VOLUME 120, NUMBER 5 FEBRUARY 11, 1998 © Copyright 1998 by the American Chemical Society
EXAFS Characterization of the Intermediate X Generated During the Assembly of the Escherichia coli Ribonucleotide Reductase R2 Diferric Tyrosyl Radical Cofactor Pamela J. Riggs-Gelasco,*† Lijin Shu,‡ Shuxian Chen,§ Doug Burdi,† Boi Hanh Huynh,§ Lawrence Que, Jr.,‡ and JoAnne Stubbe† Contribution from the Chemistry Department, Massachusetts Institute of Technology, 77 Massachusetts AVenue, Cambridge, Massachusetts 02139, Department of Chemistry, Center for Metals in Biocatalysis, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Physics Department, Emory UniVersity, Atlanta, Georgia 30322 ReceiVed June 3, 1997. ReVised Manuscript ReceiVed October 24, 1997
Abstract: The assembly of the essential diferric cluster/tyrosyl radical cofactor of the R2 subunit of Escherichia coli ribonucleotide reductase from apoR2 with Fe2+ and O2 or diferrous R2 with O2 has been studied by a variety of rapid kinetic methods. An intermediate X, formally an Fe3+/Fe4+ diiron cluster, oxidizes tyrosine 122 to the tyrosyl radical concomitant with its own reduction to the diferric cluster generating the R2 cofactor. To characterize the properties of X, rapid freeze quench methods have been used in conjunction with Mo¨ssbauer, ENDOR, and EPR spectroscopies. These studies are extended here to include rapid freeze quench EXAFS. A short, 2.5 Å Fe-Fe vector and a 1.8 Å Fe-O interaction have been identified in nine independent samples of X. These samples have been generated using both wild-type and a mutant protein in which the essential tyrosine has been replaced by phenylalanine (Y122F). The short Fe-Fe interaction is neither present in diferrous or diferric R2 nor in samples of X that have aged to decay the intermediate. Several structural models which are consistent with the data are presented.
Introduction Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides from ribonucleotides, an essential step in DNA biosynthesis. The aerobic Escherichia coli RNR is a diphosphate reductase (eq 1) and is composed of two nonidentical homodimeric subunits: R1 and R2 (nrdA and nrdB gene products). R1 contains the substrate binding domain and the five redox active cysteines required for reduction.1,2 R2 contains an essential diferric cluster and tyrosyl radical (Y122•).3 The † ‡ §
Massachusetts Institute of Technology. University of Minnesota. Emory University.
R2 cofactor is proposed to generate, via long-range coupled electron and proton transfer,4-6 a thiyl radical on R1 that initiates substrate reduction.7 Given the importance of the tyrosyl radical, the mechanism by which it is generated from diferrousor apoR2 has been studied using a variety of time-resolved physical biochemical methods.8-16 Studies reported herein describe the use of rapid freeze quenching methods (RFQ)17,18 coupled to
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850 J. Am. Chem. Soc., Vol. 120, No. 5, 1998 both EXAFS (extended X-ray absorption fine structure) and Mo¨ssbauer spectroscopy as part of an effort to characterize structurally the diiron intermediate X, which is thought to generate directly Y122• during activation of RNR. Studies of Atkin et al. in 1973 were the first to reveal that this diferric cluster and tyrosyl radical cofactor could be efficiently self-assembled by incubation of apoR2 with Fe2+, O2, and reductant.8 More recently, the stoichiometry of this reaction has been determined by several research groups (eq 2).19-21
Y122-R2 + 2Fe2+ + O2 + e- + H+ f Y122•-R2 + Fe3+(µ-O)Fe3+ + H2O (2) In Vitro, the extra electron required for the 4e- reduction of O2 to H2O can be provided by Fe2+ or by an exogenous reductant such as ascorbate.9 The ability to self-assemble the active cofactor from both apo- and diferrous R2 has allowed the study of the assembly process by various techniques such as Mo¨ssbauer, ENDOR, EPR, stopped flow absorption,8-16 and resonance Raman spectroscopies.22 In 1994, Bollinger et al. reported a detailed kinetic investigation of the reaction of apoR2 with Fe2+ and O2 that revealed an intermediate, designated X, which is kinetically competent in the oxidation of Y122 to Y122•.11-13 More recently, Tong et al. demonstrated that the same intermediate is formed during the reaction of diferrous R2 with O2.16,23 Stopped flow absorption and rapid freeze quenching methods have indicated that X has a broad absorbance maximum at 365 nm, is EPR active, and generates Y122• at 4 °C with a rate constant of ∼1 s-1. Similar studies have been carried out with (1) Stubbe, J. AdV. Enzymol. Relat. Areas Mol. Biol. 1990, 63, 349417. (2) Reichard, P. Science 1993, 260, 1773-1777. (3) Larsson, A.; Sjo¨berg, B.-M. EMBO J. 1986, 5, 2037-2040. (4) Nordlund, P.; Eklund, H. J. Mol. Biol. 1993, 232, 123-164. (5) Stubbe, J. Nature 1990, 370, 502. (6) Ekberg, M.; Sahlin, M.; Eriksson, M.; Sjo¨berg, B.-M. J. Biol. Chem. 1996, 34, 20655-20659. (7) Stubbe, J. J. Biol. Chem. 1990, 265, 5329-5332. (8) Atkin, C. L.; Thelander, L.; Reichard, P.; Lang, G. J. Biol. Chem. 1973, 248, 7464-7472. (9) Bollinger, J. M. J.; Edmondson, D. E.; Huynh, B. H.; Filley, J.; Norton, J. R.; Stubbe, J. Science 1991, 253, 292-298. (10) Bollinger, J. M. J.; Stubbe, J.; Huynh, B. H.; Edmondson, D. E. J. Am. Chem. Soc. 1991, 113, 6289-6291. (11) Bollinger, J. M. J.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024-8032. (12) Bollinger, J. M. J.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmonson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8015-8023. (13) Ravi, N.; Bollinger, J. M. J.; Huynh, B. H.; Stubbe, J.; Edmondson, D. E. J. Am. Chem. Soc. 1994, 116, 8007-8014. (14) Sturgeon, B. E.; Burdi, D.; Chen, S.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 75517557. (15) Burdi, D.; Sturgeon, B. E.; Tong, W. H.; Stubbe, J.; Hoffman, B. M. J. Am. Chem. Soc. 1996, 118, 281-282. (16) Tong, W. H.; Chen, S.; Lloyd, S.; Edmondson, D.; Huynh, B. H.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 2107-2108. (17) Ballou, D. P.; Palmer, G. Anal. Chem. 1974, 46, 1248. (18) Bollinger, J. M.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. In Methods in Enzymology; Klinman, J., Ed.; Academic Press: New York, 1995; Vol. 258. (19) Bollinger, J. M., Ph.D. Thesis, Massachusetts Institute of Technology, 1993. (20) Lynch, J. B.; Juarez-Garcia, C.; Mu¨nck, E.; Que, L., Jr. J. Biol. Chem. 1989, 264, 8091-8096. (21) Ochiai, E.; Mann, G. J.; Gra¨slund, A.; Thelander, L. J. Biol. Chem. 1990, 265, 15758-15761. (22) Ling, J.; Sahlin, M.; Sjo¨berg, B. M.; Loehr, T. M.; Sanders, L. J. J. Biol. Chem. 1994, 269, 5595-5601. (23) Tong, W. H., Ph.D. Thesis, Massachusetts Institute of Technology, 1996.
Riggs-Gelasco et al. Y122F-R2, a mutant in which the essential tyrosine residue is replaced with a less oxidizable phenylalanine, in an effort to increase the amount and lifetime of X. EPR spectroscopy demonstrated that X generated from either wild-type R2 (WTR2) or Y122F-R2 exhibits a nearly isotropic S ) 1/2 signal at g ) 2 that is broadened by carrying out the reconstitution in 17O , H 17O, and 57Fe.9,11 Mo ¨ ssbauer spectroscopy revealed that 2 2 X is composed of two inequivalent Fe sites.13 Using hyperfine values obtained from analysis of the 57Fe Q-band ENDOR spectrum of X, the Mo¨ssbauer isomer shifts have been recalculated to be 0.56 and 0.26 mm/s.14 The former is indicative of high-spin ferric ion, while the latter is intermediate between those observed for high-spin Fe3+ and Fe4+ in both model complexes and proteins.14 The smaller isomer shift and the significant hyperfine anisotropy of the latter site established by ENDOR spectroscopy have led to the latest formulation of X as an Fe3+/Fe4+ pair with significant spin delocalization onto the oxygen ligands.14 1H and 17O ENDOR studies of X generated from Y122F-R2 have also demonstrated that it contains two oxygens from O2: one as a µ-oxo bridge and a second as H2O or as a positionally disordered OH- that can exchange with solvent.24,25 In addition to providing a means to structurally characterize this reactive intermediate, the use of the discontinuous RFQ Mo¨ssbauer and EPR methods has allowed independent confirmation of the rate constants obtained with the continuous method of stopped flow absorption spectroscopy.11-14 Finally, resonance Raman studies of the reconstitution of apo-Y122F-R2 with Fe and 18O2 suggest that the µ-oxo bridge in diferric R2 is derived from O2 gas.22 Formulation of a detailed structure to X has been aided by the availability of the three-dimensional crystal structures of both diferrous R226 and met-diferric R2 (met ) Y122• is reduced).4,27 Scheme 1 summarizes the crystallographic details of the dinuclear iron site in both oxidation states. In the diferrous state, the two four-coordinate iron atoms are separated by 3.9 Å, a consequence of the bis-carboxylato (Glu238 and Glu115) bridging.26 The Fe coordination numbers for the diferrous state differ from those suggested by analysis of recent MCD data,28 where one of the iron atoms is proposed to be 5 coordinate.29 The reason for this discrepancy is presently not understood. The crystal structure4,27 of µ-oxo-bridged diferric R2 indicates that each iron is hexacoordinate with an Fe-Fe distance of 3.3 Å.30 The crystallographic data indicate that the conversion of the diferrous to diferric cluster is accompanied by reorganization of two of the carboxylate ligands. Because of carboxylate shifts, the bridging Glu238 becomes a terminal monodentate ligand and Asp84 changes to a terminal bidentate ligand. A water molecule on each iron completes the coordination spheres. The (24) Willems, J.-P.; Lee, H. I.; Burdi, D.; Doan, P. E.; Stubbe, J.; Hoffman, B. M. J. Am. Chem. Soc. 1997, 119, 9816-9824. (25) Burdi, D.; Willems, J.-P.; Stubbe, J.; Hoffman, B. Manuscript in preparation. (26) Logan, D. T.; Su, X.-D.; A° berg, A.; Regnstrom, K.; Hajdu, J.; Eklund, H.; Nordlund, P. Structure 1996, 4, 1053-1064. (27) Nordlund, P.; Sjo¨berg, B. M.; Eklund, H. Nature (London) 1990, 345, 593-598. (28) Pulver, S. C.; Tong, W. H.; Bollinger, J. M.; Stubbe, J.; Solomon, E. J. Am. Chem. Soc. 1995, 117, 12644-12678. (29) Diferrous R2 for the MCD study was prepared either by incubation of apoR2 with Fe2+ or by reduction of diferric R2 with dithionite. The samples for crystallization were prepared either by photoreduction of metR2 in the X-ray beam (100 K structure, 1.7 Å resolution) or by chemical reduction with a 3% dithionite solution (room-temperature structure, 2.1 Å resolution). (30) The crystallization was carried out at pH 6 in the presence of ethyl mercurithiosalicylate, and the consequences of these conditions on the coordination of the iron environment is presently unknown.
EXAFS Characterization of the Intermediate X Scheme 1
essential tyrosyl radical is 5.3 Å from the nearest Fe and is buried 10 Å from the surface of the protein. The unusual and unprecedented spectroscopic properties of X begs a comparison to the few relevant model systems and to the protein methane monooxygenase (MMO) which catalyzes the formation of methanol from methane with a high-valent iron cluster. Compound Q is proposed to be the kinetically competent intermediate in this oxidation, and it consists of two antiferromagnetically coupled Fe(IV) atoms, on the basis of its Mo¨ssbauer isomer shifts of δ ) 0.14-0.21.31,32 Whereas diamagnetic Q undergoes two-electron reduction to afford diferric MMO and methanol, the paramagnetic X carries out one-electron chemistry in the oxidation of tyrosine to the tyrosyl radical. The crystallographic similarities between active sites of diferrous R2 and MMO26,33 have prompted several groups to speculate that the chemistry of dioxygen activation in these two enzymes proceeds through a common mechanistic framework.34,35 Recently Que and co-workers, using the ligands tris(2pyridylmethyl)amine (TPA) and its ring-methylated derivatives, have generated a family of bis(µ-oxo)diiron compounds that may serve as models for the intermediate X and/or compound Q.34,36-39 Of these synthetic complexes, [Fe3+2(O)2(6-Me3TPA)2](ClO4)2 is crystallographically characterized and exhibits an Fe-Fe distance of 2.71 Å and an unusually small Fe-µ(31) Feig, A. L.; Lippard, S. J. Chem. ReV. 1994, 94, 759-805 and references therein. (32) Wallar, B.; Lipscomb, J. D. Chem. ReV. 1996, 96, 2625-2657 and references therein. (33) Rosenzweig, A. C.; Nordlund, P.; Takahara, P. M.; Frederick, C. A.; Lippard, S. J. Chem. Biol. 1995, 2, 409-418. (34) Que, L., Jr.; Dong, Y. Acc. Chem. Res. 1996, 29, 190-196. (35) Nordlund, P.; Eklund, H. Curr. Opin. Struct. Biol. 1995, 5, 758766. (36) Zang, Y.; Dong, Y.; Que, L., Jr.; Kauffmann, K.; Mu¨nck, E. J. Am. Chem. Soc. 1995, 117, 1169-1170.
J. Am. Chem. Soc., Vol. 120, No. 5, 1998 851 O-Fe angle of 92.5°. Related Fe3+/Fe4+ complexes have been generated by the reaction of diiron(III) precursors with H2O2. EXAFS studies indicate that the resulting products have FeFe distances of 2.9-3.0 Å, consistent with the proposed Fe2(µ-O)2 core. The high-spin Fe3+/Fe4+ 6-Me-TPA compound (6-Me-TPA ) N-(6-methyl-2-pyridylmethyl)-N,N-bis(2-pyridylmethyl)amine)38 (1) in particular exhibits an EPR spectrum that is virtually identical with that of intermediate X, though the properties of the putative Fe4+ site (δ ) 0.08 mm/s; A1, A2, A3 ) 20, 36.5, 36.5 MHz) differ somewhat from those of X (δ ) 0.26 mm/s; A1, A2, A3 ) 27.5, 36.8, 36.8 MHz).14 These spectroscopic similarities suggest that X may have an Fe2(µO)2 core, the presence of which would be manifested by the appearance of a short Fe-Fe distance in the EXAFS spectrum by analogy to those of the model complexes. We have therefore extended the series of rapid freeze quench spectroscopic studies of X to include RFQ EXAFS. Although the use of RFQ methods in conjunction with X-ray absorption spectroscopy has been reported previously, its use has been quite limited.40-43 For X, the composition of the sample can be determined prior to and after the XAS experiment with Mo¨ssbauer spectroscopy, eliminating some of the uncertainty associated with data analysis of a heterogeneous sample. In addition, a high percent composition of X has been generated in the presence of diferrous R2, rather than diferric R2, since the diferrous Fe-Fe vector is less likely to interfere with the identification of an Fe-Fe interaction of X.44 The results described herein demonstrate that the intermediate X in the assembly of the essential cofactor of E. coli RNR has a novel Fe-Fe interaction of 2.5 Å and at least one µ-oxo bridge. These results provide an essential piece to the puzzle with a goal of assigning a detailed structure to X. Experimental Section Sample Preparation. pTB2 and pTB2-Y122F plasmids12 were transformed into the E. coli host strain BL21(DE3) (Novagen). The method of protein purification has been described elsewhere.12 The overexpressed WT-R2 had a specific activity of 7000 U/mg (Units (U) ) nmol/min) using a spectrophotometric assay as previously described.45 A modification of the method of Atkin et al. was used to prepare apo enzyme.8 WT-R2 (30 mg/mL) was incubated for 15 min with 30 mM NH2OH to reduce the tyrosyl radical. The resulting metR2 was then dialyzed for 5-6 h against a solution of 50 mM lithium 8-hydroxyquinoline-5-sulfonate in 1 M imidazole hydrochloride (pH 7, 4 °C). The Fe-chelator complex was separated from the protein by Sephadex G-50 (Sigma) chromatography in 100 mM Hepes (pH 7.7, 4 °C). The Fe2+/protein ratio of the apo protein was less than 0.1 and was determined by colorimetric assay using the iron chelator (37) Dong, Y.; Fujii, H.; Hendrich, M. P.; Leising, R. A.; Pan, G.; Randall, C. R.; Wilkinson, E. C.; Zang, Y.; Que, L., Jr.; Fox, B.; Kauffman, K.; Mu¨nck, E. J. Am. Chem. Soc. 1995, 117, 2778-2792. (38) Dong, Y.; Que, L., Jr.; Kauffman, K.; Munck, E. J. Am. Chem. Soc. 1995, 117, 11377-11378. (39) Kim, C.; Dong, Y.; Que, L., Jr. J. Am. Chem. Soc. 1997, 119, 36353636. (40) George, G. N.; Bray, R. C.; Cramer, S. P. Biochem. Soc. Trans. 1986, 14, 651-652. (41) Saigo, S.; Sone, N.; Nagamura, T.; Oyanagi, H.; Iizuka, T.; Kusunoki, M.; Matsushita, T. In Biophysics and Synchrotron Radiation; Bianconi, A., Castellano, A. C., Eds.; Springer-Verlag: New York, 1986; pp 131-135. (42) Saigo, S.; Hashimoto, H.; Shibayama, N.; Nomura, M.; Nagamura, T. Biochim. Biophys. Acta 1993, 1202, 99-106. (43) Shu, L.; Nesheim, J. C.; Kaufmann, K.; Mu¨nck, E.; Lipscomb, J. D.; Que, L., Jr. Science 1997, 275, 515-518. (44) Unpublished results of L. Que and this work demonstrate the lack of a detectable Fe-Fe vector in diferrous R2. (45) Salowe, S. P.; Stubbe, J. J. Bacteriol. 1986, 165, 363-366.
852 J. Am. Chem. Soc., Vol. 120, No. 5, 1998
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Table 1. Reaction Conditions and Results to Mo¨ssbauer Fitting Analysisa 1c 2 3 4 5 6 7 8 9 10 11 12 13 7wd
sampleb
[R2]f (mM)
Fe2+ (equiv)
rxn time
ferrous (%)a
X (%)
diferric (%)
preload WT X preload WT X preload WT X preload WT X preload WT X preload Y122F X apo-Y122F X apo-Y122F X apo-Y122F X preload WT diferrous as-isolated WT diferric as-isolated Y122F diferric preload WT apo-Y122F X after warming
0.50 0.50 0.50 0.50 0.50 0.50 0.75 0.50 0.50 1.5 1.8 1.8 1.0 1.5
3.5 3.5 3.5 4.0 3.5 3.5 3.0 3.0 3.0 2.3
158 ms 158 ms 158 ms 110 ms 110 ms 195 ms 350 ms 350 ms 442 ms 28 ms
30 32 39 22 20 35 25 35 27 92
60 54 53 58 66 63 68 60 62 0
8 7 8 15 10 2 4 5 8 0
10 4
10 0
78 92
3.5 3.0
1 min
a Uncertainties in quantitation are estimated to be (1-2% and result from imperfect reference spectra and from differences in spectral resolution. Percentages may not add to 100% due to the presence of unknown species that cannot be quantitated. b Preload samples: both syringes were in 100 mM Hepes, pH 7.6; protein syringe is anaerobic. Apo samples: protein was in 100 mM Hepes, pH 7.7 and Fe2+ was in 5 mN H2SO4; both sides were O2 saturated. All samples of X except for sample 4 were generated in the presence of 5 mM sodium ascorbate (initial). c The data for this sample was collected at NSLS, beamline X-9 at 40 K. All other data were collected at 10 K on beamline 7-3 at SSRL. d Composition determined after XAS data acquisition.
ferrozine as previously described.46-48 A solution of 57Fe2+ was prepared from 57Fe0 (Advanced Materials and Technology (nuggets) or Isotec (fine dust); >95%) as described by Sturgeon et al.14 or by incubating 57Fe0 dust at ambient temperature in 1.25 N H2SO4 inside an anaerobic box for 1 week. The solution was diluted with O2-free water to bring the final H+ concentration to
