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INTRODUCTION |
Continuing interest in structure and function of iron-sulfur
proteins (1) has recently been extended to the biosynthetic problem of
how the Fe-S cluster moieties are introduced into the proteins. Genetic
studies on the biosynthesis of the nitrogenase metalloclusters in
Azotobacter vinelandii revealed nifS as essential for assembly of the [4Fe-4S] cluster of the iron protein component (2). NifS protein was subsequently identified as a
L-cysteine desulfurase, yielding alanine and sulfur as
products (3). A specific cysteinyl residue of NifS serves as the
primary sulfur acceptor (4) and has been suggested as persulfidic
sulfur donor compound for nitrogenase apo-iron protein. A more general
role for the NifS-type chemistry might be indicated by the fact that genes homologous to nifS have been found in a variety of
organisms (3, 5) and that a NifS-like protein has been isolated from Escherichia coli by its capability to restore the activity
of damaged dihydroxy acid dehydratase (6), which is an oxidation sensitive [4Fe-4S] enzyme.
Our work, designed to identify proteins involved in ferredoxin
[2Fe-2S] cluster assembly with the cyanobacterium
Synechocystis PCC 6714 as a model led to the straightforward
purification of a L-cysteine/cystine C-S-lyase
(7). This lyase, named
C-DES,1 is distinguished from
NifS in being monomeric, insensitive to thiol-alkylating reagents and
in producing pyruvate and sulfide from cysteine (instead of alanine and
sulfur). Remarkably, C-DES was isolated from Synechocystis
extract by use of a holoferredoxin formation assay (7) despite the
occurrence of 3 nifS-type sequences in the genome as proven
for the related strain Synechocystis PCC 6803 (8). With the
assay system employed, 1 mol of [2Fe-2S] ferredoxin was formed per 2 mol of cysteine utilized (Equation 1; Ref. 7).
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(Eq. 1)
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Cysteine cleavage could also be observed in the absence of
apoferredoxin which made obvious that
-elimination and cluster formation are not necessarily coupled processes. Investigation of the
lyase reaction per se revealed that C-DES strongly preferred L-cystine to L-cysteine (7).
-Elimination of
cystine should yield cysteine persulfide as an unstable,
substrate-derived S0 compound. For accumulation and
chemical characterization of the postulated persulfide molecule
substantial amounts of C-DES were required. We here report the cloning
and sequencing of the C-DES gene and the overproduction of the gene
product in E. coli. Using the recombinant enzyme we have
investigated the specificity of C-DES with respect to the cyst(e)ine
substrate. Formation of a substrate-derived persulfide was established
using desaminocystine (S-(2-carboxyethylthio)-L-cysteine). The
resultant 3-(disulfanyl)-propionic acid became stable by cyclization
and was identified as 1,2-dithiolan-3-one. Part of this work was
reported at the GBM Fall Meeting (Tübingen, Federal Republic of
Germany, Ref. 9).
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EXPERIMENTAL PROCEDURES |
Materials--
E. coli strain XL1Blue MRF' was from
Stratagene, strain PR745 was from New England Biolabs. Plasmid pUC19
and the pUC/M13 reverse sequencing primer were from
Boehringer-Mannheim. The oligonucleotide probe was synthesized by R. Frank (Zentrum für Molekulare Biologie, Heidelberg) and was
3'-end labeled by reaction with terminal transferase and
digoxigenin-ddUTP using the labeling kit from
Boehringer-Mannheim.
L-Cystine was from Serva,
L-cysteine·HCl·H2O from Fluka, and
S-ethylcysteine, S-methylcysteine,
L-djenkolic acid, and L-cystathionine were from Sigma. Other (bio)chemicals and enzymes were from
Boehringer-Mannheim, New England Biolabs, Sigma, or Aldrich.
Peptide Sequencing--
Various peptides were HPLC purified from
a tryptic digest of 200 pmol of C-DES from Synechocystis PCC
6714 and subjected to Edman degradation by R. Frank (Zentrum für
Molekulare Biologie, Heidelberg). A gas-phase sequenator and on-line
identification of the phenylthiohydantoins by HPLC was employed.
NH2-terminal sequencing of C-DES expressed in E. coli employed the chromatofocused fractions (see below).
DNA Methods and Construction of Expression Plasmid pSA16--
In
general, standard procedures as described by Sambrook et al.
(10) were used.
Starting with 50 µg of total Synechocystis DNA the 7.6-kbp
size fraction of a HindIII digest was ligated into pUC19.
After transformation of XL1Blue MRF' cells and growth on LB agar plates containing 80 µg/ml ampicillin and 10 µg/ml tetracycline, positive clones were identified on replicas using Hybond-N Nylon membranes (Amersham). Immunological detection of colonies hybridizing to the
digoxigenin-labeled oligonucleotide probe was performed following the
protocol provided by Boehringer-Mannheim.
Subcloning of a 4.5-kbp HindIII-BamHI fragment
was performed by filling in the termini followed by blunt-end ligation
into pUC19 cut with SmaI. Plasmids corresponding to both
orientations of the insert were obtained. They were used (after cutting
at the unique SphI and XbaI sites of pUC19) to
generate two subclone series by exonuclease III treatment. Partial DNA
sequences of these subclones were obtained by the Sanger method
employing the DNA sequencing kit (Sequenase version 2.0) and
[35S]dATP
S purchased from U. S. Biochemical
Corp./Amersham.
The expression plasmid pSA16 was obtained by insertion of the 1.6 kbp
SspI-AvaI fragment comprising the C-DES open
reading frame plus 142 base pairs of the 5'-flanking region and 317 base pairs of the 3'-flanking region in pUC19 oriented such that C-DES gene transcription starting from the lac promoter
was permitted (see Fig. 1).
Bacterial Growth and Isolation of C-DES--
E. coli
PR745/pSA16 cells were grown in LB medium plus 80 µg/ml ampicillin,
10 µg/ml tetracycline, and 10 µg/ml kanamycin with aeration for
16 h at 37 °C. The purification procedure described for
Synechocystis cells as starting material was used (7) but simplified as follows. The DEAE-Sephadex A-25 passage of the crude extract was replaced by precipitation of the nucleic acids using Polymin G-35 (BASF); chromatography on DE52 cellulose could be dispensed. Starting with 10 g of wet cells 64 mg of C-DES with a
purity of
75% were obtained before the final chromatofocusing step,
which partially resolved two C-DES species of about equal purity
(
95% by SDS-PAGE analysis) and specific activity (7 units/mg when
assayed for L-cystine lyase); together they amounted about 22 mg of C-DES. Protein determination of purified C-DES was by UV
absorption, using A280 (1%) = 14.5.
Coupled Optical L-Cystine Lyase Assay--
Cystine
lyase was measured in 50 mM Mops/KOH, pH 7.3, at 30 °C
by a coupled optical assay using 0.15 µmol of PLP, 0.25 µmol of
NADH, 10 µg of L-lactate dehydrogenase (500 units/mg),
and 3-20 µg of C-DES in 1 ml final volume. The reaction was
initiated by addition of 0.2 µmol of L-cystine. One unit
was defined as the amount catalyzing the formation of 1 µmol of
pyruvate/min.
Where appropriate substrates other than cystine were used to initiate
the reaction and the substrate amounts were varied as required. Lyase
assays with L-cysteine as substrate were performed with 15 nmol of PLP; with this amount the background absorbance change which
was observed after addition of L-cysteine to control samples without C-DES was avoided. Activity of C-DES with cystine as
substrate was not changed with this condition.
Chemical Synthesis of Desaminocystine, Decarboxycystine, and
Meso-Cystine--
Based on the synthetic strategy described in Ref. 11
the activated cysteine derivative
S-(2,4-dinitrophenylthio)-L-cysteine was reacted
with equimolar amounts of silver acetate and the appropriate thiol
component. The detailed procedure is given below for the synthesis of
desaminocystine using mercaptopropionic acid; essentially the same
conditions were used for cysteamine and D-cysteine.
L-Cysteine·HCl·H2O (1.56 g) was colloidly
dissolved in acetone (30 ml) under argon;
2,4-dinitrophenylsulfenylchloride (2.21 g) in 60 ml of acetone was
added and the reaction was stirred for 15 min with precipitation of the
product
S-(2,4-dinitrophenylthio)-L-cysteine·HCl. The
pale yellow cristals (3 g) melted at 181 °C.
Silver acetate (735 mg) was stirred with dimethylformamide (30 ml)
under argon. Mercaptopropionic acid (385 µl) was added and during 30 min of stirring a voluminous white solid was formed. S-(2,4-Dinitrophenylthio)-L-cysteine·HCl (1.56 g) partially dissolved in 40 ml of dimethylformamide was added which
resulted in a red solution. After 2 h the solution was mixed with
130 ml of ice-cold water, stirred on ice for 30 min, and filtered. The
filtrate was dried and the residue was dissolved (after several
washings with dimethylformamide) in 10 ml of boiling water. The sample
was filtered hot. Upon cooling, product crystals developed (80 mg)
which melted at 190-192 °C with decomposition (12). MS and
1H NMR data (not shown) confirmed the expected
structure.
Yields were 14.2% for desaminocystine, 6.1% for
decarboxycystine (m.p. 176-178 °C with decomposition, Ref. 12), and
28.6% for meso-cystine (m.p. 200-220 °C with decomposition, Ref.
13).
Chemical Synthesis of 1,2-Dithiolan-3-one--
A method
described for preparation of arylsulfane compounds (14) was adopted.
Cl2(g) was bubbled through 65 ml of CS2 at
5 °C to yield a yellow solution. 3-Mercaptopropionic acid (2.19 ml
in 10 ml of CS2) was dropwise added such that the solution remained yellowish. The reaction sample was concentrated to 20 ml and
stored at
20 °C overnight. A dense and yellowish oil (2.2 ml)
separated which was diluted with 10 ml of CS2 and added
dropwise to 10 ml of liquified H2S at
78 °C. After
stirring for 1 h with constant addition of H2S, the
H2S stream was stopped and the reaction sample was allowed
to attain room temperature. A white solid had precipitated which was
recovered by centrifugation, washed with hexane, and dried (yield
0.5 g).
The material was fractionated by preparative C18 reversed-phase HPLC on
a Waters instrument using a Hibar RT 250-10 column (Merck) and an
acetonitrile gradient in 0.1% trifluoroacetic acid (6 ml/min, 0% to
70% acetonitrile in 30 min); 3 main products (1-3, see
Scheme I) were detected via their 210-nm absorbance. These products
(130 mg of 1, 65 mg of 2 and 110 mg of
3) were recovered by ether extraction and were chemically analyzed. The data and structure assignments were as follows. 1,2-Dithiolan-3-one (1), m/z 120 (M+), IR absorption (KBr) 1688 cm
1 (C=O),
UV-VIS
max(in 0.1% trifluoroacetic acid) at 206 and 295 nm, uncharged at pH 5, m.p. 98-100 °C;
3,3'-tetrathiobispropionic acid (2), m/z
274 (M+), IR 1697 cm
1 (C=O), anionic at pH 5;
3,3'-trithiobispropionic acid (3), m/z 242 (M+), IR 1698 cm
1 (C=O), anionic at pH 5.
3,3'-Dithiobispropionic acid was identified as a further
component (m/z 210 (M+), IR 1699 cm
1 (C=O), anionic at pH 5) probably because oxidation of
mercaptopropionic acid by Cl2 was not complete (see Scheme
I).
MS was performed on a VG ZAB-2F or a Jeol JMS-700 instrument (electron
impact ionization). GC-MS analyses employed a Hewlett-Packard instrument type 5890 equipped with a HP-5 column (30 m) programmed from
40 °C (2 min) to 240 °C at 10 °C/min. The mass spectra were obtained at 70 eV. The molecular ion of 1 could be observed in GC-MS analyses only (splitless injection of
100 nmol) since the
compound decomposed upon direct MS analyses (presumably due to the
water content of the preparation). Extinction coefficients (mM
1 cm
1) in 0.1%
trifluoroacetic acid were determined as follows:
206(1) = 6.2;
210(1) = 5.6,
295(1)
0.9;
210(2) = 8.6;
210(3) = 9.4.
Product Analysis of the C-DES Reaction with
Desaminocystine--
Reactions were performed at 5 °C under argon
for 2-25 min. The reaction mixtures (0.75 ml) consisted of 0.1 to 1 mM desaminocystine in 0.1 M Mes/NaOH, pH 6, containing 0.25 mM PLP; the reaction was started by
addition of 0.02 to 0.2 mg of C-DES and terminated by addition of 13 µl of 2 N HCl followed by 1 ml of ether. Extraction was
performed for 20 min at 0 °C under argon with vigorous stirring. After centrifugation and phase separation the aqueous phase was brought
to 75 mM HClO4, neutralized with KOH, and
analyzed for its pyruvate content by use of the lactate dehydrogenase
reaction; the residue of the ether phase was dissolved in 0.5 ml of
0.1% trifluoroacetic acid and analyzed by reversed-phase HPLC (ET
250/8/4 Nucleosil 5C18 column from Macherey-Nagel, 1 ml/min, 0.1%
trifluoroacetic acid/acetonitrile gradient; see Fig. 4). Compounds were
identified by co-chromatography with chemically synthesized reference
samples and quantified via their absorbance at 210 nm. The assignment of enzymatically formed 1,2-dithiolan-3-one was verified by GC-MS analysis as described for the chemically synthesized sample.
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RESULTS |
Cloning and DNA Sequencing of the C-DES Gene--
To clone the
C-DES gene from Synechocystis PCC 6714 we attempted to gain
some sequence data for the purified protein. NH2-terminal sequencing failed, suggesting a blocked terminus, but some information could be obtained from tryptic peptides (see Fig. 2). Based on the
peptide sequence Glu-Val-Asp-Tyr-Tyr-Ala- a 64-fold degenerate pool of
oligonucleotides (17-mers) was synthesized. This probe was labeled with
digoxigenin and used to analyze Southern transfers of restricted total
Synechocystis DNA. One signal for each digest was detected
and the 7.6-kbp HindIII fragment was selected for the
cloning experiments. After ligation into the HindIII site of
pUC19 and transformation into E. coli XL1Blue MRF', about
3000 colonies were screened to obtain two positive clones. Southern analysis of the restricted plasmids showed that they contained identical HindIII fragments. The hybridization site of the
oligonucleotide probe was mapped to a 1.1-kbp EcoRI
subfragment internal to a 4.5-kbp HindIII-BamHI
fragment (Fig. 1). This 4.5-kbp segment was inserted in both orientations into pUC19. Using the resulting plasmids two subclone series were generated by exonuclease III treatment (see Fig. 1) from which the nucleotide sequence of the C-DES
gene was obtained. An open reading frame comprising 393 amino acid
residues was identified (Fig. 2), which
harbored the sequence segments obtained from tryptic peptides and
matched the molecular mass expected (43 kDa).

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Fig. 1.
Restriction map of cloned Synechocystis
PCC 6714 DNA harboring the C-DES structural gene and construction
of expression plasmid pSA16. Only relevant restriction sites are
shown. Sequenced DNA segments covered by exonuclease III-generated
subclones are indicated by the two bars above the
shown HindIII-BamHI fragment. Structural genes
and direction of transcription are indicated by arrows. The
vector portion of pSA16 is shown as thinner line and
includes the ampicillin (Ap) resistance gene.
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Fig. 2.
Deduced amino acid sequence of C-DES and
sequence alignments with the slr2143 gene product of
Synechocystis PCC 6803 (accession number D90908),
isopenicillin N-epimerase of Streptomyces
clavuligerus (CefD, accession number M32324), and one of the
E. coli NifS homologues (NifS, accession number
D90811). Known peptide segments of the C-DES sequence are
underlined. The line is dotted where
the amino acid residue could not be unambigously identified.
NH2 termini found with C-DES overexpressed in E. coli were either as deduced (but with the initiator methionine
lacking; minor species) or
Met5-Asn6-Leu7- (major species, see
text). Sequence similarities were searched with the FASTA program (15)
and aligned with the CLUSTAL program (29). The PLP binding motif is
marked with asterisks. Gaps are shown with dashes.
Shaded residues are conserved among at least 3 of the sequences
displayed. For isopenicillin N-epimerases and NifS proteins
the representative with the best score is displayed although no
biochemical data are available for the NifS homologue cited. The
cysteinyl residue corresponding to the covalent catalytic residue for
this NifS homologue (5) is indicated by an arrow.
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A search through the data bases employing a FASTA routine (15) revealed
only limited homologies except for the corresponding gene product
(slr2143) of strain Synechocystis PCC 6803 (8) which
displayed 92% identity (Fig. 2). Next to the slr2143 protein the
closest relatives detected were isopenicillin N-epimerases (gene name cefD; about 30% identity, 55% similarity) and
NifS proteins (about 25% identity, 50% similarity). In fact the C-DES gene of Synechocystis PCC 6803 had been named
cefD (8) because of this similarity, which places C-DES into
the NifS family of proteins using the relaxed definition introduced by
Ouzounis and Sander (16). However, C-DES does not show the consensus
pattern of NifS proteins in strict terms (3) because of an extra
residue inserted in the PLP binding motif and lack of a cysteinyl
residue located in the equivalent position to the covalent catalytic
residue (Fig. 2).
Heterologous Overexpression of C-DES in E. coli--
To express
C-DES in E. coli, the 1.6-kbp
SspI-AvaI fragment was cloned downstream of the
lac promoter of pUC19 in the appropriate orientation to
yield plasmid pSA16 (Fig. 1). Soluble C-DES was found to make up about
5% of the extract proteins obtained from pSA16 transformed E. coli PR745 cells; its cystine lyase activity (liberating pyruvate)
was conveniently measured by a coupled optical assay using the lactate
dehydrogenase reaction as indicator. Starting with 10 g of wet
cells about 22 mg of pure C-DES were obtained which displayed UV-VIS
absorption characteristics consistent with the expected content of PLP
(A420(1%) = 1.4; Ref. 17). However, the
preparation consisted of two closely related species which could be
partially separated by chromatofocusing.
The major species (higher pI, about 80%) showed the
NH2-terminal sequence Met-Asn-Leu-Ile-Pro-, which implies
usage of the GTG codon of Val-5 in the open reading frame as start
codon. Preceded by 5'-CT and followed by A-3', this GTG codon fits the
known preference for initiation in Synechocystis at sites
with the consensus 5'-YY(initation codon)R-3' (18). This site is most
probably also used in Synechocystis cells. The minor protein
species (about 20%) showed the NH2-terminal sequence
Ala-Asp-Pro-Val- which implies usage of the ATG codon of
Met1 in the open reading frame as start codon; the mature
protein lacks the initiator methionine.
Using a chromatofocused preparation of the major species the
A280 ratio of C-DES without or with 6 M guanidinium chloride was found to be 1.05. Based on a
molecular mass of 42,772 Da and a calculated
A280(1%) of 13.8 for a 6 M
guanidinium chloride solution (19) this gives an
A280(1%) value of 14.5 for the native C-DES (major species).
Both enzyme species were found to be equally active whether assayed for
cystine lyase in the coupled optical assay (7 units/mg) or
holoferredoxin formation (8 mg of holoferredoxin h
1
mg
1). These data are congruent with the specific activity
determined for the Synechocystis isolate (7).
-Elimination Reaction with L-Cystine and Related
Compounds--
To investigate the substrate requirements of C-DES,
several compounds related to L-cystine were examined for
pyruvate formation in the coupled optical lyase assay (Table
I). Besides L-cystine, L-djenkolic acid (which also harbors two
L-cysteinyl-moieties) finally yielded 2 mol of pyruvate per
mol of substrate. This stoichiometry indicates further enzymatic (and
possibly also spontaneous) reactions of the primary elimination
products. With our attention directed to the initial reaction of C-DES
with L-cystine, asymmetric derivatives thereof were
considered as useful. Desaminocystine, decarboxycystine, and
meso-cystine were all synthesized by reaction of the appropriate thiol
with S-(2,4-dinitrophenylthio)-L-cysteine and
were found to be readily accepted as substrates yielding 1 equivalent
of pyruvate (Table I).
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Table I
Substrate properties of L-cystine and related compounds
Data refer to the total amount or the initial rate of pyruvate
formation observed with the lactate dehydrogenase indicator reaction in
the coupled optical lyase assay of C-DES (see "Experimental
Procedures").
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Besides djenkolic acid, further non-disulfidic compounds structurally
related to L-cystine were examined (Table I). This revealed
that C-DES activity is confined to compounds with at least one
L-cysteinyl moiety and various S-substituents
but not necessarily disulfides. However, L-cysteine was by
far the least efficient of the compounds tested; with
L-cystathionine and S-(m)ethylcysteine moderate
efficiency was observed which argues against identity of C-DES with
cystathionase or S-alkylcysteine-lyase.
Identification of a Persulfide Product from Reaction of C-DES with
Desaminocystine--
Toward identification of a persulfide product
desaminocystine with its favorable kinetic properties (Table I) was
selected among the substrates tested. The stoichiometric yield of
1 equivalent of pyruvate suggested formation of
3-(disulfanyl)-propionic acid which was supposed to cyclize
spontaneously affording 1,2-dithiolan-3-one (1; see Scheme
I and Fig.
3) as a stable derivative of the linear
persulfide.

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Fig. 3.
Scheme for analysis of the C-DES reaction
with desaminocystine. The reaction mixture (0.75 ml) which
contained 0.2 mg of C-DES in 0.1 M Mes/NaOH, pH 6, 0.25 mM PLP was incubated with (initially) 0.1 mM
desaminocystine for 8 min at 5 °C under argon. Subsequent additions
of HCl and ether (for details see "Experimental Procedures")
terminated the reaction and after extraction both phases were analyzed
for their content. Each nmol of 2 and 3 probably
resulted from reaction between two persulfide molecules (see Scheme
I).
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Since only small amounts of products were available from enzymatic
reactions their identification was deduced by chromatographic analyses
referring to chemically synthesized reference compounds. Synthesis of
3-(disulfanyl)-propionic acid from mercaptopropionic acid, as outlined
in Scheme I, furnished a crude CS2-insoluble product which
was fractionated by C18 reversed-phase HPLC and found to contain
principally three components depicted in Scheme I: the dithiolanone
1, the tetrathiobis propionic acid 2, and the
trithiobis propionic acid 3. Formation of byproducts
2 and 3 can be explained by bimolecular reactions
of the persulfide molecules (see Scheme I). These latter reactions
should be largely suppressed by low temperature and dilution of the
reactants in the enzymatic reactions.
The enzymatic conversion of desaminocystine was carried out at 5 °C
and pH 6. This pH value was chosen to suppress disproportionation of
the asymmetric disulfide substrate and to favor the cyclization reaction of the presumed linear persulfide product; it compromised between the stabilizing effects mentioned and the lyase activity of
C-DES which is optimal at pH 8 and decreases to 6% at pH 6.0 or 9.6. The reactions were stopped by additions of HCl and ether. After
thorough mixing, the phases were separated and their content analyzed
(see Fig. 3). Remarkably, the sulfur-containing products (which were
routinely detected and quantified by HPLC analysis) were found to be
identical with chemically synthesized compounds 1 to
3 (Scheme I and Fig. 3) despite the entirely different
reaction conditions. To verify the identity of the enzymatically formed
key compound 1 with 1,2-dithiolan-3-one a HPLC-purified sample of 105 nmol was analyzed by GC-MS. It proved to be
indistinguishable from the chemically synthesized reference compound
affording the molecular ion m/z 120 and fragments
m/z 64 [S-S]+ and m/z 55 [O=C=CH-CH2]+ for the equivalent GC-fraction
at 8.2 min of the temperature program.
An approximately constant fractional amount (see below) of dithiolanone
1 was found in five experiments with the initial concentration of desaminocystine being varied from 0.1 to 1 mM, the concentration of C-DES varied from 0.03 to 0.27 mg/ml and with reaction times from 2 to 10 min. (Conversion of
desaminocystine was between 11 and 100% complete for these
experiments.) However, shift of the reaction temperature to 25 °C
with otherwise identical conditions lowered the fractional amount of
1 by a factor of 1.6. Control reactions without C-DES did
not yield any product.
A typical experiment performed at 5 °C is presented in Figs. 3 and
4. With this reaction an amount of
pyruvate corresponding to complete conversion of desaminocystine was
found in the aqueous phase (Fig. 3). HPLC analysis (Fig. 4) of the
ether phase revealed that 71% of the sulfur-containing products
(referring to the number of moles formed) could be recovered as
dithiolanone 1 (Fig. 3). The total amount of sulfur
represented by products 1 to 3 slightly exceeded
the amount contained initially in the substrate. This must be due to
some insufficiency of our quantification protocol but nevertheless
excludes the possibility that relevant amounts of further products had
formed.

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Fig. 4.
Separation of the dithiolanone 1 and
bis-propionic acids 2 and 3 by reversed-phase HPLC. The ether
extract of enzymatic reactions was dried, redissolved in 0.1%
trifluoroacetic acid, and chromatographed on a Nucleosil 5C18 column
(see "Experimental Procedures"). The acetonitrile gradient is
indicated by a broken line. The chromatogram displayed
refers to the experiment outlined in Fig. 3; 40% of the ether extract
was applied to the column.
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To investigate the stoichiometry of pyruvate formation
versus formation of products 1 to 3, a
kinetic experiment was performed (Table
II). The ratio of dithiolanone
1 to pyruvate was maximal for the early samples (Table II);
this indicates the direct formation of 1,2-dithiolan-3-one from the
linear persulfide 3-(disulfanyl)-propionic acid. Therefore, a
substrate-derived persulfide is generated as an obligate intermediate
along the reaction pathway of C-DES.
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Table II
Time course of product formation from desaminocystine
The reaction (4.5 ml) was performed at pH 6/5 °C with (initially)
0.1 mM desaminocystine and 0.06 mg/ml C-DES; samples (0.75 ml each) were processed after the time periods indicated and analyzed
as specified under "Experimental Procedures." The designations
1, 2, and 3 refer to 1,2-dithiolan-3-one,
3,3'-tetrathiobis propionic acid, and 3,3'-trithiobis propionic acid as
in text.
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DISCUSSION |
Using the cystine analogue desaminocystine
(S-(2-carboxyethylthio)-L-cysteine) we now
established that a persulfidic product, 3-(disulfanyl)-propionic acid,
is stoichiometrically formed by C-DES. This linear persulfide was
isolated as 1,2-dithiolan-3-one (1) exploiting cyclization
as a novel and efficient trapping reaction. Cyclization was presumably
guided by the low pH value and reaction temperature employed; these
factors are known to favor the lactonization of mercaptobutyric acid
(20) which resembles dithiolanone formation from
3-(disulfanyl)-propionic acid. The increased acidity of persulfides
when compared with thiols (21) should further facilitate cyclization at
low pH values.
The reaction with desaminocystine was studied as a model for
-elimination of cystine which should yield cysteine persulfide but
is complicated by further reactions of this suggested intermediate resulting in formation of a second molecule of pyruvate. With the
striking preference of C-DES for cystine rather than cysteine, a role
for cysteine persulfide in the overall process of Fe-S cluster
formation becomes feasible which would assign cystine to the true
sulfur-delivering substrate. In this context it is necessary to return
to the original milieu of our holoferredoxin formation assay (7) with
its predominantly reducing conditions. Excess glutathione was employed
as thiol reagent required for apoferredoxin protection and guaranteed
preponderance of cysteine instead of cystine. Apparent reaction of
C-DES with cysteine may be made possible by the presence of catalytic
amounts of cystine (Fig. 5, a + b; see also Ref. 22). The stoichiometric transfer of
sulfur to the apoprotein found with the complete system should use one
of the branches suggested in Fig. 5 (reactions c or
b' + c'). A transient transfer of sulfane sulfur
to a C-DES cysteinyl residue may additionally be involved but must be
dispensable at least for the lyase reaction with non-disulfidic
substrates.

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Fig. 5.
Possible involvement of a cysteine persulfide
intermediate in the process of sulfur incorporation into Fe-S clusters
mediated by C-DES. Cystine is depicted as the sulfur donating
amino acid substrate being required in only catalytic amounts as
compared with cysteine. Reactions a + b describe
the reaction course in the absence of apoprotein which may be extended
by reaction c with apoprotein present if H2S
were the sulfur species for incorporation. Reactions a + b' + c' describe sulfur transfer to apoprotein on
the S0 level with cysteine persulfide as immediate donor.
The reductant required for step c' may again be cysteine,
glutathione, or any other suitable compound.
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