Dinucleotide Spore Photoproduct, a Minimal Substrate of the DNA Repair Spore Photoproduct Lyase Enzyme from Bacillus subtilis
2006; Elsevier BV; Volume: 281; Issue: 37 Linguagem: Inglês
10.1074/jbc.m602297200
ISSN1083-351X
AutoresAlexia Chandor, Olivier Berteau, Thierry Douki, Didier Gasparutto, Yannis Sanakis, Sandrine Ollagnier de Choudens, Mohamed Atta, Marc Fontecave,
Tópico(s)Protist diversity and phylogeny
ResumoThe overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3′–5′)-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3′–5′)-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme. The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3′–5′)-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3′–5′)-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme. The DNA of all organisms is subject to modifications upon exposure to a wide variety of chemical and physical agents. Among them, solar ultraviolet radiation is known to induce dimerization reactions between adjacent pyrimidines (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4224) Google Scholar). In the vast majority of living systems, the resulting photoproducts are cyclobutane pyrimidine dimers (CPDs) 5The abbreviations used are: CPD, cyclobutane pyrimidine dimer; AdoMet, S-adenosylmethionine; [Fe-S], iron-sulfur cluster; DTT, dithiothreitol; TpT, thymidilyl-(3′–5′)-thymidine; SPTpT, spore photoproduct of TpT; SP lyase, spore photoproduct lyase; HPLC, high pressure liquid chromatography; MS/MS, tandem mass spectrometry; DPA, dipicolinic acid.5The abbreviations used are: CPD, cyclobutane pyrimidine dimer; AdoMet, S-adenosylmethionine; [Fe-S], iron-sulfur cluster; DTT, dithiothreitol; TpT, thymidilyl-(3′–5′)-thymidine; SPTpT, spore photoproduct of TpT; SP lyase, spore photoproduct lyase; HPLC, high pressure liquid chromatography; MS/MS, tandem mass spectrometry; DPA, dipicolinic acid. and pyrimidine (6-4) pyrimidone photoproducts (Scheme 1A) that can be generated at any of the four bipyrimidine doublets (TT, CT, TC and CC), although the yields of the lesions depend on the bases involved (2Ravanat J.L. Douki T. Cadet J. J. Photochem. Photobiol. B. Biol. 2001; 63: 88-102Crossref PubMed Scopus (700) Google Scholar). These lesions induce mutations and can be lethal because of blocking of the replication machinery. The photochemistry in bacterial spores is quite different. Indeed, in this dormant form produced by some bacteria such as Bacillus subtilis, the only photoproduct produced upon exposure to UV light corresponds to two thymines linked by the methyl group of one of the bases (3Varghese A.J. Biochem. Biophys. Res. Commun. 1970; 38: 484-490Crossref PubMed Scopus (120) Google Scholar, 4Donnellan Jr., J.E. Setlow R.B. Science. 1965; 149: 308-310Crossref PubMed Scopus (144) Google Scholar). The formation of this specific lesion, 5-thyminyl-5,6-dihydrothymine (spore photoproduct, SP) (Scheme 1A), is explained by specific features of the spores, including DNA conformation (A form), dehydration, the presence of dipicolinic acid in the core, and binding of small acid-soluble proteins to DNA (5Setlow B. Setlow P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 421-423Crossref PubMed Scopus (39) Google Scholar, 6Nicholson W.L. Setlow B. Setlow P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8288-8292Crossref PubMed Scopus (71) Google Scholar, 7Douki T. Setlow B. Setlow P. Photochem. Photobiol. 2005; 81: 163-169Crossref PubMed Scopus (49) Google Scholar, 8Douki T. Setlow B. Setlow P. Photochem. Photobiol. Sci. 2005; 4: 591-597Crossref PubMed Scopus (59) Google Scholar). The formation of SP as the unique DNA lesion in irradiated spores is proposed to account for their extreme resistance to UV radiation. Indeed, spores express a specific repair enzyme, the spore photoproduct lyase (SP lyase) that directly reverts SP to two unmodified thymines upon germination (9Setlow P. Annu. Rev. Microbiol. 1995; 49: 29-54Crossref PubMed Scopus (326) Google Scholar, 10Slieman T.A. Rebeil R. Nicholson W.L. J. Bacteriol. 2000; 182: 6412-6417Crossref PubMed Scopus (47) Google Scholar), much more efficiently than dimeric photoproducts are removed from other cell types by the classical nucleotide excision repair pathway. The specific photochemistry of DNA in spores combined with the action of SP lyase appears to be a major evolutionary advantage for spore-forming bacteria in resistance to UV radiation. In their N-terminal half, all SP lyase enzymes contain a strictly conserved amino acid sequence containing three cysteines CXXXCXXC that have been shown to be essential for activity by site-directed mutagenesis (11Fajardo-Cavazos P. Rebeil R. Nicholson W.L. Curr. Microbiol. 2005; 51: 331-335Crossref PubMed Scopus (30) Google Scholar). These cysteines have therefore been proposed to provide protein ligands for a catalytically essential [4Fe-4S]+2/+1 cluster (11Fajardo-Cavazos P. Rebeil R. Nicholson W.L. Curr. Microbiol. 2005; 51: 331-335Crossref PubMed Scopus (30) Google Scholar). This CXXXCXXC sequence is indeed the signature for a superfamily of [4Fe-4S] iron-sulfur enzymes, named "radical SAM" (12Sofia H.J. Chen G. Hetzler B.G. Reyes-Spindola J.F. Miller N.E. Nucleic Acids Res. 2001; 29: 1097-1106Crossref PubMed Scopus (764) Google Scholar), involved in a variety of biosynthetic pathways and metabolic reactions that proceed via radical mechanisms (13Fontecave M. Mulliez E. Ollagnier-de-Choudens S. Curr. Opin. Chem. Biol. 2001; 5: 506-511Crossref PubMed Scopus (81) Google Scholar, 14Cheek J. Broderick J.B. J. Biol. Inorg. Chem. 2001; 6: 209-226Crossref PubMed Scopus (132) Google Scholar, 15Frey P.A. Booker S.J. Adv. Protein Chem. 2001; 58: 1-45Crossref PubMed Scopus (82) Google Scholar). Spectroscopic and biochemical studies from Nicholson and co-workers (16Rebeil R. Nicholson W.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043Crossref PubMed Scopus (82) Google Scholar) and Broderick and co-workers (17Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (101) Google Scholar) have shown the following: (i) the protein carries a single iron-sulfur cluster (16Rebeil R. Nicholson W.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043Crossref PubMed Scopus (82) Google Scholar); (ii) the reaction is absolutely dependent on S-adenosylmethionine (AdoMet) (16Rebeil R. Nicholson W.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043Crossref PubMed Scopus (82) Google Scholar, 17Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (101) Google Scholar); and (iii) the repair mechanism is likely to involve a 5′-deoxyadenosyl radical (Ado·) generated through reductive cleavage of AdoMet (16Rebeil R. Nicholson W.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043Crossref PubMed Scopus (82) Google Scholar, 17Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (101) Google Scholar). Labeling experiments indicate that the reaction is initiated by direct C-6 hydrogen atom abstraction by Ado·, with the resulting substrate radical undergoing scission to re-generate the two initial thymines (Scheme 2) (17Cheek J. Broderick J.B. J. Am. Chem. Soc. 2002; 124: 2860-2861Crossref PubMed Scopus (101) Google Scholar). The SP lyase enzyme demonstrates a novel aspect of the diversity of DNA repair mechanisms in living organisms. In particular, it is remarkable that in the case of this enzyme, DNA repair is achieved through nucleotide radical intermediates, generally considered as precursors of DNA damage. To better understand the chemistry of this unique enzyme, we undertook this study. The first issues we address concern the substrate specificity of SP lyase. The following still are not known: (i) whether a dinucleotide SP lesion is a substrate; (ii) whether the phosphodiester bridge between the two nucleosides of the lesion is important; and (iii) whether the enzyme is stereoselective. To be able to answer these issues, it is absolutely required that well defined compounds rather than UV-irradiated plasmid DNA are used as substrates. Here we report an original preparation of the SP lesion in the form of a dinucleoside monophosphate (SPTpT) (Scheme 1B). Using a well characterized preparation of recombinant SP lyase from B. subtilis, we show that SPTpT is recognized and efficiently repaired by the enzyme (Scheme 1B). This new substrate provides a convenient assay for further enzymatic studies. Combined with a previous study addressing the question of the stereoselectivity of the enzyme (18Friedel M.G. Berteau O. Pieck J.C. Atta M. Ollagnier-de-Choudens S. Fontecave M. Carell T. Chem. Commun. (Camb.). 2006; 4: 445-447Crossref Google Scholar), this work provides new insights into the chemistry of SP lyase. Materials—The following strains were used in this study. Escherichia coli DH5α was used for routine DNA manipulations. E. coli Tuner (DE3) (Stratagene) was used for SP lyase overexpression. Enzymes, oligonucleotides, and culture media were purchased from Invitrogen. T4 DNA ligase was from Promega. Bacterial alkaline phosphatase and plasmid DNA purification kit, Flexiprep™, were from Amersham Biosciences. DNA fragments were extracted from agarose gel and purified with High Pure PCR product purification kit (Roche Applied Science), and DNA sequencing was performed by Genome Express (Grenoble, France). TpT was prepared as described previously (19Beaucage S.L. Iyer R.P. Tetrahedron. 1992; 48: 2223-2311Crossref Scopus (633) Google Scholar). Cloning and Construction of SP Lyase-overexpressing Plasmids—The SplB gene, encoding SP lyase, was amplified by a PCR-based method using B. subtilis genomic DNA as a template. The following primers were used: 5′-tgtggccatATGcagaacccatttgttccg-3′ (NdeI site underlined and ATG codon in uppercase) hybridized to the noncoding strand at the 5′ terminus of the gene and 5′-tttataaaaaagcttgctgttgatcacaac-3′ (HindIII site underlined) hybridized to the coding strand. PCR was run on a Stratagene RoboCycler Gradient 40 machine. The PCR product was digested with NdeI and HindIII and then ligated with T4 DNA ligase into the pT7-7 plasmid, which had been digested previously with the same restriction enzymes. The cloned gene was entirely sequenced to ensure that no error was introduced during the PCR. The plasmid was then named pT7-SPL. The hexahistidine linker sequence was introduced into pT7-SPL as described previously (20Rebeil R. Sun Y. Chooback L. Pedraza-Reyes M. Kinsland C. Begley T.P. Nicholson W.L. J. Bacteriol. 1998; 180: 4879-4885Crossref PubMed Google Scholar). The new plasmid was named pT7-SPL6H. Protein Expression—E. coli Tuner (DE3) were transformed by pT7-SPL6H and then grown overnight at 37 °C in LB medium (100 ml) supplemented with ampicillin (100 μg/ml). The overnight culture was used to inoculate fresh LB medium (10 liters) supplemented with the same antibiotic, and bacterial growth was allowed to proceed at 37 °C until A600 reached 0.9. To reduce formation of inclusion bodies, protein expression was performed at 18 °C and induced by adding 500 μm of isopropyl 1-thio-β-d-galactopyranoside. Cells were collected after 18 h of culture by centrifugation at 4000 × g at 4 °C for 30 min and resuspended in buffer A (50 mm Tris-HCl, pH 7.5, 200 mm KCl). The cells were disrupted by sonication and centrifuged at 220,000 × g at 4 °C for 1 h and 30 min. The solution obtained was then loaded onto a nickel-nitrilotriacetic acid-Sepharose-superflow column that had been equilibrated previously with buffer A. The column was washed extensively with the same buffer. Nonspecifically adsorbed proteins were eluted by a wash step with buffer A containing 30 mm imidazole, and then the SP lyase was eluted with buffer A containing 0.5 m imidazole. Fractions containing SP lyase were immediately concentrated in an Amicon cell fitted with a YM30 (Spectrapor) membrane, and 3 mm DTT was added before freezing. Aggregation State Analysis—Fast protein liquid chromatography gel filtration with an analytical Superdex-75 (Amersham Biosciences) at a flow rate of 0.5 ml/min, equilibrated with 0.1 m phosphate buffer, pH 7, containing 200 mm KCl, was used for size determination and was performed under strict anaerobic conditions. A gel filtration calibration kit (calibration protein II; Roche Applied Science) was used as molecular weight standards. Iron and Sulfide Binding to SP Lyase—The following procedure was carried out anaerobically in a glove box (Jacomex B553 (NMT)). ApoSP lyase (100 μm monomer) was treated with 5 mm DTT and incubated overnight with a 5-fold molar excess of both Na2S (Fluka) and (NH4)2Fe(SO4)2 (Aldrich) at 6 °C. The protein was desalted on Sephadex G-25 (80 ml, same buffer), and the colored fractions were concentrated on Nanosep 10 (Amicon). Production of SPTpT Substrates—The spore photoproduct of the dinucleoside monophosphate thymidylyl-(3′–5′)-thymidine was prepared in two different ways. It was first obtained by hydrolysis of UVC-irradiated DNA, as described previously (21Douki T. Cadet J. Photochem. Photobiol. Sci. 2003; 2: 433-436Crossref PubMed Scopus (46) Google Scholar). Briefly, dry films of calf thymus DNA (Sigma) were prepared by freeze-drying an aqueous solution and subsequently exposing them to the UVC light emitted by a germicidal lamp. Irradiated DNA was then resuspended in water and digested by sequential incubation with nuclease P1 and phosphodiesterase II, pH 5, and alkaline phosphatase and phosphodiesterase I, pH 8. Normal bases were released as nucleosides, whereas bipyrimidine photoproducts were found as modified dinucleoside monophosphates. SPTpT was purified by HPLC. The concentration of the final solution was determined after acidic hydrolysis of an aliquot and comparison of the intensity of the HPLC signal of the released thymine spore photoproduct with that of an authentic standard. SPTpT was also prepared by exposure of dry films of TpT and dipicolinic acid (DPA) to UVC radiations (Scheme 1B). For this purpose, 300 ml of 1 mm TpT solution containing 10 mm DPA under its sodium form (pH 7) was prepared. The solution was divided in six equal fractions that were poured in six 15-cm diameter Petri dishes and freeze-dried overnight. The dry films were then exposed for 1 h to UVC light. The dry residues were made soluble in 30 ml of water. The freeze-drying/irradiation/solubilization cycle was repeated six times. After the last irradiation, the whole dry residue was made soluble in 10 ml of water. SPTpT was isolated by reverse phase HPLC in the gradient mode by using triethyl ammonium acetate, pH 6.5, and acetonitrile as solvents. Identity and purity of the collected photoproduct were inferred from the comparison of its HPLC-MS/MS features with those of authentic SPTpT isolated from DNA. In addition, an aliquot fraction (100 μl) of the purified SPTpT was mixed with pure formic acid. The resulting solution was heated at 120 °C for 2 h and then dried under vacuum. The residue was made soluble in 100 μl of water and characterized by HPLC-MS/MS. Irradiation conditions were optimized by irradiating 1 × 1-cm dry films prepared from 1 ml of solutions containing 0.3 mm TpT and increasing concentration of either the Ca2+ or the Na+ salt of DPA. SP Lyase Activity—Typically, the reaction mixture contained wild-type SP lyase (1 μm), SPTpT substrate (10 μm), AdoMet (1 mm), and DTT (2 mm) in a final volume of 50 μl of 100 mm Tris-HCl, pH 8, containing 200 mm KCl. In addition, either dithionite (3 mm) or the physiological reducing system (Fldx/Fldx red/NADPH, 4:1:1 μm) was added as a reductant. The reactions were carried out under anaerobic conditions at 37 °C for various periods of time. At each time point (0, 10, 30, 60, 120, and 240 min) 5 μl of the solution was transferred to an Eppendorf tub, and the reaction stopped by flash-freezing in liquid nitrogen. Each sample was then diluted in 45 μl of 2 mm triethyl ammonium acetate and analyzed by HPLC coupled to mass spectrometry for their TpT and SPTpT content. When irradiated calf thymus DNA was used as a substrate, 10 μm enzyme was incubated with AdoMet (1 mm), dithionite (2 mm), DTT (2 mm) in a final volume of 50 μl of 100 mm Tris-HCl, pH 8, containing 200 mm KCl. HPLC-Mass Spectrometry Analysis—Conversion of the spore photoproduct (SPTpT) into the unmodified dinucleoside monophosphate (TpT) in SP lyase-treated samples was quantified by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS). Samples (20 μl from the initial 50 μl) were injected onto an Uptisphere ODB (particle size 3 μm, 150 × 2 mm inner diameter) octadecylsilyl silica gel column (Interchim, Montluçon, France) connected to a series 1100 Agilent chromatographic system. A gradient of acetonitrile (maximum proportion 10%) in 2 mm aqueous triethylammonium acetate was used at a flow rate of 200 μl/min. The eluent was mixed with methanol (100 μl/min) at the outlet of the HPLC column, and the mixture was directed on-line toward the inlet of an API 3000 triple quadrupolar mass spectrometer (SCIEX/Applied Biosystems, Toronto, Canada). The mass spectrometer was used in the multiple reaction monitoring mode. Under these conditions, the ions produced in the electrospray source were filtered in the first quadrupole to select only ions of mass-to-charge ratio of 545 ([M – H]–). The ions were then directed toward a second quadrupole used as a collision cell. Specific fragments, corresponding to the major peaks of the fragmentation spectrum of the compounds of interest, were quantified after selection in a third quadrupole. The monitored transitions were 545 to 251 (loss of the two 2-deoxyribose units and the phosphate group) for SP and 545 to 419 (loss of a thymine moiety) for TpT. A third transition, 545 to 195 (release of phosphorylated 2′-deoxyribose), was also used and gave a signal for both compounds. HPLC-MS/MS was also used to quantify the level of cyclobutane dimers and (6-4) photoproducts within the DNA (22Douki T. Cadet J. Biochemistry. 2001; 40: 2495-2501Crossref PubMed Scopus (257) Google Scholar). For HPLC-MS identification purposes, the molecular mass of the compounds was first determined with the mass spectrometer recording full mass spectra in the 200–600 mass range. In a second step, the pseudo-molecular ion (545 for SPTpT and 251 for its hydrolysis product) was fragmented. Fragmentation spectra were recorded in the 200–600 and 30–260 mass range, respectively. Protein Analysis—Protein concentration (by monomer) was determined by the method of Bradford (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar). Protein-bound iron (24Fish W.W. Methods Enzymol. 1988; 158: 357-364Crossref PubMed Scopus (523) Google Scholar) and labile sulfide (25Beinert H. Anal. Biochem. 1983; 131: 373-378Crossref PubMed Scopus (394) Google Scholar) were determined according to standard procedures. Light Absorption Spectroscopy—UV-visible absorption spectra were recorded with a Cary 1 Bio (Varian) spectrophotometer. EPR—Reconstituted SP lyase (250 μm, 3.9 iron/protein) was reduced with 3 mm dithionite under anaerobic conditions for 20 min and frozen inside the glove box. X-band EPR spectra were recorded on a Bruker Instruments ESP 300D spectrometer equipped with an Oxford Instruments ESR 900 flow cryostat (4.2–300 K). Spectra were quantified under nonsaturating conditions by double integration against a 1 mm Cu-EDTA standard. Mössbauer Spectroscopy—For Mössbauer measurements, reconstituted SP lyase (6.23 mg) was prepared as described above in a total volume of 2.6 ml. After cluster reconstitution (3.9 iron/protein), the mixture was desalted on a NAP-25 column (Amersham Biosciences) and concentrated by ultrafiltration using a Nanosep 30 membrane (Amicon) to a final volume of 400 μl. The protein solution was transferred into a Mössbauer cup and frozen in liquid nitrogen. 57Fe-Mössbauer spectra were recorded at zero magnetic field on a spectrometer operating in constant acceleration mode using an Oxford cryostat that allowed temperatures from 1.5 to 300 K and a 57Co source in rhodium. Isomer shifts are reported relative to metallic iron at room temperature. Characterization of the SP Lyase Enzyme—The enzyme was an N-terminal His-tagged protein purified from extracts of an E. coli strain overexpressing the splB gene from B. subtilis as described under "Experimental Procedures." Analysis by denaturing gel electrophoresis (SDS-PAGE) showed that SP lyase was obtained as nearly pure protein with an apparent molecular mass of 41 kDa, as expected from the amino acid sequence (data not shown). The as-isolated pure protein, which lacked the light absorption bands in the visible spectrum characteristic for [Fe-S] clusters and did not contain measurable amounts of iron and sulfur, had a marked propensity to precipitate that was partly prevented by using buffers containing large concentrations of salt (200 mm KCl). Addition of DTT and glycerol also helped in this matter. Nevertheless, even under these conditions, only 30% of the SP lyase remained soluble after 48 h at 4 °C. We prepared the holo-form of the enzyme by reconstituting the [Fe-S] cluster by treatment of the as-isolated apoform with iron and sulfide salts under anaerobic conditions in accordance with procedures described previously (26Tamarit J. Mulliez E. Meier C. Trautwein A. Fontecave M. J. Biol. Chem. 1999; 274: 31291-31296Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). After reconstitution, the enzyme was found to contain ∼3.5–3.9 iron and sulfur atoms per protein monomer. In the holo-form, and under anaerobic conditions, the protein no longer precipitated and could resist several cycles of freezing-thawing without precipitation, loss of the cluster, or inactivation. Size exclusion chromatography analysis of the reconstituted enzyme under anaerobic conditions showed that SP lyase exists predominantly as a dimer in solution, as shown previously (16Rebeil R. Nicholson W.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9038-9043Crossref PubMed Scopus (82) Google Scholar). The light absorption spectrum of the reconstituted protein, shown in Fig. 1A, is very different from that reported previously (20Rebeil R. Sun Y. Chooback L. Pedraza-Reyes M. Kinsland C. Begley T.P. Nicholson W.L. J. Bacteriol. 1998; 180: 4879-4885Crossref PubMed Google Scholar) and is most similar to spectra of other [Fe-S] proteins of the radical SAM enzyme superfamily (ribonucleotide reductase, MiaB, and HemN) (26Tamarit J. Mulliez E. Meier C. Trautwein A. Fontecave M. J. Biol. Chem. 1999; 274: 31291-31296Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 27Pierrel F. Hernandez H.L. Johnson M.K. Fontecave M. Atta M. J. Biol. Chem. 2003; 278: 29515-29524Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 28Layer G. Verfurth K. Mahlitz E. Jahn D. J. Biol. Chem. 2002; 277: 34136-34142Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It is thus consistent with the presence of a large proportion of polypeptides carrying a [4Fe-4S] cluster in solution. If [2Fe-2S] clusters are present, the UV-visible spectrum indicates that they account for a minor proportion of total iron. Exposure of the protein to air resulted in a rapid degradation of the cluster, as measured by the decrease of the absorption band at 420 nm (data not shown). This explained why the as-isolated protein was in the apo-form. During anaerobic reduction of the reconstituted protein with an excess of sodium dithionite, the solution bleached with a concomitant decrease of the absorption band at 420 nm. The resulting reduced solution displayed an axial EPR spectrum with g values of 2.03 and 1.93 (Fig. 1B). The temperature dependence and microwave power saturation properties of the EPR signals were characteristic for the S = 1/2 ground state of a [4Fe-4S]+1 cluster (data not shown). Integration of the signal indicated that only 10% of total iron present in the sample was in the form of a reduced [4Fe-4S]+1 cluster. This likely results from incomplete reduction of the cluster because of a low redox potential, as generally observed in the radical SAM family of enzymes, and may also be related to partial degradation of the protein under reducing conditions. The following provides the first characterization of the SP lyase iron centers by Mössbauer spectroscopy, using a protein sample that had been anaerobically reconstituted with 57Fe and sulfide. This study was carried out because we are aware of examples for which Mössbauer spectroscopy contradicted UV-visible spectroscopy regarding the nature and diversity of protein-bound clusters. The Mössbauer spectra, obtained at 78 and 4.2 K, are displayed in Fig. 2. At both temperatures, quadrupole doublets were observed. Inspection of the spectra indicates that the protein sample was not homogeneous and contained at least four different species labeled A–D, respectively. In Fig. 2 the solid lines are simulations obtained with four different doublets using the parameters and relative amounts listed in Table 1. Because the left line of the four quadruple doublets coalesces at approximately +0 mm/s, the determination of the hyperfine parameters of these species suffers from a rather large degree of uncertainty. Moreover, there is a significant overlap between doublets A and B, and therefore their relative area ratios cannot be determined accurately. For C and D, the determination of the area is more reliable because the high energy absorption peaks are well resolved. The 4.2 and 78 K spectra are almost identical except for an apparent small decrease of the area attributed to doublet B. The ratios quoted in Table 1 are derived from the spectrum at 4.2 K.TABLE 1Parameters and relative amounts of Mössbauer doublets obtained by simulationsSiteδΔEQAreamm/s%A0.44 (4)1.06 (12)40 (5)B0.33 (5)0.66 (10)27 (5)C0.70 (10)1.98 (20)12 (2)D1.26 (10)2.88 (20)21 (2) Open table in a new tab Doublet D is attributed to octahedral Fe2+ (S = 2) impurities accounting for ∼20% of total iron. Doublet C can also be assigned to ferrous iron (about 10% of total iron), but whether it belongs to an [Fe-S] complex cannot be established. The hyperfine parameters of the major doublet A (Table 1) are consistent with a [4Fe-4S]2+ (S = 0) cluster, accounting for 40% of total iron, and are comparable with parameters reported for clusters of other radical SAM enzymes (29Krebs C. Broderick W.E. Henshaw T.F. Broderick J.B. Huynh B.H. J. Am. Chem. Soc. 2002; 124: 912-913Crossref PubMed Scopus (118) Google Scholar, 30Layer G. Grage K. Teschner T. Schunemann V. Breckau D. Masoumi A. Jahn M. Heathcote P. Trautwein A.X. Jahn D. J. Biol. Chem. 2005; 280: 29038-29046Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 31Mulliez E. Ollagnier-de Choudens S. Meier C. Cremonini M. Luchinat C. Trautwein A.X. Fontecave M. J. Biol. Inorg. Chem. 1999; 4: 614-620Crossref PubMed Scopus (21) Google Scholar, 32Ollagnier-de Choudens S. Sanakis Y. Hewitson K.S. Roach P. Munck E. Fontecave M. J. Biol. Chem. 2002; 277: 13449-13454Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). As is often the case with such enzymes, [2Fe-2S]2+ (S = 0) clusters are also present in the SP lyase preparations. The parameters of doublet B are consistent with such a cluster, and they are in the range of those obtained for [2Fe-2S]2+ clusters found in ribonucleotide reductase-activating enzyme and in biotin synthase (31Mulliez E. Ollagnier-de Choudens S. Meier C. Cremonini M. Luchinat C. Trautwein A.X. Fontecave M. J. Biol. Inorg. Chem. 1999; 4: 614-620Crossref PubMed Scopus (21) Google Scholar, 32Ollagnier-de Choudens S. Sanakis Y. Hewitson K.S. Roach P. Munck E. Fontecave M. J. Biol. Chem. 2002; 277: 13449-13454Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Furthermore, we exclude the presence of [3Fe-4S]+ clusters in SP lyase preparations on the basis of the absence of characteristic S = 1/2 signals in the EPR spectrum (data not shown). As a conclusion the Mössbauer spectra from preparations from anaerobically reconstituted SP lyase indicate a large degree of inhomogeneity regarding the nature of the [Fe-S] clusters, despite a well defined UV-visible spectrum. Apart from the presence of high spin ferrous impurities, the results are consistent with the presence of [2Fe-2S]2+ and [4Fe-4S]2+
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