Evidence for Cysteine Persulfide as Reaction Product of l-Cyst(e)ine C-S-Lyase (C-DES) fromSynechocystis

The pyridoxal phosphate-dependent monomericl-cysteine/cystine C-S-lyase (C-DES), previously isolated from Synechocystis PCC 6714 by its capacity to direct [2Fe-2S] cluster assembly of ferredoxinin vitro (Leibrecht, I., and Kessler, D. (1997)J. Biol. Chem. 272, 10442–10447), has now been cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence of C-DES was found to be nearly identical (92% identity) to the open reading frame slr2143 ofSynechocystis PCC 6803 and showed a more distant relationship to the NifS family of proteins (about 27% identity). Recombinant C-DES displayed activities equal to the isolate fromSynechocystis in terms of the cyst(e)ine lyase reaction and holoferredoxin formation which recommended its use for functional and mechanistic studies. Investigation of the substrate spectrum for β-elimination found l-cysteine to be a poor substrate (k cat ≈ 0.15 s−1) in contrast tol-cystine (k cat = 36 s−1) and several related compounds. Of these compounds, desaminocystine (S-(carboxyethylthio)-l-cysteine) was used for C-DES-mediated persulfide generation. Stabilization of the linear persulfide 3-(disulfanyl)-propionic acid was achieved by cyclization as a novel intramolecular trapping reaction; this yielded 1,2-dithiolan-3-one which was isolated and identified by chemical analyses.

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 S 0 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 (Tü bingen, Federal Republic of Germany, Ref. 9).

EXPERIMENTAL PROCEDURES
Materials-E. coli strain XL1Blue MRFЈ 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 fü r Molekulare Biologie, Heidelberg) and was 3Ј-end labeled by reaction with terminal transferase and digoxigenin-ddUTP using the labeling kit from Boehringer-Mannheim. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF061964.
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 fü r Molekulare Biologie, Heidelberg). A gas-phase sequenator and on-line identification of the phenylthiohydantoins by HPLC was employed. NH 2 -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 g of total Synechocystis DNA the 7.6-kbp size fraction of a HindIII digest was ligated into pUC19. After transformation of XL1Blue MRFЈ cells and growth on LB agar plates containing 80 g/ml ampicillin and 10 g/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 [ 35 S]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 5Ј-flanking region and 317 base pairs of the 3Ј-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 g/ml ampicillin, 10 g/ml tetracycline, and 10 g/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 Ն75% were obtained before the final chromatofocusing step, which partially resolved two C-DES species of about equal purity (Ն95% 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 A 280 (1%) ϭ 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 mol of PLP, 0.25 mol of NADH, 10 g of L-lactate dehydrogenase (500 units/mg), and 3-20 g of C-DES in 1 ml final volume. The reaction was initiated by addition of 0.2 mol of L-cystine. One unit was defined as the amount catalyzing the formation of 1 mol 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.
Silver acetate (735 mg) was stirred with dimethylformamide (30 ml) under argon. Mercaptopropionic acid (385 l) was added and during 30 min of stirring a voluminous white solid was formed. S-(2,4-Dinitrophe-nylthio)-L-cysteine⅐HCl (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 Chemical Synthesis of 1,2-Dithiolan-3-one-A method described for preparation of arylsulfane compounds (14) was adopted. Cl 2 (g) was bubbled through 65 ml of CS 2 at Ϫ5°C to yield a yellow solution. 3-Mercaptopropionic acid (2.19 ml in 10 ml of CS 2 ) was dropwise added such that the solution remained yellowish. The reaction sample was concentrated to 20 ml and stored at Ϫ20°C overnight. A dense and yellowish oil (2.2 ml) separated which was diluted with 10 ml of CS 2 and added dropwise to 10 ml of liquified H 2 S at Ϫ78°C. After stirring for 1 h with constant addition of H 2 S, the H 2 S 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) 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 Ն100 nmol) since the compound decomposed upon direct MS analyses (presumably due to the water content of the preparation). Extinction coefficients (mM Ϫ1 cm Ϫ1 ) in 0.1% trifluoroacetic acid were determined as follows: 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 l 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 HClO 4 , 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.

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. NH 2 -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 64fold 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 MRFЈ, about 3000 colonies were screened to obtain two positive clones. Southern analysis of the restricted plasmids showed that they contained identical Hin-dIII 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).
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. The line is dotted where the amino acid residue could not be unambigously identified. NH 2 termini found with C-DES overexpressed in E. coli were either as deduced (but with the initiator methionine lacking; minor species) or Met 5 -Asn 6 -Leu 7 -(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. two closely related species which could be partially separated by chromatofocusing.
The major species (higher pI, about 80%) showed the NH 2terminal 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 5Ј-CT and followed by A-3Ј, this GTG codon fits the known preference for initiation in Synechocystis at sites with the consensus 5Ј-YY(initation codon)R-3Ј (18). This site is most probably also used in Synechocystis cells. The minor protein species (about 20%) showed the NH 2 -terminal sequence Ala-Asp-Pro-Val-which implies usage of the ATG codon of Met 1 in the open reading frame as start codon; the mature protein lacks the initiator methionine.
Using a chromatofocused preparation of the major species the A 280 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 A 280 (1%) of 13.8 for a 6 M guanidinium chloride solution (19) this gives an A 280 (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 Ϫ1 mg Ϫ1 ). 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 Lcysteinyl-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).
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.
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 CS 2 -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] ϩ and m/z 55 [OϭCϭCH-CH 2 ] ϩ 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.
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.  Fig. 3; 40% of the ether extract was applied to the column.

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 1,2dithiolan-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 ϩ 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 bЈ ϩ cЈ). 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.
It should be noted that cysteine persulfide was formerly recognized as a product of the ␥-cystathionase reaction with L-cystine, a secondary substrate. Reactions run in the presence of iodoacetate produced some S-(carboxymethylthio)-L-cysteine (22).
Through generation of cysteine persulfide C-DES shares the occurrence of a S 0 compound along its reaction pathway with rhodanese, mercaptopyruvate sulfur transferase, and NifS, proteins implicated in Fe-S cluster synthesis (23,24,3). This may indicate that persulfidic compounds are the physiologically relevant sulfur donors. For rhodanese and NifS a protein persulfide is formed during catalysis which is located at a catalytically essential cysteinyl residue (Cys-247 for bovine mitochondrial rhodanese, Ref. 25; Cys-325 for NifS from A. vinelandii, Ref. 4). However, the protein persulfide equilibrates with thiols contained in the medium to yield the persulfide derivatives of the thiols (6,26) and the regenerated sulfhydryl form of the enzyme. For mercaptopyruvate sulfur transferase a close relationship to rhodanese has recently been suggested from sequencing and mutagenesis studies (27).
All the experiments reported were performed using overexpressed C-DES isolated from E. coli. Recombinant C-DES proved to be indistinguishable from the original Synechocystis isolate with respect to activity. However, a minor difference was noted upon NH 2 -terminal sequencing. Whereas the NH 2 terminus of C-DES isolated from Synechocystis appeared to be blocked, two species with different NH 2 termini were found for the enzyme expressed in E. coli. Presumably lack of a typical Shine-Dalgarno sequence upstream of the C-DES gene enables the translation machinery of E. coli to use alternative start sites. Overexpression did not impair the growth rate of the host cells, thus excluding any toxic effect.
Sequence comparisons revealed that C-DES is distantly related to the NifS family of proteins; best matches were obtained for isopenicillin N-epimerases and NifS proteins in the narrow sense. Although the C-DES homologue in Synechocystis PCC 6803 has been named CefD, an isopenicillin N-epimerase function for C-DES is quite unlikely. Evidence for penicillin synthesis in cyanobacteria is lacking and a homologue for isopenicillin synthase has not been detected in the genome of Synechocystis PCC 6803 (8). The highest score for the NifS-like proteins contained in the data bases was obtained by comparison with a putative E. coli NifS protein (Fig. 2). This protein has been described as similar to the NifS-type cysteine sulfinate desulfinase of E. coli which yields alanine and sulfite as products (5). Interestingly, no cysteinyl residue of cysteine sulfinate desulfinase was found to be essential for activity by mutagenic replacement against alanine (5). Different members of the NifS sequence family evidently make use of different types of PLP chemistry.
Using recombinant C-DES it should be possible to further follow the path of sulfur from cysteine persulfide to apoferredoxin by analysis of sulfur-containing (protein-)intermediates. This might reveal a persulfidic derivative of apoferredoxin (see Fig. 5) as already discussed for apoproteins in general (6) and perhaps exemplified by the Y13C mutant of ferredoxin I of A. vinelandii (28), where the cysteinyl persulfide might represent a dead end or side product. By mutagenesis of C-DES, residues 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, 3,3Ј-tetrathiobis propionic acid, and 3,3Јtrithiobis propionic acid as in text.  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 ϩ b describe the reaction course in the absence of apoprotein which may be extended by reaction c with apoprotein present if H 2 S were the sulfur species for incorporation. Reactions a ϩ bЈ ϩ cЈ describe sulfur transfer to apoprotein on the S 0 level with cysteine persulfide as immediate donor. The reductant required for step cЈ may again be cysteine, glutathione, or any other suitable compound. essential for catalysis of the ␤-elimination reaction and possibly for protein-protein interaction could be identified. Along these lines we hope to gain comprehension of the perfect stoichiometry of sulfur incorporation into apoferredoxin by the catalytic action of C-DES.