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• W2034989917 abstract "We report an EPR study of the iron-sulfur enzyme, anaerobic ribonucleotide reductase activase from Lactococcus lactis. The activase (nrdG gene) together withS-adenosyl-l-methionine (AdoMet) give rise to a glycyl radical in the NrdD component. A semi-reduced [4Fe-4S]+ cluster with an axially symmetric EPR signal was produced upon photochemical reduction of the activase. Air exposure of the reduced enzyme gave a [3Fe-4S]+ cluster. The Fe3S4 cluster was convertible to the EPR-active [4Fe-4S]+ cluster by renewed treatment with reducing agents, demonstrating a reversible [3Fe-4S]+- to-[4Fe-4S]+ cluster conversion without exogenous addition of iron or sulfide. Anaerobic reduction of the activase by a moderate concentration of dithionite also resulted in a semi-reduced [4Fe-4S]+cluster. Prolonged reduction gave an EPR-silent fully reduced state, which was enzymatically inactive. Both reduced states gave the [3Fe-4S]+ EPR signal after air exposure. The iron-sulfur cluster interconversion was also studied in the presence of AdoMet. The EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was independent of which reductant was applied, whereas the EPR signal of the [3Fe-4S]+ cluster after air exposure was unchanged. The results indicate that an AdoMet-mediated [4Fe-4S]+center is the native active species that induces the formation of a glycyl radical in the NrdD component. We report an EPR study of the iron-sulfur enzyme, anaerobic ribonucleotide reductase activase from Lactococcus lactis. The activase (nrdG gene) together withS-adenosyl-l-methionine (AdoMet) give rise to a glycyl radical in the NrdD component. A semi-reduced [4Fe-4S]+ cluster with an axially symmetric EPR signal was produced upon photochemical reduction of the activase. Air exposure of the reduced enzyme gave a [3Fe-4S]+ cluster. The Fe3S4 cluster was convertible to the EPR-active [4Fe-4S]+ cluster by renewed treatment with reducing agents, demonstrating a reversible [3Fe-4S]+- to-[4Fe-4S]+ cluster conversion without exogenous addition of iron or sulfide. Anaerobic reduction of the activase by a moderate concentration of dithionite also resulted in a semi-reduced [4Fe-4S]+cluster. Prolonged reduction gave an EPR-silent fully reduced state, which was enzymatically inactive. Both reduced states gave the [3Fe-4S]+ EPR signal after air exposure. The iron-sulfur cluster interconversion was also studied in the presence of AdoMet. The EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was independent of which reductant was applied, whereas the EPR signal of the [3Fe-4S]+ cluster after air exposure was unchanged. The results indicate that an AdoMet-mediated [4Fe-4S]+center is the native active species that induces the formation of a glycyl radical in the NrdD component. ribonucleotide reductase S-adenosyl-l-methionine deazaflavin derivative (10-methyl-10H-pyrimido[4,5-b]quinoline-2,4-dione) milliwatt(s) Ribonucleotide reduction for the synthesis of deoxyribonucleotides for DNA replication or repair is catalyzed by RNR1 and involves free radical chemistry that requires a protein-bound radical (1.Sjöberg B.-M. Reichard P. Gräslund A. Ehrenberg A. J. Biol. Chem. 1977; 252: 536-541Abstract Full Text PDF PubMed Google Scholar) for substrate activation (2.Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (626) Google Scholar, 3.Stubbe J. van der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1368) Google Scholar, 4.Sjöberg B.-M. Struct. Bonding. 1997; 88: 139-173Crossref Google Scholar). RNR is therefore an essential enzyme in all living organisms. An emerging group of RNR enzymes, operating only during anaerobiosis, has recently been identified in facultative and strictly anaerobic microorganisms (5.Fontecave M. Eliasson R. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2147-2151Crossref PubMed Scopus (59) Google Scholar, 6.Reichard P. J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar). These enzymes, coded by thenrdDG genes (7.Sun X. Harder J. Krook M. Jörnvall H. Sjöberg B.-M. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 577-581Crossref PubMed Scopus (98) Google Scholar, 8.Young P. Öhman M. Sjöberg B.-M. J. Biol. Chem. 1994; 269: 27815-27818Abstract Full Text PDF PubMed Google Scholar), are known as class III RNRs (9.Reichard P. Science. 1993; 260: 1773-1777Crossref PubMed Scopus (502) Google Scholar). Up to now, the activating protein (designated activase with an iron-sulfur cluster) of anaerobic RNR from Gram-negative Escherichia coli is the only nrdG gene product that has been investigated at a pure protein level. The activase of anaerobic RNR from Gram-positive Lactococcus lactis (10.Leenhouts K. Buist G. Bolhuis A. ten Berge A. Kiel J. Mierau I. Dabrowska M. Venema G. Kok J. Mol. Gen. Genet. 1996; 253: 217-224Crossref PubMed Scopus (262) Google Scholar) is a new activating enzyme that has recently been isolated (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The goal of this investigation of the activase from L. lactis in comparison with the activase from Gram-negative E. coli was to gain insight into the mechanism of the formation of a catalytically active glycyl radical in anaerobic ribonucleotide reductases. The two bacteria L. lactis and E. coli are extremely distantly related in evolution and are believed to have a common ancestor >2 billion years ago (cf. Ref. 11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Comparison of the functional features of their anaerobic ribonucleotide reductases gives an interesting insight into how evolution has kept the principal features of the iron-sulfur/free radical chemistry more or less intact for such an enormous length of time. The nrdD and nrdG genes form an operon that codes for an active anaerobic RNR. The large NrdD component carries the catalytic and allosteric sites. The small NrdG component is an iron-sulfur protein that participates in the formation of the glycyl radical (12.Sun X. Eliasson R. Pontis E. Andersson J. Buist G. Sjöberg B.-M. Reichard P. J. Biol. Chem. 1995; 270: 2443-2446Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13.Ollagnier S. Mulliez E. Gaillard J. Eliasson R. Fontecave M. Reichard P. J. Biol. Chem. 1996; 271: 9410-9416Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The molecular mass of the activase from L. lactis RNR is 23.3 kDa (199 amino acids)/polypeptide, whereas the molecular masses of the activating enzymes from anaerobic E. coli and bacteriophage T4 are 17.5 and 18 kDa, 2The amino acid sequence of anaerobic RNR activase from L. lactis can be accessed in the GenBankTM/EBI Data Bank under accession number U73336. The molecular mass of the activase (NrdG) from bacteriophage T4 was predicted from the amino acid sequence deposited in the GenBankTM/EBI Data Bank. The gene product of NrdD (but not that of nrdG) from bacteriophage T4 has been purified to homogeneity and investigated at a pure protein level. respectively. A protein-linked glycyl free radical responsible for initiation of physiological catalysis is located in the metal-free NrdD component (6.Reichard P. J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar). The glycyl radical is formed by a complicated activation reaction requiring activase, AdoMet, NADPH, and an auxiliary reducing enzyme system (14.Harder J. Eliasson R. Pontis E. Ballinger M.D. Reichard P. J. Biol. Chem. 1992; 267: 25548-25552Abstract Full Text PDF PubMed Google Scholar). It has been found that photochemically reduced deazaflavin can successfully substitute for the reducing enzyme system and NADPH (15.Mulliez E. Fontecave M. Gaillard J. Reichard P. J. Biol. Chem. 1993; 268: 2296-2299Abstract Full Text PDF PubMed Google Scholar). However, AdoMet is exclusively required for the purified activase to function (16.Eliasson R. Fontecave M. Jörnvall H. Krook M. Pontis E. Reichard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3314-3318Crossref PubMed Scopus (52) Google Scholar). Anaerobic RNR thus falls into a super-group of AdoMet-dependent enzymes (17.Frey P.A. FASEB J. 1993; 7: 662-670Crossref PubMed Scopus (75) Google Scholar), some of which carry protein-bound glycyl free radicals (18.Wagner A.F.V. Frey M. Neugebauer F.A. Schafer W. Knappe J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 996-1000Crossref PubMed Scopus (306) Google Scholar, 19.Frey M. Rothe M. Wagner A.F.V. Knappe J. J. Biol. Chem. 1994; 269: 12432-12437Abstract Full Text PDF PubMed Google Scholar, 20.Ollagnier-de Choudens S. Fontecave M. FEBS Lett. 1999; 453: 25-28Crossref PubMed Scopus (62) Google Scholar, 21.Sawers G. Hesslinger C. Muller N. Kaiser M. J. Bacteriol. 1998; 180: 3509-3516Crossref PubMed Google Scholar). It has been shown that homolytic cleavage of AdoMet is associated with an iron-sulfur complex in the activase from E. coli RNR and leads to formation of 5′-deoxyadenosine and methionine concomitant with the activation of the NrdD component for catalysis (14.Harder J. Eliasson R. Pontis E. Ballinger M.D. Reichard P. J. Biol. Chem. 1992; 267: 25548-25552Abstract Full Text PDF PubMed Google Scholar). Previous studies on anaerobic RNR from E. coli have implied that an Fe4S4 cluster is crucial for radical formation to occur (22.Ollagnier S. Mulliez E. Schmidt P.P. Eliasson R. Gaillard J. Deronzier C. Bergman T. Gräslund A. Reichard P. Fontecave M. J. Biol. Chem. 1997; 272: 24216-24223Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The activase from E. coli is a mixture of monomer and dimer in the absence of NrdD (23.Tamarit 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). It forms a tight α2β2-complex with NrdD, whereas such a tightly bound complex is not found in L. lactis anaerobic reductase (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). L. lactis anaerobic RNR contains a glycyl radical when activated (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The activase is virtually an apoprotein when purified to homogeneity. After incorporation of exogenous ferrous iron and sulfide, the activase is inactive and EPR-silent under O2-free conditions. Direct exposure of this iron-containing protein to air leads to a small fraction of EPR-observable species, due to a cuboidal [3Fe-4S]+ cluster that is associated with a minor fraction of iron in the protein. The majority of the iron-sulfur species remains EPR-silent. In previous work, we found that when the activase is photochemically reduced, it shows an EPR-observable [4Fe-4S]+ center (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Here we report that chemically reduced activase can exist in at least two different oxidation states. An interconversion between [3Fe-4S]+ and [4Fe-4S]+ clusters induced without adding exogenous iron has been followed by EPR spectroscopy in the presence and absence of AdoMet. E. coli strain IG017 (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) containing the nrdG gene of anaerobic RNR fromL. lactis to be overexpressed and lacking active hostnrdDG genes was generously provided by Professor P. Reichard (Karolinska Institute, Stockholm, Sweden). In a typical experiment, cells were grown aerobically in Luria-Bertani medium containing kanamycin (75 μg/ml) at 30 °C and induced by isopropyl-β-d-thiogalactopyranoside at 0.4∼0.5A 600. Protein purification was carried out at 4 °C in 50 mm Tris-HCl (pH 8.0) containing 50 mm KCl, 5 mm dithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride. The packed cells were thawed together with lysozyme from egg white (1 mg/ml; Sigma) and the above buffer. Prior to streptomycin sulfate (3%) and ammonium sulfate (80% saturation) precipitation, the lysate was frozen three times in liquid nitrogen. After a 20-min centrifugation at 18,000 ×g, the pellet was resuspended and dialyzed in 30 mm Tris-HCl (pH 8.0) containing 50 mm KCl and 1 mm dithiothreitol. The sample was diluted to 5 mg/ml and then applied to a DEAE-cellulose column (DE52, Whatman). The column was rinsed with 1 column volume of the same buffer containing 50 mm KCl and 1 mm dithiothreitol. The active fractions were pooled with the elution buffer containing 0.2m KCl and further chromatographed on a Superdex 75 column (Amersham Pharmacia Biotech). The activase-containing fractions were concentrated and dialyzed in an Amicon concentrator with a YM-3 membrane. All further steps were carried out anaerobically. The activase fraction from the Superdex column was incubated with Na2S and (NH4)2Fe(SO4)2·6H2O for 6 h in an ice bath under argon protection in a glove box continuously purged with argon (95%) and hydrogen (5%). Anaerobic desalting by a Sephadex G-25 column (30 ml, superfine; Amersham Pharmacia Biotech) and dialysis were carried out to remove unbound inorganic ions. The final solvent was 50 mm Tris-HCl (pH 8.0) containing 50 mm KCl and 1 mmdithiothreitol. The iron and sulfur assays have been described elsewhere (15.Mulliez E. Fontecave M. Gaillard J. Reichard P. J. Biol. Chem. 1993; 268: 2296-2299Abstract Full Text PDF PubMed Google Scholar). The activase samples prepared by this method, after reconstitution with iron and sulfide, contained 4∼5 iron and 1.4∼2 sulfide ions/polypeptide. Activase concentrations are given per polypeptide dimer, unless otherwise indicated. The reconstituted NrdG component served as starting material for the biophysical studies. The enzyme assay has been described elsewhere (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Iron-sulfur determinations and enzyme assays were carried out by P. Reichard and R. Eliasson as described (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Reduction of the activase protein was accomplished by additions of aliquots of argon-saturated stock solution of sodium dithionite (10∼100 mm) using gas-tight Hamilton syringes and a manifold. Activase was made anaerobic by multiple rounds of gentle degassing and flushing with argon while on ice. Chemical reduction was allowed to proceed under a strictly argon-protected atmosphere at 22 ± 1 °C. The EPR tubes were frozen in liquid nitrogen for measurements by EPR spectroscopy. A deazaflavin derivative (dFl), 10-methyl-10H-pyrimido[4,5-b]quinoline-2,4-dione, was generously provided by Dr. J. Coves (University Joseph Fourier, Grenoble, France). 100 μm dFl was introduced into activase samples (50∼200 μm) in the dark. The samples were anaerobically irradiated by either a daylight lamp at 20 ± 1 °C or a halogen lamp from a Royal AF slide projector (ZETT, Germany) at 0 °C (ice bath) on a time scale from 0 to 80 min. X-band EPR spectra were recorded on a Bruker ESP 300 or Elexsys E-500 spectrometer, both equipped with continuous flow liquid helium cryostats (Oxford Instruments). A dual-mode resonator (ER4116DM) was used in the low temperature measurements. The microwave source operated in dual modes (9.45 and 9.62 GHz, corresponding to parallel and perpendicular mode EPR, respectively). A calibrated frequency counter and a Bruker ER035M NMR gaussmeter were used for the g-value determinations. Quantitation of the EPR signals was performed by double integration of the spectra recorded under nonsaturating conditions and by comparison of the double integrals with that of a standard sample of 1 mm CuCl2 and 10 mm EDTA. Microwave power saturation experiments were carried out by measuring the amplitude of the central component of the EPR signals at 10 K. Saturation curves were evaluated to yield a P 12parameter as described previously (24.Sahlin M. Gräslund A. Ehrenberg A. J. Magn. Reson. 1986; 67: 135-137Google Scholar). EPR powder simulations were achieved using the program SimFonia (Version 1.25, Bruker). This program allows interactive fitting of the simulated resonance pattern to an experimental one. It is based on the perturbation theory, assuming that the electronic Zeeman interaction is the largest, followed by zero-field splitting, the hyperfine interaction, and the nuclear quadrupole interaction. The nuclear hyperfine interaction is the smallest among the five considered interactions. L. lactis RNR activase, after reconstitution with iron and sulfide under strictly anaerobic conditions, is essentially EPR-silent (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). When the reconstituted activase was irradiated by light in the presence of dFl (pH 8.0), it gave an S = 1/2 EPR signal (Fig.1 A, upper trace) detectable up to ∼38 K. The signal had fast relaxation properties and axial symmetry with g ∥ = 2.040 andg ⊥ = 1.935 (TableI). All of these EPR properties are typical of a [4Fe-4S]+ cluster (25.Cammack R. Adv. Inorg. Chem. 1992; 38: 281-322Crossref Scopus (294) Google Scholar, 26.Guigliarelli B. Bertrand P. Adv. Inorg. Chem. 1999; 47: 421-497Crossref Scopus (69) Google Scholar). The spinS = 1/2 ground state of the [4Fe-4S]+cluster arises from an Fe2+-Fe2+ pair and a localized or delocalized (27.Beinert H. Holm R.H. Münck E. Science. 1997; 277: 653-659Crossref PubMed Scopus (1530) Google Scholar) mixed-valence Fe2+-Fe3+ pair. Strong light from a commercial projector was found to be more effective than that of daylight lamps for generation of the EPR signal. After the signal intensity reached a maximum, further illumination did not bring more signal intensity (Fig.1 B). We observed up to 1.1 mmclusters/mm activase protein (polypeptide dimer). Since these samples had a total of 4∼5 iron and 1.4∼2 sulfide ions/polypeptide, this corresponds to about the maximal amount that could be formed in this reduced form (the number varied slightly between different preparations).Table IEPR properties of the iron-sulfur centers of anaerobic ribonucleotide reductase activase in L. lactisIron-sulfur speciesSpin state (S)gvalueP 12 at 10 KSpectrum widthaFrom the maximum of the low-field peak to the minimum of the high-field peak.TemperatureMaximum yield (per activase)g xxg yyg zzOptimalMaximummWmilliteslasK[3Fe-4S]+, [3Fe-4S]+-AdoMet1/22.0042.0152.0330.134.85∼10<300.8[3Fe-4S]+ (E. coli)bFrom Refs. 13, 22, 23, and 45. Part of the E. coli activase data were estimated from figures therein.1/2g⊥ = 2.00, g ∥ = 2.026NDcND, not determined.3.75NDND<0.2[3Fe-4S]+distorted5/24.27 and 9.5713.8ND>52ND[4Fe-4S]+-DTdDT, dithionite.1/2g ⊥= 1.934, g ∥ = 2.024>1018.7112320.25[4Fe-4S]+(E. coli)bFrom Refs. 13, 22, 23, and 45. Part of the E. coli activase data were estimated from figures therein.1/2g ⊥= 1.92, g ∥ = 2.02>1018.26∼10350.7∼0.8[4Fe-4S]+-DT/AdoMet1/21.861.922.000.4325.4310<400.23[4Fe-4S]+-DT/AdoMet (E. coli)bFrom Refs. 13, 22, 23, and 45. Part of the E. coli activase data were estimated from figures therein.1/2g ⊥= 1.91, g ∥ = 2.00ND20.25NDNDND[4Fe-4S]+-dFl1/2g ⊥= 1.935, g ∥ = 2.04014.123.0412381.1[4Fe-4S]+-dFl/AdoMet1/21.861.922.000.4225.4310350.74[4Fe-4S]+-dFl/glycerol1/21.861.922.000.4525.4310310.14[4Fe-4S]+-DT/glycerol (E. coli)1/21.881.922.02∼1021.8∼1030ND[2Fe-2S]+ (E. coli)eFrom Ref. 45, under less complete conditions of anaerobicity (23).1/21.881.932.04ND31.44370NDa From the maximum of the low-field peak to the minimum of the high-field peak.b From Refs. 13.Ollagnier S. Mulliez E. Gaillard J. Eliasson R. Fontecave M. Reichard P. J. Biol. Chem. 1996; 271: 9410-9416Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22.Ollagnier S. Mulliez E. Schmidt P.P. Eliasson R. Gaillard J. Deronzier C. Bergman T. Gräslund A. Reichard P. Fontecave M. J. Biol. Chem. 1997; 272: 24216-24223Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 23.Tamarit 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, and 45.Ollagnier S. Meier C. Mulliez E. Gaillard J. Schuenemann V. Trautwein A. Mattioli T. Lutz M. Fontecave M. J. Am. Chem. Soc. 1999; 121: 6344-6350Crossref Scopus (56) Google Scholar. Part of the E. coli activase data were estimated from figures therein.c ND, not determined.d DT, dithionite.e From Ref. 45.Ollagnier S. Meier C. Mulliez E. Gaillard J. Schuenemann V. Trautwein A. Mattioli T. Lutz M. Fontecave M. J. Am. Chem. Soc. 1999; 121: 6344-6350Crossref Scopus (56) Google Scholar, under less complete conditions of anaerobicity (23.Tamarit 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). Open table in a new tab Exposure of the photochemically reduced activase to air resulted in the EPR signal shown in Fig. 1 C, observable only below 30 K (Table I). The EPR signal is very similar to those of the extensively studied [3Fe-4S]+ clusters in c-aconitase (28.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar,29.Beinert H. Kennedy M.C. Stout C.D. Chem. Rev. 1996; 96: 2335-2373Crossref PubMed Scopus (484) Google Scholar) and endonuclease III (30.Cunningham R.P. Asahara H. Bank J.F. Scholes C.P. Salerno J.C. Surerus K. Münck E. McCracken J. Peisach J. Emptage M.H. Biochemistry. 1989; 28: 4450-4455Crossref PubMed Scopus (203) Google Scholar). This signal is characteristic of an oxidized [3Fe-4S]+ cluster and accounts for >0.8 mm clusters in 1 mm activase. Therefore, the reduced [4Fe-4S]+ cluster almost quantitatively converted to the [3Fe-4S]+ cluster. In addition, a weakg = 4.3 signal attributed to adventitious ferric ions was also observed (data not shown). In comparison, the oxidizedE. coli anaerobic RNR contains a [3Fe-4S]+cluster with a similar but not identical EPR signal (22.Ollagnier S. Mulliez E. Schmidt P.P. Eliasson R. Gaillard J. Deronzier C. Bergman T. Gräslund A. Reichard P. Fontecave M. J. Biol. Chem. 1997; 272: 24216-24223Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), which resembles that of m-aconitase (28.Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar, 29.Beinert H. Kennedy M.C. Stout C.D. Chem. Rev. 1996; 96: 2335-2373Crossref PubMed Scopus (484) Google Scholar). The reduced [4Fe-4S]+ EPR signal reappeared after photochemical reduction in the absence of exogenous iron and sulfide. A second exposure of the reduced sample to air again gave the [3Fe-4S]+ cluster, but with a lower yield than the first exposure (60∼80% yield retained in different experiments). These results demonstrate that the 3-iron cluster must be able to easily pick up an endogenous iron ion upon reduction and assemble a [4Fe-4S]+ cluster in exceptionally high yield. After anaerobic incubation of the reconstituted pure enzyme with a 100-fold excess of dithionite (10 mm, pH 8.0) for 1 h, there was no EPR-detectable species in the L. lactis activase in either perpendicular or parallel mode EPR. Under similar reduction conditions, the activase from the E. coli anaerobic enzyme has been shown to have an S= 1/2 EPR-visible [4Fe-4S]+ center (22.Ollagnier S. Mulliez E. Schmidt P.P. Eliasson R. Gaillard J. Deronzier C. Bergman T. Gräslund A. Reichard P. Fontecave M. J. Biol. Chem. 1997; 272: 24216-24223Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). To explore possible reasons for this apparent deviation between the two activases, a lower concentration of dithionite (1 mm, 10-fold excess) was incubated with the reconstituted L. lactis activase. The mixture was frozen in liquid N2 after various reaction times for the EPR measurements. This series of experiments gave EPR-observable results below 32 K (Fig. 1 B). A representative spectrum (g ∥ = 2.024 andg ⊥ = 1.934) is shown in Fig. 1 A(lower trace). The EPR signal originates from anS = 1/2 ground state species that has fast relaxing characteristics. The optimum temperature for the EPR signal is ∼12 K (Table I). It was broadened at high temperatures, typical of a [4Fe-4S]+ cluster (25.Cammack R. Adv. Inorg. Chem. 1992; 38: 281-322Crossref Scopus (294) Google Scholar, 26.Guigliarelli B. Bertrand P. Adv. Inorg. Chem. 1999; 47: 421-497Crossref Scopus (69) Google Scholar). The EPR signal was observed at a maximum of 0.25 clusters/activase. The signal intensity decayed with continued reduction (Fig. 1 B), indicating the existence of a further, fully reduced form of the activase. This fully reduced form should be the one formed also in the 100-fold excess reduction, where it was reached much more rapidly. We hypothesized that the short-lived semi-reduced form had probably been quickly bypassed under high dithionite concentration (10 mm). In contrast to the case for the E. colianaerobic RNR enzyme, a relatively low concentration of dithionite and short reaction time must therefore be applied to obtain the EPR-active [4Fe-4S]+ cluster-containing form of L. lactisRNR activase. This indicates a considerable difference of redox potentials between the two activating enzymes. Exposure of the semi-reduced activase (0.25 clusters/activase) to air rapidly led to a large EPR signal (∼0.7–0.8 spins/activase), identical to the spectrum shown in Fig.1 C, and so did the fully reduced form. The characteristicg = 2.015 EPR signal has been assigned to a [3Fe-4S]+ cluster. The different signal intensities of the two successively formed EPR-active [4Fe-4S]+ and [3Fe-4S]+ clusters (0.25 and 0.7∼0.8 mmclusters/mm protein, respectively) suggest that both the semi-reduced and fully reduced forms of the activase can be oxidized by air and produce the g = 2.015 EPR signal characteristic of the [3Fe-4S]+ cluster. In contrast, the reconstituted activase (the starting material before the reduction) gave EPR signals corresponding to <0.1 cluster/activase for [3Fe-4S]+ upon admission of air (11.Torrents E. Buist G. Liu A. Eliasson R. Kok J. Gibert I. Gräslund A. Reichard P. J. Biol. Chem. 2000; 275: 2463-2471Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). A direct conclusion is that the majority of the FeS clusters in the dithionite-reduced sample differ distinctly from those of the reconstituted protein, despite the fact that both are EPR-silent. After returning to the anaerobic status, the dithionite-reduced and subsequently air-oxidized sample was incubated with 10-fold excess dithionite for a second time. Again, the semi-reduced S= 1/2 signal ([4Fe-4S]+ cluster) and the fully reduced EPR-silent state were reproduced in succession. The second exposure of the activase to air again restored the EPR-active [3Fe-4S]+ cluster, but with lower yield than the first exposure (60∼80% yield retained). Concomitant with the appearance of less [3Fe-4S]+, we observed an enlarged g= 4.3 EPR signal. A striking observation is that no extra iron or sulfide was required for the [3Fe-4S]+-to-[4Fe-4S]+ cluster interconversion to proceed. Addition of equimolar exogenous Fe2+ resulted in precipitation of the samples. Scheme FS1 summarizes the relationship between different redox forms that have been observed in L. lactis activase and, in particular, their reversibility. The [4Fe-4S]+-containing activase had a significant activating activity together with AdoMet in terms of making the essential glycyl radical in the NrdD protein, whereas the fully reduced activase had no such activity (data not shown). The fully reduced form, derived from prolonged dithionite treatment, is therefore an inactive state of the L. lactis enzyme. A completely new EPR signal with rhombic symmetry was observed in the activase reduced by a moderate concentration of dithionite or dFl plus light in the presence of equimolar AdoMet (Fig.2, traces B and D). Surprisingly, in the absence of AdoMet, an identical EPR signal could be produced by dithionite reduction in the presence of 30% glycerol (Fig. 2, trace E). Similar to the EPR signals in the absence of AdoMet in the reduced activase, the new rhombic signal started to broaden at high temperatures and was best observed around 10 K (TableI). The accumulation of the new signals in the presence of AdoMet was relatively slower (Fig. 3) compared with the data in Fig. 1 B in the absence of AdoMet and glycerol under otherwise identical conditions. The new rhombic EPR signal obtained in the presence of AdoMet or glycerol had relatively slow relaxation properties at 10 K with P 12 ≈ 0.4 mW compared with P 12 > 10 mW for the axial signals seen with only reductants present. The signal is assigned to a [4Fe-4S]+ moiety. The spectra in Fig. 2 (traces A and B) were reproduced by simulation, usingS 1 = S 2 =S 3 = 2 and S 4 = 5/2 for the individual nuclear spins of the iron ions in conjunctio" @default.
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• W2034989917 title "Electron Paramagnetic Resonance Evidence for a Novel Interconversion of [3Fe-4S]+ and [4Fe-4S]+Clusters with Endogenous Iron and Sulfide in Anaerobic Ribonucleotide Reductase Activase in Vitro" @default.
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