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• W2149189957 abstract "l-Serine deaminases catalyze the deamination of l-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of l-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g⊥ = 1.93. Mössbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical Mössbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with l-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster. l-Serine deaminases catalyze the deamination of l-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5′-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5′-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of l-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g∥ = 2.03 and g⊥ = 1.93. Mössbauer spectra of the 57Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S]2+ clusters, as evidenced by the typical Mössbauer parameters-isomer shift, δ = 0.47 mm/s, quadrupole splitting of ΔEQ = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with l-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster. l-Serine can be deaminated by a variety of enzymes of varying degrees of specificity, producing pyruvate and ammonia (see Scheme 1). As described in the Swiss-Prot data base, these enzymes fall into two families, the serine/threonine deaminases (EC 4.3.1.19) and the bacterial l-serine deaminases (EC 4.3.1.17). The first of these, the serine/threonine deaminases, require pyridoxal 5′-phosphate as a cofactor in catalysis. Enzymes within this family that have been characterized to any significant extent include the mammalian liver l-serine deaminase, which serves a gluconeogenic function (1Mudd S.H. Finkelstein J.D. Irreverre F. Laster L. Biochem. Biophys. Res. Commun. 1965; 19: 665-670Google Scholar, 2Ogawa H. Gomi T. Konishi K. Date T. Nakashima H. Nose K. Matsuda Y. Peraino C. Pitot H.C. Fujioka M. J. Biol. Chem. 1989; 264: 15818-15823Google Scholar), and the biosynthetic threonine deaminase (EC 4.3.1.19), which is encoded by the ilvA gene of Escherichia coli. This enzyme deaminates either serine or threonine (3Umbarger H.E. Adv. Enzymol. Relat. Areas Mol. Biol. 1973; 37: 349-395Google Scholar) and is essential for isoleucine biosynthesis. A second threonine deaminase, the biodegradative enzyme, which is encoded by the tdcB gene of E. coli, uses the same cofactor, as does d-serine deaminase of E. coli (4.3.1.18) (4Dowhan Jr., W. Snell E.E. J. Biol. Chem. 1970; 245: 4629-4635Google Scholar, 5Dowhan Jr., W. Snell E.E. J. Biol. Chem. 1970; 245: 4618-4628Google Scholar, 6Marceau M. McFall E. Lewis S.D. Shafer J.A. J. Biol. Chem. 1988; 263: 16926-16933Google Scholar). Moreover, serine is deaminated by enzymes of varying physiological function, including cystathionine γ-synthase (7Brown E.A. D'Ari R. Newman E.B. J. Gen. Microbiol. 1990; 136: 1017-1023Google Scholar) and the β subunit of tryptophan synthase (8Yanofsky C. Crawford I.P. Boyer P.D. 3rd Ed. The Enzymes. 7. Academic Press, New York1972: 1-31Google Scholar). The mechanism of all these enzymes is well understood. The role of the pyridoxal 5′-phosphate cofactor is to facilitate removal of the α-proton of the bound amino acid, allowing for a β-elimination of the hydroxyl group as water. Tautomerization of the resulting α-aminoacrylate results in 2-iminopropionic acid, which is hydrolyzed to ammonia and pyruvate (9Davis L. Metzler D.E. Boyer P.D. The Enzymes. 7. Academic Press, New York1972: 33-74Google Scholar). By contrast, the bacterial l-serine deaminases (LSDs) 1The abbreviations used are: LSD, l-serine deaminase; BSA, bovine serum albumin; DTT, dithiothreitol; EPPS, N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonate; EPR, electron paramagnetic resonance; Fe/S, iron-sulfur; LDH, lactate dehydrogenase; SAM, s-adenosyl-l-methionine; PFL, pyruvate formate-lyase; Tricine, N-[2-hydroxy-1,1-bis-(hydroxymethyl)ethyl]glycine. are activated by iron and are not as well characterized (10Grabowski R. Hofmeister A.E. Buckel W. Trends Biochem. Sci. 1993; 18: 297-300Google Scholar, 11Flint D.H. Allen R.M. Chem. Rev. 1996; 96: 2315-2334Google Scholar). This family of enzymes is exclusively prokaryotic and wide-spread in bacteria, although not omnipresent. In fact, many bacteria elaborate one to three LSDs. In E. coli these proteins are encoded by the sdaA, sdaB, and tdcG genes (12Hesslinger C. Fairhurst S.A. Sawers G. Mol. Microbiol. 1998; 27: 477-492Google Scholar, 13Su H.S. Lang B.F. Newman E.B. J. Bacteriol. 1989; 171: 5095-5102Google Scholar, 14Shao Z. Newman E.B. Eur. J. Biochem. 1993; 212: 777-784Google Scholar); the corresponding protein products are designated as LSD1, LSD2, and TdcG, respectively. All three proteins are very similar in primary structure; LSD2 and TdcG exhibit 77 and 78% identity with LSD1. In contrast to LSD1 and LSD2, TdcG is synthesized only during anaerobic growth (12Hesslinger C. Fairhurst S.A. Sawers G. Mol. Microbiol. 1998; 27: 477-492Google Scholar). The manner in which expression and/or function of these enzymes is regulated is known in some detail, but the mechanism of catalysis is not. LSD1 has been moderately characterized (13Su H.S. Lang B.F. Newman E.B. J. Bacteriol. 1989; 171: 5095-5102Google Scholar, 15Su H. Moniakis J. Newman E.B. Eur. J. Biochem. 1993; 211: 521-527Google Scholar, 16Newman E.B. Walker C. Ziegler-Skylakakis K. Biochem. Cell Biol. 1990; 68: 723-728Google Scholar). It contains 454 amino acids, including 9 cysteines (13Su H.S. Lang B.F. Newman E.B. J. Bacteriol. 1989; 171: 5095-5102Google Scholar). When purified aerobically, the enzyme is inactive until incubated with iron and DTT under aerobic conditions. These characteristics make it very similar to the LSDs from Clostridium acidiurici (17Carter J.E. Sagers R.D. J. Bacteriol. 1972; 109: 757-763Google Scholar), Peptostreptococcus asaccharolyticus (18Grabowski R. Buckel W. Eur. J. Biochem. 1991; 199: 89-94Google Scholar), and Clostridium propionicum (19Hofmeister A.E.M. Grabowski R. Linder D. Buckel W. Eur. J. Biochem. 1993; 215: 341-349Google Scholar), which are all activated in the same manner. Electron paramagnetic resonance (EPR) spectroscopic studies on the P. asaccharolyticus enzyme indicate the presence of [3Fe-4S]+ clusters (20Hofmeister A.E.M. Albracht S.P. Buckel W. FEBS Lett. 1994; 351: 416-418Google Scholar). It is believed, however, that the catalytically active form of the enzyme contains a [4Fe-4S] cluster, in light of the striking similarities between the proposed reaction mechanism of the iron-dependent LSDs and aconitase, which has been studied in detail using a variety of spectroscopic techniques (10Grabowski R. Hofmeister A.E. Buckel W. Trends Biochem. Sci. 1993; 18: 297-300Google Scholar, 21Beinert H. Kennedy M.C. Stout C.D. Chem. Rev. 1996; 96: 2335-2373Google Scholar). Herein, we use a variety of spectroscopic methods to show that treatment of LSD1 with iron and sulfide results in formation of a [4Fe-4S] cluster on the protein and that this state of the protein is essential for catalysis. Reconstituted LSD1 catalyzes the deamination of l-serine with a first-order rate constant (kcat) of 436 s–1, and a Km value of 2.67 ± 0.25 mm. It will also deaminate l-threonine and l-allo-threonine, albeit with greatly reduced efficiency; however, d-serine and l-cysteine are not substrates but serve as competitive inhibitors. Additionally, analyses of EPR spectra recorded on the dithionite-reduced protein in the presence of various substrates and inhibitors are consistent with a direct role in catalysis for the iron-sulfur (Fe/S) cluster as opposed to a simple structural element. Materials—l-Serine and dithiothreitol (DTT) were obtained from ICN Biomedicals Inc. l-Threonine, l-cysteine, lactate dehydrogenase (LDH), sodium sulfide (nonahydrate), molecular weight standards for gel filtration, and ferric chloride were obtained from Sigma. Sodium hydrosulfite (dithionite), potassium ferricyanide, and l-allo-threonine were obtained from Aldrich. Bovine serum albumin (BSA) standard and the Bradford reagent for protein quantification were purchased from Pierce (Rockford, IL). Nicotinamide adenine dinucleotide (reduced form) was obtained from Roche Applied Science. Tryptone and yeast extract were obtained from Marcor Development (Carlstadt, NJ) and Biospringer USA (Milwaukee, WI), respectively. 57Fe metal (97–98%) was obtained from Pennwood Chemicals (Great Neck, NY). It was washed with CHCl3 and dissolved with heating in an anaerobic solution of 1 m H2SO4 (1.5 mol of H2SO4 per mol of 57Fe). Upon dissolution, the pH of the solution was raised to ∼4.5 by the addition of solid sodium bicarbonate immediately before the reconstitution procedure. Spectroscopic Methods—UV-visible spectra were recorded on Cary 50 or Cary 300 spectrometers (Varian, Walnut Creek, CA), employing the associated WinUV software package. The Cary 300 was equipped with a 6 × 6 Peltier-thermostattable multicell holder and an associated temperature controller. Low temperature X-band EPR spectra were recorded in perpendicular mode on a Bruker (Billerica, MA) ESP300 spectrometer equipped with an ER 041 MR microwave bridge and a 4102ST resonator. Sample temperature was maintained with an ITC503S temperature controller and an ESR900 liquid helium cryostat (Oxford Instruments, Concord, MA). Spin concentration was determined by double integration of the sample spectrum obtained under non-saturating conditions and comparing the resulting intensity to that of a standard (1 mm CuSO4, 10 mm EDTA) obtained under identical conditions. General spectral manipulations were performed on personal computers using the WinEPR software package (Bruker) or the IGOR Pro software package (Wavemetrics). Mössbauer spectra were recorded on spectrometers from WEB research (Edina, MN) operating in the constant acceleration mode in a transmission geometry. Spectra were recorded with the temperature of the sample maintained at 4.2 K. For low field spectra, the sample was kept inside an SVT-400 Dewar from Janis (Wilmington, MA), and a magnetic field of 40 millitesla was applied parallel to the γ-beam. For high field spectra, the sample was kept inside a 12SVT Dewar (Janis), which houses a superconducting magnet that allows for application of variable magnetic fields between 0 and 8 tesla parallel to the γ-beam. The quoted isomer shifts are relative to the centroid of the spectrum of a metallic foil of α-Fe at room temperature. Data analysis was performed using the program WMOSS from WEB research. Analytical Procedures and Assays—Protein concentration was determined by the Bradford dye-staining procedure with BSA as the standard (22Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). As determined in this work, this method overestimates the true concentration of LSD1 by a factor of 1.35. Routine chemical analyses of iron and sulfide were carried out by the methods of Beinert (23Kennedy M.C. Kent T.A. Emptage M. Merkle H. Beinert H. Münck E. J. Biol. Chem. 1984; 259: 14463-14471Google Scholar, 24Beinert H. Methods Enzymol. 1978; 54: 435-445Google Scholar, 25Beinert H. Anal. Biochem. 1983; 131: 373-378Google Scholar). The method of Gill and von Hippel (26Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Google Scholar) was used to establish an extinction coefficient for apo-LSD1, which in turn was used to correct the Bradford dye-staining protein assay. Routine gel-filtration of LSD1 was carried out in a Coy Laboratory Products (Grass Lake, MI) anaerobic chamber using Sephadex G-25 resin (Amersham Biosciences). When it was necessary to concentrate the protein, it was carried out using an Amicon stirred-cell ultrafiltration device (Millipore, Billerica, MA) in combination with a YM-10 membrane, which has a molecular mass cutoff of 10 kDa. Ultrafiltration was carried out inside of the anaerobic chamber by threading the tubing from the nitrogen source through one of the chamber's ports. Overexpression and Purification of LSD1—A 200-ml overnight culture of E. coli BL21(DE3) containing plasmid psdaAH6 in terrific broth (100 μg ml–1 ampicillin) was used to inoculate 16 liters of the same media, which was divided evenly among four 6-liter Erlenmeyer flasks. Plasmid psdaAH6 contains the sdaA gene cloned as a fusion construct with a carboxyl-terminal 6× histidine tag that is separated from the last amino acid of the protein by a linker of 2 amino acid residues. The cultures were grown at 37 °C with gentle shaking (200 rpm) and induced at an A600 of 0.5 by addition of solid isopropyl-β-d-thiogalactopyranoside to a final concentration of 400 μm. At induction, solid FeCl3 was also added to each flask to a final concentration of 50 μm. Expression of the sdaA gene was allowed to proceed for 4 h at 37 °C, after which, the cultures were placed in an ice-water bath for 30 min. The bacteria were harvested by centrifugation for 20 min at 10,000 × g and 4 °C, yielding ∼30 g of cells from 16 liters of cell culture after freezing in liquid nitrogen. LSD1 was purified by immobilized metal affinity chromatography using a nickel-nitrilotriacetic acid matrix (Qiagen, Valencia, CA). All steps were carried out inside of the anaerobic chamber under an atmosphere of N2 and H2 (95%/5%), wherein the O2 concentration was maintained below 1 ppm via the use of palladium catalysts. Steps involving centrifugation were carried out outside of the anaerobic chamber; however, samples were loaded into appropriate centrifuge bottles and tightly sealed before removing them. In a typical purification, 30 g of cells was thawed in 60 ml of Buffer A (50 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm DTT, 20 mm imidazole) and allowed to incubate with lysozyme (1 mg ml–1) for 30 min at room temperature. Subsequent to chilling on ice, the cells were lysed by sonic disruption in 4 × 1-min bursts, and the lysate was centrifuged at 50,000 × g for 1 h at 4 °C. The supernatant was loaded onto a nickel-nitrilotriacetic acid column (2.5 × 7 cm) that was equilibrated in Buffer A, and the column was washed with 50 ml of Buffer A containing 10% glycerol and 40 mm imidazole. Finally, the protein was eluted with Buffer A containing 10% glycerol and 250 mm imidazole. Fractions that displayed a brown color were pooled and concentrated using an Amicon stirred-cell and exchanged into Buffer B (50 mm EPPS, pH 8.0, 100 mm NaCl, 10% glycerol, 1 mm DTT) by anaerobic gel filtration. The protein was routinely frozen in small aliquots and stored in liquid N2 until ready for use. Reconstitution of Serine Deaminase—Reconstitution of LSD1 with iron and sulfide was carried out inside of the anaerobic chamber with buffers and solutions that were prepared with deoxygenated water. A typical reconstitution reaction contained 100 μm LSD1, and 8-fold molar excesses of FeCl3 and Na2S in a final volume of 2 ml. The protein was initially treated with a 50-fold molar excess of DTT for 10 min on ice. The FeCl3 was added, and then a solution of Na2S was added dropwise over 10 min. The mixture was allowed to stir gently on ice for 4 h in the anaerobic chamber. It was then placed in centrifuge tubes, capped, and centrifuged at 20,000 × g for 20 min at 4 °C. It was brought back into the anaerobic chamber, and the supernatant was removed and exchanged into Buffer B by anaerobic gel filtration. Activity Determination for Serine Deaminase—LSD1 was first diluted to a final concentration of 5 μm in Buffer B containing 50% glycerol. A typical reaction contained in a final volume of 1 ml: 0.2 mm NADH, 10 units of LDH, 0–20 mm l-serine, 100 mm EPPS, pH 8.0, and 55 nm LSD1. The reaction mixture was prepared inside the anaerobic chamber; all of its components were anaerobic except for LDH. However, the volume of LDH added to the reaction mixture was 1% of the total volume. The reaction mixture, excluding LSD1, was incubated for 3–5 min at 37 °C in a septum-sealed anaerobic cuvette (Starna, Atascadero, CA), and the reaction was initiated by injection of LSD1 via a gas-tight syringe. Enzyme turnover was monitored by a time-dependent decrease in absorption at 340 nm (ϵ340 = 6.22 × 103m–1 cm–1), which is due to NADH oxidation. One unit of activity is defined as 1 μmol of NAD+ produced per minute. When turnover with l-threonine, l-cysteine, l-allo-threonine, or d-serine was monitored, the concentration of LSD1 in the assay was increased to 5.5 μm. The kinetic parameters Km and Vmax were obtained from fits of initial rate data as a function of serine concentration according to Equation 1. In some instances, data were fitted to Equation 2, which accounts for substrate cooperativity. When assays were carried out in the presence of inhibitors, the data were fitted to Equation 3 by multiple non-linear regression using the GraFit software program (27Leatherbarrow R.J. GraFit Version 5. Erithacus Software Ltd., Horley, UK2001Google Scholar). This equation describes competitive inhibition. v=Vmax·[S]/(Km+[S]) (Eq. 1) v=Vmax·[S]n/(K+[S]n) (Eq. 2) v=Vmax·[S]/(Km+(1+(I/KI))·[S])) (Eq. 3) Molecular Sieve Chromatography of LSD1—Molecular sieve chromatography of LSD1 was performed under anaerobic conditions with an ΔKTA (Amersham Biosciences) fast-performance liquid chromatography system, which was maintained inside a Coy anaerobic chamber. The fast-performance liquid chromatography system was equipped with a HiPrep 16/60 Sephacryl S-200 HR column (Amersham Biosciences) and was controlled with the associated software program UNICORN, which was also used for data collection and analysis. The column was equilibrated in an anaerobic solution of Buffer B, and samples (250 μm) and standards (2–10 mg ml–1) were chromatographed at a flow rate of 1 ml min–1 over a time span of 120 min. The proteins, cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and β-amylase (200 kDa), were used to generate a standard curve of known molecular masses, and the void volume (V0) of the column was determined using blue dextran (2000 kDa). The elution volumes (Ve) of the standards were obtained, and the ratios of VeV0–1 were plotted against the log of their respective molecular masses. The standard curve was then used to extrapolate the apparent molecular weight of reconstituted LSD1 from its elution volume. Preparation of Samples for EPR and Mössbauer Spectroscopy—Samples of LSD1 suitable for EPR or Mössbauer spectroscopy were prepared inside of the anaerobic chamber. EPR samples contained 200–300 μm enzyme in Buffer B, were treated with 2 mm (final concentration) sodium dithionite, and frozen immediately in cold isopentane (–150 °C) sitting in a Dewar of liquid nitrogen. In some samples, l-serine, l-threonine, or l-cysteine (20 mm each) was added to the reduced protein before freezing. In all cases, the amount of time that elapsed between the addition of dithionite (and the substrate when appropriate) and the freezing of the sample was less than 30 s. Mössbauer spectra were recorded on proteins that contained 57Fe in place of natural-abundance iron. In all samples, the 57Fe was incorporated into the protein via reconstitution, as described above, and samples contained 250–350 μm protein, and 20 mm l-threonine when appropriate. Samples were frozen within 30 s after adding the substrate to the enzyme. Purification and Characterization of Serine Deaminase—The iron-dependent serine deaminases have been reported to be unstable, losing activity quickly during purification, and to some extent, even in the cell (28Hofmeister A.E.M. Textor S. Buckel W. J. Bacteriol. 1997; 179: 4937-4941Google Scholar, 29Newman E.B. Kapoor V. Can. J. Biochem. 1980; 58: 1292-1297Google Scholar, 30Newman E.B. Dumont D. Walker C. J. Bacteriol. 1985; 162: 1270-1275Google Scholar). To maximize purification of active enzyme, the protein was expressed as a fusion construct with a carboxyl-terminal 6× histidine appendage and purified under anaerobic conditions inside an anaerobic chamber. The eluted protein was light brown in color, and its UV-visible spectrum displayed features, although not prominent, that are consistent with the presence of an Fe/S cluster (Fig. 1, solid line). Among these features, the shoulder at 320 nm and the hump at 416 nm are the most defining, as is the broad tailing that extends beyond 700 nm. An extinction coefficient was determined using apo-LSD1 and the procedure of Gill and von Hippel and was used to correct the Bradford dye-binding protein assay. It was found that the Bradford assay, using BSA as a standard, overestimates the concentration of LSD1 by a factor of 1.35. Analysis of subsequent aliquots of the as-isolated protein indicated the presence of 1.86 ± 0.46 eq of iron and 0.670 ± 0.019 eq of sulfide per polypeptide using the Bradford correction factor. Reconstitution of Serine Deaminase—Treatment of as-isolated LSD1 with 5 mm DTT and 8 eq each of FeCl3 and Na2S resulted in a pronounced change in its color after anaerobic gel filtration, affording a protein that was intensely brown. Analytical methods revealed that the reconstituted protein contained (8.07 ± 0.03) iron and (7.44 ± 0.46) S2– per monomer. The quoted uncertainties are the standard deviation of three independent analyses, and they reflect the individual uncertainties of the protein concentration and the iron and sulfide concentration. The UV-visible spectrum of the reconstituted protein is also shown in Fig. 1 (dashed line). It displays prominent features at 316 and 407 nm. Importantly, the ratio of the feature at 278 nm to that at 407 nm (2.56) decreased significantly from its value in the as-isolated protein (8.33). These features, in combination with the overall spectral envelope of the protein, are most consistent with the formation of [4Fe-4S] clusters upon reconstitution, although it must be emphasized that the types and relative amounts of different Fe/S cluster species cannot be determined with great accuracy from UV-visible spectra. To quantitatively determine the composition of the Fe/S cluster(s) in reconstituted LSD1, Mössbauer and EPR spectroscopic experiments were carried out as described below. Spectroscopic Characterization of Reconstituted Serine Deaminase—Shown in Fig. 2A is the Mössbauer spectrum of 57Fe-reconstituted LSD1 recorded at 4.2 K in an externally applied magnetic field of 40 millitesla oriented parallel to the γ-beam. The prominent features of this spectrum are two sharp lines of similar intensity at –0.13 and +1 mm/s, and a broad peak at 2.7 mm/s. In addition, there is a featureless component extending from –6 to +6 mm/s, and a pronounced shoulder at 0.55 mm/s, which is denoted by an arrow. The two lines at –0.13 and +1 mm/s belong to a quadrupole doublet of isomer shift, δ = 0.47 mm/s, and quadrupole splitting, ΔEQ = 1.14 mm/s. These parameters are consistent with the presence of [4Fe-4S]2+ clusters associated with the protein. The shoulder at +0.55 mm/s is at a position that is typical of the high energy line of a quadrupole doublet originating from [2Fe-2S]2+ clusters, thus raising the possibility that this cluster form is also present in the sample. The broad component ranging from –6 to +6 mm/s, and the peak at 2.7 mm/s, are often caused by adventitiously bound iron species in samples of reconstituted Fe/S proteins. Alternatively, these features could arise from paramagnetic Fe/S cluster species, such as [2Fe-2S]+, [3Fe-4S]+, and [4Fe-4S]+ clusters. It is not possible to distinguish and quantify small amounts of these Fe/S cluster forms using Mössbauer spectroscopy alone if they are present. However, the [2Fe-2S]+, [3Fe-4S]+, and [4Fe-4S]+ clusters all have an S = 1/2 electronic ground state and exhibit characteristic EPR spectra. Consequently, we recorded the EPR spectrum of reconstituted LSD1 in the absence of dithionite. The spectrum reveals the presence of a small fraction of [3Fe-4S]+ clusters, which amounts to 0.02 eq of spin per polypeptide, corresponding to 0.7% of the total iron in the sample (Fig. 3, inset). Such small quantities are beyond the detection limit of Mössbauer spectroscopy and can be neglected for the data analysis. The characteristic features of [2Fe-2S]+ and [4Fe-4S]+ clusters are not observed in the EPR spectrum of reconstituted LSD1 in the absence of dithionite. Therefore, the broad absorption in the Mössbauer spectrum is attributed to adventitiously bound Fe.Fig. 3X-band EPR spectra of reconstituted LSD1 reduced with 2 mm dithionite. Conditions of measurement were: microwave power, 5 milliwatts; receiver gain, 2 × 104; modulation amplitude, 10 G; temperature, 13 K; microwave frequency, 9.65 GHz. Inset: reconstituted LSD1 before reduction with dithionite, obtained under identical conditions.View Large Image Figure ViewerDownload (PPT) To determine the parameters and relative amount of the [4Fe-4S]2+ cluster, and to assess the presence of the [2Fe-2S]2+ clusters more rigorously, the spectral envelope was analyzed by fitting the region from –1 to +2 mm/s. Initially, we fitted the data to only one quadrupole doublet; however, the fit becomes poor at ∼0.5 mm/s, which corresponds to the location of the shoulder. Inclusion of a second quadrupole doublet significantly improves the quality of the fit. The more intense quadrupole doublet accounts for 50 ± 5% of the total absorption and has parameters, δ = 0.47 mm/s and ΔEQ = 1.14 mm/s, that are indicative of the configuration [4Fe-4S]2+. The less intense quadrupole doublet (10 ± 5% of the total absorption) has parameters, δ = 0.28 mm/s and ΔEQ = 0.59 mm/s, that are indicative of the configuration [2Fe-2S]2+. We emphasize that the parameters for the latter component have a greater uncertainty, because its spectral features are overshadowed by the absorption features emanating from the [4Fe-4S]2+ clusters as well as the adventitiously bound Fe, of which the exact shape is not known. Shown as a solid line overlaid with the experimental data in Fig. 2A is the result of the least squares fit; the individual spectral components of the [4Fe-4S]2+ and [2Fe-2S]2+ clusters are plotted above the data as dashed and dotted lines, respectively. To substantiate the presence of [4Fe-4S]2+ and [2Fe-2S]2+ clusters in reconstituted LSD1, we recorded a Mössbauer spectrum in an externally applied field of 8 tesla oriented parallel to the γ-beam (Fig. 2B). [4Fe-4S]2+ and [2Fe-2S]2+ clusters have diamagnetic (S = 0) ground states, resulting in no internal magnetic fields at 4.2 K. Therefore, it is expected that the effective magnetic field at the 57Fe nuclei would equal the externally applied field. The solid line overlaid with the experimental data in Fig. 2B is a simulation using the parameters described above and assuming diamagnetism (S = 0). The individual components are shown as dashed ([4Fe-4S]2+) and dotted lines ([2Fe-2S]2+) above the data. The quality of the simulation is excellent, and it corroborates the presence of [4Fe-4S]2+ and [2Fe-2S]2+ clusters. EPR spectra of the reconstituted enzyme are consistent with the configurations and stoichiometries of Fe/S species determined by Mössbauer spectroscopy. In the absence of dithionite, the enzyme displays a weak and fairly isotropic signal centered at g = 2.019, which accounts for 0.02 eq of spin and which is not observed at temperatures above 50 K at 5-milliwatt power (Fig. 3, inset), suggesting that the signal emanates from [3Fe-4S]+ clusters. Other Fe/S cluster species are not detected. From the analyses of the Mössbauer and EPR spectra, it is possible to determine the stoichiometries of the Fe/S clusters present in recon" @default.
• W2149189957 created "2016-06-24" @default.
• W2149189957 creator A5009467609 @default.
• W2149189957 creator A5029872141 @default.
• W2149189957 creator A5045998043 @default.
• W2149189957 creator A5050467129 @default.
• W2149189957 creator A5065723233 @default.
• W2149189957 creator A5069952389 @default.
• W2149189957 date "2004-07-01" @default.
• W2149189957 modified "2023-10-17" @default.
• W2149189957 title "Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis" @default.
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