(Received for publication, September 9, 1996, and in revised form, February 5, 1997)
From the Institut für Biologische Chemie, Universität Heidelberg, D-69120 Heidelberg, Federal Republic of Germany
Iron-sulfur proteins acquire their clusters by posttranslational assembly. To identify components involved in this process an in vitro assay for holoprotein formation was established using the [2Fe-2S] ferredoxin of the cyanobacterium Synechocystis as a model. Conversion of apoferredoxin to the holo- form was observed in an anaerobic reaction medium containing Fe(NH4)2(SO4)2, L-cysteine, glutathione, and catalytic amounts of Synechocystis extract, specifically depleted of endogeneous ferredoxin. An approximate 2500-fold purification of the converter activity yielded a monomeric, 43-kDa, pyridoxal phosphate-containing enzyme, which catalyzed the breakdown of L-cysteine to yield sulfide (assembled in ferredoxin), pyruvate, and ammonia; 1 mol of [2Fe-2S] ferredoxin was formed per 2 mol of cysteine utilized.
The purified enzyme also catalyzed the -elimination reaction with
cysteine in the absence of apoferredoxin. An increased reactivity was
found with cystine instead of cysteine, which should yield cysteine
persulfide as the primary product.
These results provide a function-based identification of a cysteine/cystine C-S-lyase as a participant in ferredoxin Fe-S cluster formation. A substrate-derived cysteine persulfide could be involved in this reaction.
Iron-sulfur (Fe-S) clusters are found as functional units in numerous electron-transferring proteins. They are essential in basic biological processes such as oxidative phosphorylation, photosynthesis, and nitrogen fixation. More recently, they have also been identified as components of enzymes not concerned with redox reactions (1). Lately they have been implicated in gene regulation (2).
Despite these important and diverse functions and thorough biochemical investigations of Fe-S proteins, the biosynthetic steps leading to Fe-S cluster assembly have not been established. While it seems likely that enzyme-catalyzed reactions are involved in the formation of Fe-S clusters in vivo, the chemical reconstitution of apoproteins with free ferrous ion and sulfide in the presence of thiols (3) has been most intensively exploited for cluster build-up in vitro. Nevertheless, several proteins have been suggested as candidates in the biosynthetic process. Rhodanese (4) and 3-mercaptopyruvate sulfurtransferase (5) were effective in purified in vitro systems, comprising apoprotein, thiol, ferrous ion, and the respective sulfur-containing substrate, thiosulfate, or mercaptopyruvate. Analysis of yeast respiration-deficient mutants suggested a role for the BCS1 gene product (6) in the synthesis of functional Rieske protein. Studies on nif mutants of Azotobacter vinelandii (7) identified nifS as essential for nitrogenase activity. More recently, nifS was cloned and expressed in Escherichia coli; the gene product (NifS) was characterized as a pyridoxal phosphate-containing L-cysteine desulfurase, which yielded alanine and elemental sulfur as products (8); with dithiothreitol present in the reaction mixture, sulfide was produced instead of sulfur. Further work showed that the apo- form of the nitrogenase iron protein component could be activated by NifS-catalyzed reassembly of its [4Fe-4S] cluster in vitro (9). NifS was also successfully used for the in vitro reconstitution of the [4Fe-4S] cluster of a mutant FNR protein (10) as well as of the [2Fe-2S] cluster of SoxR (11), both E. coli proteins. Most recently, a NifS-like protein was isolated from E. coli and found to reactivate apodihydroxy acid dehydratase in vitro (12).
The significance of these various findings depends on the nature of the in vivo sulfur source for Fe-S clusters. Feeding experiments with E. coli assigned this role to cysteine (13). Cysteine sulfur was also incorporated into the [2Fe-2S] cluster of ferredoxin in isolated, intact chloroplasts (14). In the chloroplast system, ferredoxin cluster formation was found to be a stroma-located process, consisting of two separate steps: first, the NADPH-stimulated liberation of sulfide from cysteine followed by the ATP-dependent sulfide incorporation into ferredoxin (15).
With the cyanobacterium Synechocystis as a procaryotic model for the chloroplast organelle we now established an apo- to holoferredoxin conversion assay. The procaryotic system was chosen because of the relative ease of obtaining large quantities of cell material without the problem of compartmentalization. The endogeneous [2Fe-2S] ferredoxin was adopted as the Fe-S protein studied because of its abundance and the well characterized chemical procedures of reversible cluster removal (16). Moreover, the physiological existence of apoferredoxin has been demonstrated for the chloroplast system, where cluster assembly occurs in the stroma after proteolytic processing of preferredoxin that is imported from the cytosol (17).
We here report on the set up of the assay, the purification and characterization of the converter protein (provisionally named C-DES because of its ysteine ulfhydrase activity), and the stoichiometry of the cluster formation reaction.
L-Cysteine and L-propargylglycine were from Fluka; L-allylglycine, L-vinylglycine, and pyridoxal phosphate were from Sigma; D-cysteine was from Novabiochem; and L-cystine was from SERVA. Glutathione and glutathione reductase were purchased from Boehringer Mannheim. Other chemicals were of the highest purity commercially available. L-[U-14C]Cystine (302.2 mCi/mmol) was obtained from Du Pont NEN. Sephadex G-25 and G-75, DEAE-Sephadex A-25, Q-Sepharose FF, Mono P HR5/5, and Polybuffer 74 were from Pharmacia; Ultrogel AcA 44 and Dowex 50 WX8 were from SERVA; and DE52 cellulose was from Whatman. Ferredoxin-NADP reductase was purified from frozen spinach leaves (18); the final crystallization step was omitted.
Unless otherwise stated, proteins were concentrated by ultrafiltration using PM 10 membranes or Centricon 10 units, both of which were supplied by Amicon. Buffer exchanges were performed using a Sephadex G-25 column.
Growth of SynechocystisSynechocystis PCC 6714 (ATCC 27178) was grown at 30 °C in BG-11 medium (19) supplemented
with 0.9 g/liter KHCO3 in an 18-liter fermenter at pH
7.5-8.5. Cultures were illuminated with fluorescent tubes and gassed
continuously with 5% CO2. After 4 days of growth, typically 15 g of cell paste was harvested by centrifugation, shock-frozen, and stored at 70 °C.
30 g of frozen cells were
thawed, resuspended in 130 ml of 50 mM
Mops1/NaOH, pH 8.0, containing 5 mM
dithiothreitol and 0.5 mM phenylmethanesulfonyl fluoride,
and disrupted by sonication. After centrifugation (105 × g, 90 min) the extract (1500 mg of protein) was
concentrated, transferred into 0.15 M Tris/Cl, pH 7.8, containing 0.3 M NaCl (final volume 80 ml), and loaded onto
a DEAE-Sephadex A-25 column (20 cm2 × 5 cm, 10 cm/h). The
column was washed with 80 ml of starting buffer; all unadsorbed protein
(1450 mg) was collected and reduced for 30 min at 30 °C with 10 mM dithiothreitol. Then the buffer was exchanged for 50 mM Mops/NaOH, pH 7.6, and this protein preparation was used
for experiments requiring deferredoxinized extract.
Bound ferredoxin was eluted from the DEAE-Sephadex column with
190 ml of 0.15 M Tris/Cl, pH 7.8, containing 0.5 M NaCl. The eluate was treated with DNase and RNase (15 mg
each) in the presence of 10 mM MgCl2 (48 h,
4 °C). Ferredoxin was purified to homogeneity (yield 7-8 mg) by
ammonium sulfate fractionation and DE52 cellulose chromatography
according to Ref. 20. It was converted to the apo- form by double
precipitation with 0.5 N HCl under argon according to Ref.
16. The pellet was dissolved in 50 mM Mops/KOH, pH 8, containing 20 µM EDTA and kept under argon. Apoferredoxin
concentration (usually ~60 µM) was determined by the
Bradford method (21) calibrated with holoferredoxin, which was
spectrophotometrically quantified (Mr = 10,259 (Ref. 22); 276 nm = 12,550 M
1
cm
1,
423 nm = 6400 M
1 cm
1 (Ref. 23)).
For purification of C-DES, deferredoxinized extract (80 ml) was gel-filtered on Ultrogel AcA 44 (20 cm2 × 85 cm; 5 cm/h) in 50 mM Mops/NaOH, pH 7.6, containing 20 µM EDTA. C-DES was collected in fractions corresponding to Kav values between 0.525 and 0.575. These fractions were diluted with 4 volumes of 50 mM Tris/Cl, pH 7.8, containing 1 mM dithiothreitol and 20 µM EDTA and loaded onto DE52 cellulose (20 cm2 × 3.5 cm; 8 cm/h) equilibrated with this buffer. A linear gradient (560 ml) to 160 mM Tris/Cl, pH 7.8, containing 320 mM NaCl, 1 mM dithiothreitol, and 20 µM EDTA was applied. C-DES was eluted in the trailing shoulder of the major protein peak. This eluate fraction was diluted with 7 volumes of 50 mM Tris/Cl, pH 7.4, containing 1 mM dithiothreitol and 20 µM EDTA and pumped onto Q-Sepharose FF (5 cm2 × 1.5 cm; 20 cm/h) equilibrated with this buffer. A linear gradient (100 ml) to 150 mM Tris/Cl, pH 7.4, containing 300 mM NaCl, 1 mM dithiothreitol, and 20 µM EDTA was applied. C-DES was eluted just in front of the main protein peak. This fraction was transferred into 25 mM 1-methylpiperazine/iminodiacetic acid, pH 5.7, and applied to a Mono P HR 5/5 column (5 cm/min). Homogeneous C-DES was eluted at a pH of 4.4 using Polybuffer 74 (diluted 1:29 (v/v) and adjusted to pH 4.0 with iminodiacetic acid) as eluant. Separation of C-DES from Polybuffer and transfer into 50 mM Mops/NaOH, pH 7.6, was achieved by gel filtration on Sephadex G-75.
All operations were carried out at 4 °C except for chromatofocusing,
which was performed at room temperature. The final C-DES preparation
was stored at 70 °C; its activity proved to be stable for several
months.
The standard assay for holoferredoxin formation, run at 30 °C under argon, contained 10 µM apoferredoxin, which was incubated with samples of deferredoxinized Synechocystis extract (or purified fractions thereof) in 55 mM Mops/KOH, pH 7.5, 9 mM glutathione, 0.15 mM pyridoxal phosphate, and 90 µM L-cysteine. The reaction was started by the addition of Fe(NH4)2(SO4)2 to a final concentration of 0.4 mM (final volume 30 µl to 0.35 ml).
The reaction was stopped, routinely after 1 h, by the addition of
1.7 mM EDTA and 10% glycerol. The holoferredoxin content of the sample was analyzed by native PAGE (20% resolving gel, Laemmli
buffer system (24) with SDS omitted); the same result was obtained
whether the sample was analyzed immediately or after freezing. The gels
were stained with the carbocyanine dye "stains all" (4, 5, 4,
5
-dibenzo-3, 3
-diethyl-9-methylthiacarbocyanine bromide) (25); the
amount of holoferredoxin (0.1-1 µg/lane) was estimated by comparison
with known standards of ferredoxin.
Exact quantities of holoferredoxin were determined (after transfer into
50 mM Mops/NaOH, pH 7.6) by activity measurements made
according to Ref. 26; the rate of cytochrome c reduction by
NADPH via ferredoxin, which is mediated by ferredoxin-NADP reductase,
was measured, and 1 µg of ferredoxin in 0.5 ml gave a
A550 of 0.2 min
1
cm
1 under the conditions employed.
Where specified, substrate concentrations in the standard assay were varied as follows. Free sulfide (up to 0.4 mM), mercaptopyruvate (up to 5 mM), coenzyme A (0.5 mM), or thiosulfate (0.5 mM) was used instead of cysteine; the concentration of cysteine employed was varied from 20 µM to 2 mM, and the concentration of Fe(NH4)2(SO4)2 was varied from 20 µM to 0.5 mM. ATP and NADPH (up to 5 mM each) were added to some reactions.
Quantification of Pyruvate Formation from CysteineThe standard assay composition was modified as follows. 45 µM [14C]cystine (adjusted to 11,200 dpm/nmol), reduced in situ prior to the addition of apoferredoxin by a 100-fold excess of glutathione to yield [14C]cysteine (5600 dpm/nmol) was employed. [14C]Pyruvate formed during the reaction was quantified both by Dowex 50 WX8 passage and phenylhydrazine derivatization. Following the addition of 2 N HCl to a final concentration of 285 mM, a 30-µl aliquot (13,000 dpm) of the assay mix was diluted to 1 ml with H2O and applied to a 1-ml column of Dowex 50 WX8 (H+ form), equilibrated with 5 mM HCl. The 14C content of the total eluate, including a 1.5-ml wash with 5 mM HCl, was determined and valued as pyruvate. A further 30-µl aliquot was mixed with 10 µmol of unlabeled pyruvate; after the addition of HClO4 (final concentration 75 mM), 50 µmol of phenylhydrazine were added to give a final volume of 1 ml. The precipitated pyruvate phenylhydrazone was collected, and its specific radioactivity was determined. Each nmol of [14C]pyruvate (in the sample) raised the value obtained by 560 dpm/µmol.
Measurement of the Glutathione Content of Synechocystis CellsAbout 50 mg (wet weight) of freshly grown
Synechocystis cells were suspended in 0.5 ml of 0.1 M HClO4 containing 10 mM EDTA and
disrupted by vigorous shaking (7 min) with 500 mg of glass beads
(125-200 µm). The suspension was neutralized with 0.1 M KOH and centrifuged. Glutathione contained in the supernatant was
determined using the kinetic glutathione reductase assay coupled to the
reduction of 5,5-dithiobis(2-nitrobenzoate) (27).
Native separations at pH 7-8 were performed using 8% resolving gels and the buffer system of Williams and Reisfeld (28). After electrophoresis (1.3 watts, 3 h) the gel was sliced in horizontal strips of 2 mm × 7 cm, and proteins were eluted with gentle shaking overnight in 2 ml of 50 mM Mops/NaOH, pH 7.6, containing 1 mM dithiothreitol and 20 µM EDTA.
SDS-PAGE according to Laemmli (24) was routinely performed with 12% acrylamide gels; proteins were visualized by Coomassie staining. The following molecular mass standards were used: bovine serum albumin (66 kDa), aldolase (40 kDa), carboanhydrase (30 kDa), and cytochrome c (12.5 kDa).
To perform the apo- to
holoferredoxin conversion assay, it was necessary to resolve the crude
extract of Synechocystis wild type cells into two fractions:
one containing specifically the abundant endogenous (holo)ferredoxin
and the other comprising all remaining proteins. The strategy pursued
is outlined in Fig. 1. The extract proteins were
transferred into a high salt buffer (0.43 M chloride) and
then applied to a DEAE-Sephadex A-25 column. The stringent buffer
conditions and the small pore size of the gel matrix (exclusion limit
30 kDa) permitted essentially all proteins to pass through the column
(Table I, Fig. 2). However, a very small
amount of protein was retained, namely all of the ferredoxin and a few
other polypeptides that could be detected by SDS-PAGE after elution
from the column with 0.63 M chloride (Fig. 2, lane
C). The ferredoxin in this fraction was purified to homogeneity
and converted to the apo- form via acidic precipitation. This
preparation was used for studying holoferredoxin formation mediated by
deferredoxinized extract.
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Apo- to holoferredoxin conversion was analyzed routinely by native PAGE
(Fig. 3) making use of the dramatic increase in
electrophoretic mobility of ferredoxin upon acquisition of its
[2Fe-2S] cluster. Deferredoxinized extract (up to 150 µg of
protein/lane) contained no other protein that could have interfered by
migrating in a similar position (Fig. 3, lane 0). This
method also displayed the redox status of residual apoprotein; the
intact species, with its 6 cysteinyl residues, which was the only one
that could be reconstituted, had the lowest mobility compared with the
oxidized species, containing internal disulfide bonds (Fig. 3).
The assay composition was optimized for holoferredoxin formation with deferredoxinized Synechocystis extract as enzyme source. Basic features of the system were the absolute requirement for extract proteins and free ferrous ion for conversion to occur. All assays were performed under argon and with glutathione present as reductant to protect apoferredoxin from any oxidation. The addition of any further sulfur compound (cysteine, sulfide, mercaptopyruvate, coenzyme A, thiosulfate; see "Experimental Procedures") was not needed. However, later in purification L-cysteine was required (Table I), and sulfide (favored initially because of the findings reported in Ref. 15) as well as the other sulfur compounds tested was ineffective. With sulfide-supplemented assays, the order of the additions was critical; our standard assay protocol, with ferrous ion added last, did not result in any spontaneous chemical reconstitution.
The addition of pyridoxal phosphate, ATP, or NADPH had no significant effect. Substitution of deferredoxinized extract by a corresponding amount of the resuspended membranous pellet did not support apo- to holoferredoxin conversion.
It should be noted that the occurrence of glutathione (reported for several cyanobacteria; Ref. 29) in Synechocystis cells was verified; per gram wet cells, 1.8 µmol of glutathione were found, corresponding to an intracellular concentration of about 4 mM.
Purification of C-DES from Deferredoxinized ExtractThe purification procedure is outlined in Fig. 1 and detailed in Table I; included in the table is the dependence of holoferredoxin formation, mediated by the various protein fractions, on the presence of L-cysteine.
An initial approach to estimate the number and size of proteins
involved in conversion was to perform a frontal analysis experiment; deferredoxinized extract was continuously applied to an Ultrogel AcA 44 column. Effluent fractions were assayed; holoferredoxin formation
started from a Kav value of 0.5. About the same
Kav value was determined from a zonal gel
filtration run (Fig. 4); only protein(s) with a
molecular mass of 40 ± 5 kDa should therefore be involved.
Further purification employed ion exchange chromatography on DE 52 cellulose. However, recovery of conversion activity was rather poor (about 10%) without an extra sulfur compound added. Therefore, again the assay mixture was supplemented with the various sulfur compounds (see above). Only L-cysteine was found to restore holoferredoxin formation (recovery 60-70%), whereby a concentration of 90 µM proved to be sufficient; pyridoxal phosphate stimulated slightly, whereas ATP did not exert any effect. These properties were retained after chromatography on Q-Sepharose FF. Thus, by using ion exchange-purified C-DES preparations, the standard assay conditions with 90 µM L-cysteine, 0.15 mM pyridoxal phosphate, and 0.4 mM Fe(NH4)2(SO4)2 could be established. Nevertheless, the active fractions were still rather impure.
Therefore, Q-Sepharose fractions were carried through native PAGE or chromatofocusing. Proteins eluted from slices of native gels were tested for conversion and analyzed by SDS-PAGE, which revealed a 43-kDa polypeptide contained in the active eluates (data not shown). Detection of the same single polypeptide correlated with conversion activity recovered after separation of Q-Sepharose fractions by chromatofocusing on a Mono P column (Fig. 2, lane D; Table I). Since both strategies independently identified the same 43-kDa polypeptide, this protein was considered as the active component.
Stoichiometry of the Apo- to Holoferredoxin Conversion Reaction Catalyzed by C-DESWith homogenous C-DES, L-cysteine was an absolute requirement for holoferredoxin formation (Table I), implicating cysteine as the relevant sulfur source for this enzyme. The omission of pyridoxal phosphate from the standard assay mixture approximately halved the reaction rate, suggesting that C-DES is pyridoxal phosphate-dependent. From the literature, the following product patterns are known to result from cysteine cleavage by pyridoxal phosphate-dependent enzymes: sulfide, pyruvate, and ammonia (31) or sulfur and alanine (8).
For every 2 mol of cysteine utilized, approximately 1 mol of holoferredoxin and 2 mol of pyruvate were produced (Fig. 5); residual cysteine was quantified after derivatization with vinylpyridine (data not shown), and ammonia was not measured. To obtain these quantitative data, holoferredoxin activity was determined by the cytochrome c reduction assay. The fate of cysteine was traced by employing the 14C-labeled compound. [14C]Pyruvate formed was distinguished from alanine by passage through Dowex 50 at pH 2 and further identified by coderivatization with unlabeled pyruvate using phenylhydrazine (Fig. 5). During the time course of the reaction, 37 nmol of ferredoxin were formed per nmol of C-DES employed (Fig. 5). Therefore, C-DES serves as catalyst that mediates the following overall reaction.
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Reactivity of C-DES with L-Cysteine and L-Cystine as Alternative Substrates
Having
established the stoichiometry of the complete reaction system (Reaction
1), we investigated whether cysteine was processed by C-DES in the
absence of apoferredoxin. In fact, pyruvate formation occurred at the
same rate as before (28 nmol min1 mg
1 with
90 µM cysteine; see Figs. 5 and 6). In
this context we also examined reactions with L-cystine
included instead of cysteine (glutathione omitted; Fig. 6).
Surprisingly, pyruvate formation occurred readily; with 45 µM cystine instead of 90 µM cysteine an
approximately 15-fold higher rate was observed. When the reaction was
run until substrate depletion, finally 2 mol of pyruvate were yielded
per mol of cystine employed. The sulfur-containing product, possibly
H2S2, was not characterized. In light of these
findings, C-DES should more properly be considered as a
cysteine/cystine C-S-lyase.
The question of whether cystine is also a better substrate in the holoferredoxin formation assay could not be investigated, since in the presence of excess glutathione (required for apoferredoxin protection) cystine is predominantly reduced to cysteine.
Structural Properties of C-DESThe molecular mass of C-DES was determined as 40 ± 5 kDa by gel filtration (Fig. 4) and as 43 ± 2 kDa by SDS-PAGE (Fig. 2). These data identified the enzyme as a monomer.
Stimulation of C-DES activity by the addition of pyridoxal phosphate in
context with the -elimination reaction observed with either cysteine
or cystine as substrate was taken as evidence for C-DES being a
pyridoxal phosphate-dependent enzyme whereby about 50% of
the molecules retain their coenzyme after purification. Given the small
amount of protein available, this assumption could not be directly
tested by chemical or spectroscopic analyses. However,
NaBH4 treatment of C-DES (10 mM
NaBH4 for 1 h at 30 °C, pH 7.5-7.2) resulted in
complete inactivation of the pyridoxal phosphate-loaded enzyme,
indicating susceptibility to Schiff base reduction (32).
NifS protein (producing sulfur and alanine from cysteine) has been
reported to be particularly sensitive to inactivation by various
thiol-alkylating agents including unsaturated amino acid derivatives
(8, 33). We therefore tested the susceptibility of C-DES toward
N-ethylmaleimide (0.1 mM), iodoacetamide (1 mM), L-allylglycine (5 mM), and
L-vinylglycine (5 mM). After an incubation period of 30 min at 30 °C, C-DES was separated from excess reagent and assayed for apo- to holoferredoxin conversion. Its activity proved
to be virtually unimpaired. A further experiment was conducted employing 5 mM L-propargylglycine, which is an
irreversible inhibitor of -cystathionase (34). Again C-DES activity
remained stable.
Based on a self-contained assay system derived from the cyanobacterium Synechocystis we pursued a systematic search for a protein capable of directing [2Fe-2S] cluster assembly of ferredoxin. Purification identified a monomeric, 43-kDa, pyridoxal phosphate containing lyase named C-DES, which specifically and efficiently used L-cysteine as precursor for cluster sulfide.
From our purification results, the cellular concentration of ferredoxin is estimated to be 60-120 µM, and the concentration of C-DES is estimated to be 0.15-1 µM. The resulting molar ratio of about 150:1 is of the same order as that employed in our assays (e.g. 120:1 for the experiment outlined in Fig. 5). Taking into account the doubling time of about 10 h under the growth conditions employed, Synechocystis cells synthesize approximately 1 mg of ferredoxin/30 g of wet cells/h. The endogenous ferredoxin [2Fe-2S] cluster-forming activity detected by our assay was more than sufficient to meet the cells' requirements (Table I).
In the initial purification stages, the addition of L-cysteine was not required for in vitro cluster formation. Obviously, some enzyme component(s) of the crude extract supplied the sulfur source, probably by utilizing glutathione. Free sulfide did not exert any effect in our system at any stage of purification, which is in contrast to the chloroplast system (15). A further difference is the lack of any requirement for ATP.
The product pattern resulting from L-cysteine is identical
to that of cysteine desulfhydrase of Salmonella typhimurium
(31). However, in contrast to C-DES the S. typhimurium
protein is tetrameric and is a more typical catabolic enzyme, with its
high specific activity (450 µmol min1 mg
1
with 2 mM cysteine as substrate; Ref. 35); furthermore,
this protein was reported not to attack cystine (31). From a functional point of view, isolation of a NifS-like protein might have been expected, especially when considering the widespread occurrence of
NifS-type sequences (8, 12), which has recently been extended to
Synechocystis: analysis of the total genome sequence of
strain PCC 6803 identified three homologues (36). Indeed, C-DES and NifS are both pyridoxal phosphate-containing enzymes, degrading L-cysteine with similar rates (89 nmol min
1
mg
1 for NifS with 0.5 mM cysteine (8), 28 nmol min
1 mg
1 for C-DES with 90 µM cysteine). Both catalyze cysteine cleavage in the
absence as well as in the presence of apoprotein; therefore, the
release of sulfur from cysteine and its insertion into the Fe-S cluster
do not seem to be necessarily coupled processes with these enzymes.
However, it should be emphasized that cysteine sulfur released by C-DES
was quantitatively found assembled in the [2Fe-2S] cluster of
ferredoxin (see Fig. 5), indicating interaction between C-DES and the
apoprotein substrate. Distinct from NifS, C-DES is monomeric, produces
pyruvate, ammonia, and sulfide instead of alanine and sulfur, and
cannot be inhibited by thiol-alkylating reagents.
A striking feature of C-DES is its reactivity with cystine, which
finally yields two equivalents of pyruvate. This reaction is known as a
secondary activity of certain - and
-cystathionases (37, 38),
which are all homooligomeric enzymes. Including cysteine in the
presence of excess glutathione in our reaction medium, we initially
thought of this reduced compound as the primary sulfur donor species
for [2Fe-2S] ferredoxin formation. By the increased reactivity of
C-DES with cystine, we became aware of the possibility that the
equilibrium concentration of cystine during conversion might be
crucial.
-Elimination starting with cystine should yield cysteine
persulfide as substrate-derived sulfane carrier compound. Whether this
persulfidic intermediate is formed during catalysis by C-DES and is
involved in sulfur transfer to apoferredoxin awaits further studies.
Work on such mechanistic problems will be greatly aided by the
availability of substantial amounts of purified C-DES. Cloning,
sequencing of the gene, and overexpression of C-DES in E. coli are in progress. Based on the sequence information, targeted
inactivation should reveal the physiological consequences of a
defective C-DES gene in Synechocystis. Using this approach
we hope to examine the in vivo role of C-DES in ferredoxin
Fe-S cluster formation.
We are grateful to Prof. Joachim Knappe for encouragement, stimulating discussions, and continuous support throughout this work. We thank Dr. A. F. Volker Wagner for productive discussions and Dr. Patricia Ellison (Max Planck Institut für Medizinische Forschung) for valuable comments on the manuscript.