react (Steuber et al., 1994). In the present study, we compare the membranous and soluble sulfite reductase of D. desulfuricans with desulfoviridin from D. vulgaris (Hildenborough). Cofactor analysis, EPR properties, and both N-terminal sequences and immunological cross-reactivity of the subunits are reported. We will present evidence that the 11-kDa protein subunit) is not an integral part of the dissimilatory sulfite reductase from D. desulfuricans but can be isolated from the soluble fraction and the soluble sulfite reductase. In addition, the influence of Triton X-100 on the attachment of the 11-kDa protein to the membranous sulfite reductase will be discussed. (71
MATERIALS AND METHODS Cultivation of bacteria. D. desuljiuricuns strain Essex 6 (DSM 642), was grown in 20-1 batch cultures in a basal mineral medium, containing 30 mM lactate and 15 mM sulfate (Cypionka and Pfennig, 1986; Steuber et al., 1994). D. vulgaris strain Hildenborough (NCIB 8303) was grown in 300-1 batch cultures according to Van der Westen et al. (1978). Sulfite reductase from D. desulfuricuns (Essex). Unless specified all manipulations were performed at 4°C in the presence of air. Desulfoviridin was obtained from the soluble fraction after cell rupture by a French press or treatment of cells by lysozyme. Soluble sulfite reductuse (sSiR). The soluble fraction was dialyzed against 50 mM potassium dihydrogenphosphate, pH 6.6, and loaded onto a DE 52 column (4cmX4.2cm, Whatman). A linear KCI gradient (from 0 to 0.5 M) in SO mM potassium dihydrogen phosphate, pH 6.6, led to the elution of the enzyme at approximately 0.3 M KCI. The green fractions were concentrated by ultrafiltration (10-kDa cutoff) and loaded onto a highresolution Fractogel-TMAE column (Merck) connected to a Pharmacia FPLC system. A linear KCl gradient (from 0 to 1.O M) in 50 mM potassium dihydrogenphosphate, pH 6.6, led to the elution of sulfite reductase at approximately 0.4 M KCI. Finally, the green sulfite reductase fraction was concentrated, desalted and loaded onto a Pharmacia Superdex 200 gel-filtration column. Membranous sulfite reductuse (mSiK). The membrane fraction was added to 0.5 M potassium dihydrogenphosphate, pH 7.0, containing 4 % (by mass) Triton X-100, proteiddetergent 1 :4 (by mass). After stirring for 45 min at room temperature the suspension was centrifuged at lOOOOOXg, 1 h, giving the solubilized membrane fraction as supernatant. This fraction was dialyzed against water to a final concentration of 50 mM potassium dihydrogenphosphatc, and the pH was adjusted to 6.6. The enzyme was purified to apparent homogeneity following the procedure described for sSiR but in the presence of 0.05% (by mass) Triton X-100.
Dissimilatory sulfite reductase from D. vuZguris (Hildenborough). Desulfoviridin was obtained from the soluble fraction after cell rupture by a Manton Gaulin press according to Pierik and Hagen (1991). Sulfite reductase activity. The sulfite reductase activity was determined in the presence of methyl viologen or cytochrome c3 as mediators (Schedel et al., 1975; Pierik and Hagen, 1991; Steuber et al., 1994) using the manometric method by Umbreit et al. (1972). In experiments with whole cells, redox mediators were absent. Analytical procedures. Protein was determined by the bicichoninic acid method (Smith et al., 1985), or with the microbiuret method at 550nm (Goa, 1953) after trichloroacetic a c i d deoxycholate precipitation (Bensadoun and Weinstein, 1976). BSA served as the standard. Iron was determined colorimetrically using 3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazine, disodium salt trihydrate (ferene; Pierik et al., 1992b). Acid-labile sulfide was measured according to Brumby et al. 1965). Siroheme was extracted with an HCUacetone mixture and subsequently determined as the pyridine hemochrome (Siege1 et al., 1978). Electrophoresis and blotting. SDS/PAGE was carried out with the Protean I1 electrophoresis system (22OX2OOXO.75 mm, BioRad), or with the Mighty Small system (100XSOX0.75 mm, Hoefer Scientific Instruments), using 15 % acrylamide gels (Laemmli, 1970). Gels were stained with silver (Rabilloud, 1990). Isoelectric focusing was carried out on PhastGel IEF 3-9 gels with a flat-bed Ultraphor electrophoresis unit (Pharmacia). IEF standards (PI 4.6-9.6) were obtained from BioRad. The blotting of the 50-kDa and 45-kDa subunits of the sulfite reductase was performed as previously described (Pierik et al., 1992a). Since the 11-kDa component passed through the nitrocellulose or poly(viny1idene fluoride) membranes, it was blotted for 20 min with the Biometra Fast-Blot device (75 mA). Immunostaining of the 50-, 45- and 11-kDa components was obtained with antisera raised against the subunits of desulfoviridin from D. vulgaris (Hildenborough) as described (Pierik et al., 1992a). For N-terminal sequencing, approximately 30 nmol sulfite reductase, or of the 1LkDa protein, were separated on a lOOXSOX0.75 mm SDS/polyacrylamide gel and blotted onto a poly(viny1idene difluoride) Immobilon-P support; Coomassie R-250 was used for staining. Sequencing of the polypeptides on the Immobilion support was carried out with the Applied Biosystems 477A gas-phase system. Spectroscopy. Ultraviolet/visible spectra were recorded on the Aminco DW-2000 spectrophotometer. X-band EPR spectra were obtained with a Bruker EPR 200 D instrument with peripheral equipment and data handling as described by Pierik and Hagen (1991).
RESULTS Purification of the soluble and membranous sulfite reductase from D. desulfuricans (Essex). The soluble and the membranous sulfite reductase from D. desulfuricuns were purified to apparent homogeneity by a three-step procedure (Table 1). Triton X-100 was used to solubilize mSIR from the membrane fraction. Both sSiR and mSiR were eluted from the high-resolution TMAE anion-exchange column in one fraction each, in contrast to the enzyme from D. vulgaris where a slow and fast form had been observed (Liu et al., 1979; Pierik et al., 1992a). Isoelectric focusing of sSiR and mSiR from D. desulfuricans revealed one major band after Coomassie staining at pl less than 4.65 (phycocyanin standard) similar to the fast form of D. vulgaris desulfoviridin (Liu et al., 1979; Pierik et al., 1992a). After silver staining
875
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Table 1. Purification of the membranous (mSir) and the soluble @Sir)sulfite reductase from D. desulfuricans (Essex). Activity was determined manometrically with methyl viologen or, in the case of the mSiR Superdex fraction, with cytochrome c3,as described in Materials and Methods. ~~
Enzyme
Fraction
Protein
Activity
Specific activity
Purification factor
~
mSiR
mg
pmol H, min
TMAE Superdex
850 75 55 20
30.6 3.75 3.60 2.56l2.20
soluble fraction DE 52 TMAE Superdex
740 66 32 18
22.2 2.18 1.22 0.75
membranes, Triton X-100 DE 52
sSiR
of the gels with the enzymes purified from D. desuljiuricans several bands appeared between pH 6.5 and the acidic front of the gel. These extra bands might represent forms of sulfite reductase differing in PI. Alternatively, the bands could result from partially degraded enzyme as observed for the IEF standards. The specific activity of mSIR from D. desulfuricans was 100 ? 20 nmol H, min- mg-' with cytochrome c3 as redox mediator versus 4 0 5 10 nmol H, min-' mg-' of sSIR and methyl viologen as redox mediator. The sulfite reductase activity of freshly harvested cells of D. desulfuricans was 142 nmol H, min-' mg protein (manometric assay). The specific activity of the D. vulgaris enzyme was 150-300 nmol H, min-' mg-' with methyl viologen as redox mediator.
'
nmol H, min-' mg-'
-fold
36 50 65 1281110
1 1.4 1.8 3.513.0
30 33 38 41
2.1
1 1.1 1.3 1.4
2.2
2.3
2.4
2.5
2.6
Ve/Vo
Molecular composition of sulfite reductase from D. desulfuricans (Essex) and properties of the 11-kDa component. After gel filtration of sSiR from D. desulfuricans on a Superdex 200 column in SO mM potassium dihydrogenphosphate, pH 6.6, a fraction was obtained containing two proteins with molecular masses of approximately 11 kDa (Fig. 1). In the presence of 0.05 % Triton X-100 but otherwise identical conditions mSiR did not yield the fraction with the 1I-kDa proteins (Fig. 1). The molecular mass of both sSiR and mSiR was 200% 10 kDa by gel filtration in the absence of Triton X-100. The 50 (a), 45 (/3) and 11 kDa (y) protein components were present in the peak fractions obtained after gel filtration of sSiR and mSiR from D. desulfuricans as demonstrated by protein blots ; for comparison the data for the D. vulgaris enzyme are also given (Fig. 2). Note that larger protein quantities had to be applied in these blots in order to detect the 1I-kDa component; most of this small protein passed through the membrane under the conditions necessary to transfer the 50-kDa and the 45-kDa subunits. Both peak fractions with either sSiR or mSiR exhibited cross-reactivity towards the antisera raised against the 50- (a), 45- (p) and the 11-kDa ( y ) subunits from the D. vulgaris enzyme (Fig. 2) demonstrating the presence of the three subunits in sulfite reductase from D. desu(furicans. The N-terminal sequences of the corresponding subunits of sSiR and mSiR were identical, and there were only minor differences with regard to the N-terminal sequences of the D. vulgaris enzyme (Table 2). In the 50-kDa subunit, a lysine at position seven in the D. vulgaris sulfite reductase was replaced by a leucine in the D. desulfuricans enzyme. Furthermore, in the 11-kDa protein, a valine at position three in the D. vulgaris reductase was replaced by isoleucine in the D. desulfuricans enzyme. The 11-kDa protein component was not only present in sulfite reductase from D. desulfuricuns and D. vulgaris but also in the low-molecular-mass protein fraction obtained by non-denaturating gel filtration of sSiR from D. desuljiuricans (Fig. 2). After separation on SDS/PAGE (220X200X0.75 mm gel, Fig. 3) and blotting on nitrocellulose, immunostaining with anti-
Elution volume (ml) Fig. 1. Molecular mass and subunit composition of the dissimilatory sulfite reductase from Desulfovibrio desulfuricans (Essex). Below, non-denaturing gel filtration of soluble (sSiR, 27 mg, A) and membranous (mSiR, 20 mg, B); 27 mg sSiR or 20 mg mSiR were loaded on a Superdex 200 column (buffer SO mM potassium dihydrogenphosphate, pH 6.6, flow 1 ml min - I ) . The increase of absorbance at V , = 305 ml (trace B) results from Triton X-100 (0.05% by mass in buffer). Above,
molecular masses of sSiR and mSiR. Calibration with Sigma standards (200.0. 150.0, 66.0, 29.0 and 12.4 kDa). serum against the y subunit of D. vulgaris desulfoviridin revealed cross-reactivity of the band at approximately 11 kDa (Fig. 3). N-terminal amino acid analysis of the proteins present in this band revealed that the y subunit with the sequence A-EI-T-Y-K-G-K constituted approximately 10% of the total protein.
876
Steuber et al. (Eur: J. Biockem. 233)
Table 2. N-terminal amino acid sequences of protein components (a, p, y subunits) of D. desulfuricans (Essex) and D. vulgaris (Hildenborough) sulfite reductase. X, residue not identified; *, residue introduced to preserve alignment with other sequences. Protein component
Organism
Sequence
Reference
SO kDa (a) SO kDa (a) mSiR SO kDa (a) sSiR
D. vulgar. D. desulf:
A K H A T P K L D Q L E S G P W P S F V S D I K Q A K H A T P L L D Q L E S G P W X * F V
Karkhoff-Schweizer et al. (1995) this study
A K H A T P L L D Q L E S G P W P S F V
this study
(m
45 kDa 45 kDa (fi) mSiR 45 kDa (/J) sSiR
D. vulgur. D. desulj:
A F I S S G Y N P E K P M A N R I T D I G P R K F A F X P T G Y N P
Karkhoff-Schweizer et al. (299.5) this study
D. desulf.
A F I P T G Y N P X K P M
this study
11 kDa ( y ) 11 kDa ( 7 ) mSiR 11 kDa (1,) sSiR
D. vulgar. D. desulf.
A E V T Y K G K S F E V D E D G F L L R F D D W C A E I T Y K G K
Karkhoff-Schweizer et al. (2993) this study
D. de.\uy
A E I T Y K G K
this study
ANTI a
1
2
ANTI
3
1
2
p 3
ANTI y
1
2
3
Protein Blot k
D
a
1
2
3
ANTI y
SDS-PAGE 4
1
2
kDa
1
2
Fig.2. Western blots and protein blots of the 50-, 45- and 11-kDa protein component of the membranous and the soluble dissimilatory sulfite reductase from D. desulfuricans (Essex). To the left is shown an immunostain of sulfite reductase (0.10 pg, respectively) from D. vulgaris (Hildenborough) (lane 1) and D. de.su(fiirican.s (sSiR, lane 2; and mSiR, lane 3) with antiserum raised against the corresponding subunits of the D. vulgaris enzyme. To the right is shown a protein blot (50 pg, respectively) of D. vulgaris sulfite reductase (lane l ) , sSiR (lane 2), mSiR (lane 3), and isolated 11-kDa protein (1 S pg, lane 4).
Cofactors of sulfite reductase from D. desulfuricans (Essex). Both mSiR and sSiR from D. desulfuricans contained 24 t 3 Fe and 18 2 3 S2-/200 kDa, in close agreement with the data reported for desulfoviridin from D. vulgaris (Pierik and Hagen, 1991). The pyridine hemochrome spectra of mSiR and sSiR were essentially the same and comparable to those of the enzyme from D. vulgaris. We estimated that only approximately 5 % of the siroheme moiety was metallated based on the following assumptions: the absorption at 550-700 nm solely results from heme-iron, and; the absorption coefficient ess7 -750 = 1.57 X lo4M-'cm ' as described for the assimilatory sulfite reductase from E. coli (Siege1 et al. 1978). ~
Electron paramagnetic resonance spectra. The EPR signals
recorded at X-Band, 10 K, of as-isolated mSiR and sSiR from D. desulfuricans were essentially identical and comparable to the signals for the enzyme from D. vulgaris (Pierik and Hagen, 1991). Thus, in all three sulfite reductase samples, the high-spin Fe(II1) siroheme exhibited resonances in the region g = 6 (g, and g, components) and around g = 2 (g, components). Quantification of the siroheme high-spin EPR resonances with the help
Fig. 3. SDSmAGE and immunodetection of isolated 11-kDa protein from D. desulfuricans (Essex) with antiserum raised against the y subunit of D. vulgaris (Hildenborough) dissimilatory sulfite reductase. Lane 1, 11 kDa protein, 20 pg; lane 2, prestained molecular-mass markers (116.0-38.8 kDa, BioRad).
of computer simulations of the complete spectra (Arendsen et al., 1993), yielded 14% (mSiR) and 16% (sSiR) for D. desuljuricans sulfite reductase compared to 26 % for the enzyme from D. vulgaris, when an ideal stoichiometry of two siroheme/mol sulfite reductase is assumed. At low magnetic field, resonances with apparent g values around 9 and 17 were present in the EPR spectra of mSiR and sSiR which again were similar to those described for sulfite reductase from D. vulgaris (Pierik and Hagen, 1991), and related dissimilatory sulfite reductases (Arendsen et al., 1993).
DISCUSSION Purification and activity of sulfite reductase from D. desulfuricans (Essex). An active membranous sulfite reductase (mSiR) was solubilized (Triton X-100) and purified to homo-
877
Steuber et al. (ELK.I. Biochem. 233)
geneity from the sulfate-reducing bacterium D. desulfuricuns (Essex 6 ) . A soluble sulfite reductase (sSiR) was also obtained in pure form from this organism (Table 1). mSiR and sSiR are desulfoviridin-type enzymes and contained 24 2 3 Fe/200 kDa compared to 2 2 t 4 Fe for the soluble desulfoviridin from D. vulgaris (Hildenborough) (Pierik et al., 1991). Both sSiR and mSiR were eluted from the high-resolution TMAE anion-exchange column in one distinct fraction each. This is in contrast to the enzyme from D. vulgaris (Hildenborough) where a slow and a fast form had been observed (Liu et al., 1979; Pierik et al., 1992a). Earlier, Seki and coworkers separated desulfoviridin from D. vulgaris (Miyazaki) into two forms by DEAE-Sephadex chromatography (Seki et al., 1979; Seki and Ishimoto, 1979). Most recently, three distinct forms of D. vulgaris (Hildenborough) sulfite reductase were obtained after anion-exchange chromatography on a FPLC Mono Q column. The separation was extremely dependent on buffer and gradient conditions as the three fractions differed slightly in pl (Wolfe et al., 1994). Two of these enzyme forms (DSir-I and DSiR-11) could be isolated in sufficient quantity. The two forms contained 11 ? 1 Fe/ mol enzyme and 10 t 1 Fe/mol enzyme compared to approximately 24 Fe/mol enzyme prior to the FPLC step. A contaminant fraction was observed in the Mono Q chromatogram that carried up to 83 Fe/mol enzyme. Note that the authors could not determine whether these three forms of SIR represent true natural forms of dissimilatory sulfite reductase. Another point of interest is concerned with the specific activity of sulfite reductase. In our enzyme samples (=24 Fe/mol enzyme), especially in the case of D. desulfuricans mSiR, the specific activity increased during the purification procedure (Table l), and was highest after gel filtration in the assay
-
H2
hydrogenase
-
cytochrome c1
- SIR
HSO;
(-100 nmol H, min-' mg-I). The specific activity was even slightly higher (128 nmol H, min ' mg-') with methyl viologen as electron carrier (Steuber et al., 1994). In the purification procedure described by Wolfe et a]. (1994) the specific activity (methyl viologen assay) of the purified enzyme from D. vulgaris (-12 Fe/mol enzyme) was always greater than or equal to that prior to the FPLC step. With sulfite as the electron acceptor, we measured an hydrogen uptake of up to 140 nmol H, min-' mg- ' protein with freshly harvested cells of D. desulfuricans in the manometric assay. By means of the sulfide electrode, a maximum rate of 210 nmol H, oxidized min-' mg-' protein was found for washed cells of D. vulgaris (Marburg) with HSO; as electron acceptor (Fitz and Cypionka, 1991). In studies on the uptake of sulfite by D. desulfziricarzs, Cypionka (1987) reported an average rate of 8 nmol HS- produced min-' mg-' protein after addition of pulses of HSO; to H,-saturated non-buffered cell suspensions. In D. desulfuricans dissimilatory sulfite reductase represents approximately 1 o/o of the total cell protein as reported for D. vulgaris (Marburg) (Badziong and Thauer, 1978). Thus, from these data the in tiivo activity of dissimilatory sulfite reductase from D. desLiljkricans was calculated to approximately 10 pmol HSO; HS- min-' mg-' protein which is 300-times the experimentally observed value of approximately 35 nmol HSO, H S - min-' mg ' protein in the assay H2 hydrogenase --t cytochrome c 3-+ SIR -+ HSO;. The reason for the decrease in activity upon isolation and purification of the enzyme remains unclear. Note that the activity of freshly harvested cells of D. desulfuricuns decreased to 5% of the activity of cells measured in chemostat culture (Cypionka, 1987). According to Karkhoff-Schweizer et al. ( 1 993) the steady-state level of CI and /3 subunits expressed in D. vulgaris (Hildenborough) cells was rather constant, while that of the j~ subunit increased strongly in the stationary growth phase. Growing
-
-
-
batch cultures of D. desulfuricam (Essex) showed maximum formation of thiosulfate and trithionate (up to 100 pM) towards the end of the growth phase (Sass et al., 1992). We did not achieve any significant increase in sulfite reductase activity upon addition of the fraction containing the two low-molecular-mass proteins including the 1' subunit (Fig. 1) to either mSiR or sSiR from D. desulfuricans. Furthermore, chromatography of sSiR on Superdex 200 led to the loss of thiosulfate-dependent uptake of HSO, + HS-, thiosulfate dihydrogen (S20:- + 2e + 2H' reductase activity, 2.4 nmol H, min-' mg-' protein) which was still present after TMAE chromatography (Kroder, M., unpublished results).
-
Molecular composition of sulfite reductase from D. desulfuricans (Essex) and properties of the low-molecular-mass component. The dissimilatory sulfite reductases mSiR and sSiR from D. desulfuricans (Essex), and the soluble enzyme from D. vulgaris (Hildenborough), were closely related with regard to their epitope maps and the N-terminal amino acid sequences of their a,/j', and y subunit (Figs 2 and 3). However, the three enzymes, as isolated, may have significant differences in their subunit composition, especially with regard to the y subunit. A hexameric a2p2y2subunit composition had been proposed earlier for desulfoviridin from D. vulgaris (Hildenborough) (Pierik et al., 1992a) and was found more recently after a more rigorous purification procedure (Wolfe et al., 1994). Gel filtration of D. desulfuricans sSiR under non-denaturing conditions led to the separation of a low-molecular-mass fraction carrying the y subunit and a second small protein. Up to 36% of the expected 1' subunit (assuming a u,-.,y2 subunit composition, approximately 200 kDa) was not tightly bound to sSiR from D. desulfuricans but dissociated during gel filtration. This finding was supported by Western blot analysis revealing the presence of the j , subunit in fractions obtained earlier during the purification of sSiR from D. desulfuricans (data not shown). Karkhoff-Schweizer et al. (1993) showed that the y subunit of desulfoviridin from D. vulgaris (Hildenborough), with a calculated molecular mass of 11.859 kDa, was not encoded on the same operon used for the CI and p subunits. The complete sequence of the D. desuljuricans subunit is not available at present in contrast to that of D. vulgaris sulfite reductase (Karkhoff-Schweizer et al., 1993, 1995). Nevertheless, we think it is reasonable to assume a similar structure for the D. desulfuricans j , subunit in view of the immunological properties and N-terminal amino acid sequence. Hence, we conclude that the j , subunit is not an integral part of the soluble desulfoviridin from D. desulfuricans, but appears loosely associated and will be lost in part during purification. With regard to the subunit stoichiometry a&y2 proposed earlier (Pierik et al., 1992a; Wolfe et al., 1994) a composition a2p,y,, may be envisaged, with n greater than 2 in the native enzyme. Alternatively, since the gene for the j , subunit is expressed separately from the genes encoding for the CI and /3 subunit, the composition could still be as a result of disregulation of the two transcription units (Karkhoff-Schweizer et al., 1993). The second low-molecular-mass protein observed during gel filtration of sSiR from D. desulfuricans (Fig. 1) may be related to the DsvD protein described previously by Karkhoff-Schweizer and colleagues (1995) from the analysis of the structural gene of dissimilatory sulfite reductase from D. vulgaris and Archaeoglobus ,ful,yidus. ;J
Electron paramagnetic resonance properties of sulfite reductase from D. desulfuricans (Essex). The membrane-bound and the soluble sulfite reductase give essentially identical EPR spectra that are very similar to those of the D. vulgaris enzyme. The prediction that dissimilatory sulfite reductases exhibit S of
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Steuber et al. (Eur: J . Biochem. 233)
912 EPR features (Arendsen et al., 1993) seems to hold for another desulfoviridin-type dissimilatory sulfite reductase purified by a different purification procedure. Dissimilatory sulfite reductases contain siroheme and Fe-S centers as prosthetic groups. No high-resolution X-ray structure exists for sulfite reductase. Thus, structural models strongly depend on EPR and Mossbauer spectroscopic data. Basic questions concern, the type of Fe-S cluster present in desulfoviridin, and, the distance between the Fe-S center and the siroheme moiety, and the nature of the bridge between these sites if one exists. Following the earlier proposal by Christner et al. (1981), and Wilkerson et al. (1983), the active site of the assimilatory sulfite reductase from E. coli (Siegel et al., 1973), the nitrite reductase from spinach (Wilkerson et al., 1983) and the assimilatory (Huynh et al., 1984; Tan and Cowan, 1991) and dissimilatory sulfite reductase from sulfate-reducing bacteria (Moura et al., 1988; Wolfe et al., 1994) are modeled as diamagnetic [4Fe-4SI2+cubanes exchange-coupled to the siroheme prosthetic groups. Recently, Pierik et al. (1991) challenged this model for the high-spin dissimilatory sulfite reductases from sulfate-reducing bacteria. These enzymes differ from the other siroheme and Fe-S-containing enzymes in a functional aspect, since the reduction of sulfite in sulfate-reducing bacteria is part of an anaerobic respiration process that generates a proton motive force (Badziong and Thauer, 1980; Fitz and Cypionka, 1989). According to Pierik et al. (1991), the Fe-S clusters of dissimilatory sulfite reductases contain more than four iron atoms that are not necessarily exchange-coupled to the siroheme prosthetic group. This is documented by a set of EPR signals at high g values (Pierik et al., 1991) that are due to a new type of superspin state (S = 912) Fe-S cluster. S = 912 EPR signals are also found in desulforubidin, a dissimilatorytype sulfite reductase that contains, unlike desulfoviridin, fully metallated sirohemes (Arendsen et al., 1993). Furthermore, Arendsen and colleagues confirmed the absence of S = 912 EPR signals in the assimilatory enzymes from E. coli and D. vulguris (Hildenborough), as predicted earlier by Pierik et al. (1991). This work was supported by Deutsche Forschungsgemeinschaft and European Science Foundation (Human Capital and Mobility, project MASIMO). We thank H. Cypionka, M. Kroder, F. Neese and A. J. Pierik for valuable discussions.
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Supplementary material. Molecular properties of the dissimilatory sulfite reductase from Desulfovibrio desulfuricans (Essex) and comparison with the enzyme from Desulfovibrio desulfuricans (Hildenborough). Fig. S1. EPR spectra of S = 912 ironsulfur clusters in isolated dissimilatory sulfite reductase. This information is available, upon request, from the Editorial Office.
